Rabies lyssavirus (RABV) is enzootic in raccoons across the eastern United States. Intensive management of RABV by oral rabies vaccination (ORV) has prevented its spread westward and shown evidence of local elimination in raccoon populations of the northeastern US. The USDA, Wildlife Services, National Rabies Management Program (NRMP) collaborates with other agencies to implement broad-scale ORV and conducts extensive monitoring to measure the effectiveness of the management. Enhanced Rabies Surveillance (ERS) was initiated during 2005 and updated in 2016 to direct surveillance efforts toward higher-value specimens by assigning points to different methods of encountering specimens for collection (strange-acting, roadkill, surveillance-trapped, etc.; specimen point values ranged from 1 to 15). We used the 2016-2019 data to re-evaluate the point values using a dynamic occupancy model. Additionally, we used ERS data from 2012-2015 and 2016-2019 to examine the impact that the point system had on surveillance data. Implementation of a point system increased positivity rates among specimens by 64%, indicating a substantial increase in the efficiency of the ERS to detect wildlife rabies. Our re-evaluation found that most points accurately reflect the value of the surveillance specimens. The notable exception was that samples from animals found dead were considerably more valuable for rabies detection than originally considered (original points = 5, new points = 20). This work demonstrates how specimen prioritization strategies can be used to refine and improve ERS in support of wildlife rabies management.

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

Kaposi-sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is the causative agent of several malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). Active KSHV replication has also been associated with a pathological condition called KSHV inflammatory cytokine syndrome (KICS), and KSHV may play a role in rare cases of post-transplant polyclonal lymphoproliferative disorders. Several commonly used herpesviral DNA polymerase inhibitors are active against KSHV in tissue culture. Unfortunately, they are not always efficacious against KSHV-induced diseases. To improve the outcome for the patients, new therapeutics need to be developed, including treatment strategies that target either viral proteins or cellular pathways involved in tumor growth and/or supporting the viral life cycle. In this review, we summarize the most commonly established treatments against KSHV-related diseases and review recent developments and promising new compounds that are currently under investigation or on the way to clinical use.

In sporadic Creutzfeldt-Jakob disease, molecular subtypes are neuropathologically well identified by the lesioning profile and the immunohistochemical PrPd deposition pattern in the grey matter (histotypes). While astrocytic PrP pathology has been reported in variant CJD and some less frequent histotypes (e.g., MV2K), oligodendroglial pathology has been rarely addressed. We assessed a series of sCJD cases with the aim to identify particular histotypes that could be more prone to harbor oligodendroglial PrPd. Particularly, the MM2C phenotype, in both its more "pure" and its mixed MM1+2C or MV2K+2C forms, showed more frequent oligodendroglial PrP pathology in the underlying white matter than the more common MM1/MV1 and VV2 histotypes, and was more abundant in patients with a longer disease duration. We concluded that the MM2C strain was particularly prone to accumulate PrPd in white matter oligodendrocytes.

All dating studies involving SARS-CoV-2 are problematic. Previous studies have dated the most recent common ancestor (MRCA) between SARS-CoV-2 and its close relatives from bats and pangolins. However, the evolutionary rate thus derived is expected to differ from the rate estimated from sequence divergence of SARS-CoV-2 lineages. Here, I present dating results for the first time from a large phylogenetic tree with 86,582 high-quality full-length SARS-CoV-2 genomes. The tree contains 83,688 genomes with full specification of collection time. Such a large tree spanning a period of about 1.5 years offers an excellent opportunity for dating the MRCA of the sampled SARS-CoV-2 genomes. The MRCA is dated 16 August 2019, with the evolutionary rate estimated to be 0.05526 mutations/genome/day. The Pearson correlation coefficient (r) between the root-to-tip distance (D) and the collection time (T) is 0.86295. The NCBI tree also includes 10 SARS-CoV-2 genomes isolated from cats, collected over roughly the same time span as human COVID-19 infection. The MRCA from these cat-derived SARS-CoV-2 is dated 30 July 2019, with r = 0.98464. While the dating method is well known, I have included detailed illustrations so that anyone can repeat the analysis and obtain the same dating results. With 16 August 2019 as the date of the MRCA of sampled SARS-CoV-2 genomes, archived samples from respiratory or digestive tracts collected around or before 16 August 2019, or those that are not descendants of the existing SARS-CoV-2 lineages, should be particularly valuable for tracing the origin of SARS-CoV-2.

Genetic prion disease accounts for 10-15% of prion disease. While insertion of four or more octapeptide repeats are clearly pathogenic, smaller repeat insertions have an unclear pathogenicity. The goal of this case series was to provide an insight into the characteristics of the 2-octapeptide repeat genetic variant and to provide insight into the risk for Creutzfeldt-Jakob disease in asymptomatic carriers. 2-octapeptide repeat insertion prion disease cases were collected from the National Prion Disease Pathology Surveillance Center (US), the National Prion Clinic (UK), and the National Creutzfeldt-Jakob Disease Registry (Australia). Three largescale population genetic databases were queried for the 2-octapeptide repeat insertion allele. Eight cases of 2-octapeptide repeat insertion were identified. The cases were indistinguishable from the sporadic Creutzfeldt-Jakob cases of the same molecular subtype. Western blot characterization of the prion protein in the absence of enzymatic digestion with proteinase K revealed that 2-octapeptide repeat insertion and sporadic Creutzfeldt-Jakob disease have distinct prion protein profiles. Interrogation of large-scale population datasets suggested the variant is of very low penetrance. The 2-octapeptide repeat insertion is at most a low-risk genetic variant. Predictive genetic testing for asymptomatic blood relatives is not likely to be justified given the low risk.

Human rotaviruses (HuRVAs) are highly important causes of acute gastroenteritis in infants and young children worldwide. A lack of reliable and reproducible reverse genetics systems for HuRVAs has limited a proper understanding of HuRVA biology and also the rational design of live-attenuated vaccines. Since the development of the first reverse genetics system for RVAs (partially plasmid-based reverse genetics system) in 2006, there have been many efforts with the goal of generating infectious recombinant HuRVAs entirely from cloned cDNAs. However, the establishment of a HuRVA reverse genetics system was very challenging until 2019. This review article provides an overview of the historical background of the recent development of long-awaited HuRVA reverse genetics systems, beginning with the generation of recombinant human-simian reassortant RVAs with the aid of a helper virus in 2006 and the generation of recombinant animal (simian) RVAs in a helper virus-free manner in 2017, and culminating in the generation of recombinant HuRVAs entirely from plasmid cDNAs in 2019. Notably, the original HuRVA reverse genetics system has already been optimized to increase the efficiency of virus generation. Although the application of HuRVA reverse genetics systems has only just been initiated, these technologies will help to answer HuRVA research questions regarding viral replication and pathogenicity that could not be addressed before, and to develop next-generation vaccines and intestine-specific rotaviral vectors.

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.

Ebola virus (EBOV), one of the most infectious human viruses and a leading cause of viral hemorrhagic fever, imposes a potential public health threat with several recent outbreaks. Despite the difficulties associated with working with this pathogen in biosafety level-4 containment, a protective vaccine and antiviral therapeutic were recently approved. However, the high mortality rate of EBOV infection underscores the necessity to continuously identify novel antiviral strategies to help expand the scope of prophylaxis/therapeutic management against future outbreaks. This includes identifying antiviral agents that target EBOV entry, which could improve the management of EBOV infection. Herein, using EBOV glycoprotein (GP)-pseudotyped particles, we screened a panel of natural medicinal extracts, and identified the methanolic extract of Perilla frutescens (PFME) as a robust inhibitor of EBOV entry. We show that PFME dose-dependently impeded EBOV GP-mediated infection at non-cytotoxic concentrations, and exerted the most significant antiviral activity when both the extract and the pseudoparticles are concurrently present on the host cells. Specifically, we demonstrate that PFME could block viral attachment and neutralize the cell-free viral particles. Our results, therefore, identified PFME as a potent inhibitor of EBOV entry, which merits further evaluation for development as a therapeutic strategy against EBOV infection.

On 2 February 2017, Epidemiological Surveillance Services were notified of an outbreak of acute gastroenteritis (AGE) among schoolchildren who had taken part of a school trip from 30 January to 3 February 2017 at a holiday camp in Catalonia. A retrospective cohort study was performed to identify the causative agent, estimate the magnitude of the outbreak and identify its source, as well as to determine the route of transmission. Data collected by standardised questionnaires identified 41 episodes of AGE among 174 individuals who attended the camp. Cases had mainly symptoms of abdominal pain (73.8%), nausea (64.3%), vomiting (54.8%), diarrhoea (45.2%) and headache (42.9%). Consumption of water was associated with gastroenteritis (crude RR: 1.72, 95%CI: 1.01-2.92; adjusted RR: 1.88, 95%CI 1.03-3.56). NoV GII was detected in faeces (5 out of 13) and water samples. Additionally, faecal indicator bacteria and protozoa were detected in water samples. The outbreak showed a high attack rate and was caused by a natural water fountain not properly treated and not monitored for safety quality. There could have been a discharge of wastewater at a point close to the fountain; however, the source of contamination of the water could not be identified. Health education may be useful to eliminate risks associated with the consumption of untreated water from natural fountains.

The incidence of dengue in Latin America has increased dramatically during the last decade. Understanding the pathogenic mechanisms in dengue is crucial for the identification of biomarkers for the triage of patients. We aimed to characterize the profile of cytokines (IFN-γ, TNF-α, IL-1β, IL-6, IL-18 and IL-10), chemokines (CXCL8/IL-8, CCL2/MCP-1 and CXCL10/IP-10) and coagulation mediators (Fibrinogen, D-dimer, Tissue factor-TF, Tissue factor pathway inhibitor-TFPI and Thrombomodulin) during the dengue-4 epidemic in Brazil. Laboratory-confirmed dengue cases had higher levels of TNF-α (p < 0.001), IL-6 (p = 0.005), IL-10 (p < 0.001), IL-18 (p = 0.001), CXCL8/IL-8 (p < 0.001), CCL2/MCP-1 (p < 0.001), CXCL10/IP-10 (p = 0.001), fibrinogen (p = 0.037), D-dimer (p = 0.01) and TFPI (p = 0.042) and lower levels of TF (p = 0.042) compared to healthy controls. A principal component analysis (PCA) distinguished between two profiles of mediators of inflammation and coagulation: protective (TNF-α, IL-1β and CXCL8/IL-8) and pathological (IL-6, TF and TFPI). Lastly, multivariate logistic regression analysis identified high aspartate aminotransferase-to-platelet ratio index (APRI) as independent risk factors associated with severity (adjusted OR: 1.33; 95% CI 1.03-1.71; p = 0.027), the area under the receiver operating characteristics curve (AUC) was 0.775 (95% CI 0.681-0.869) and an optimal cutoff value was 1.4 (sensitivity: 76%; specificity: 79%), so it could be a useful marker for the triage of patients attending primary care centers.

HPV-related head and neck squamous cell carcinoma (HNSCC) has emerged as a diverse clinical and biological disease entity, mainly in young patients with oropharyngeal tumors who are nonsmokers and nondrinkers. Indeed, during the past few years, the pendulum has shifted towards a new epidemiological reality, the "HPV pandemic", where the majority of oropharyngeal squamous cell carcinomas (OPSCCs) are attributed to HPV. The oncogenic potential of the virus is associated to its capacity of integrating oncogenes E6 and E7 into the host cell, leading to the inactivation of several tumor suppressor genes, such as Rb. HPV status can affect prognosis in OPSCC, but its role as a predictive biomarker remains to be elucidated. Given the favorable prognosis associated with HPV-positive disease, the concept of de-escalation treatment strategies has been developed with the primary intent being the reduction of treatment-related long-term toxicities. In this review, we aim to depict current data regarding treatment de-escalation in HPV-associated OPSCC and discuss ongoing clinical trials.

Comment in Indian J Med Res. 2022 May-Jun;155(5&6):575-577.

Phage therapy is an experimental therapeutic approach used to target multidrug-resistant bacterial infections. A lack of reliable data with regard to its efficacy and regulatory hurdles hinders a broad application. Here we report, for the first time, a case of vancomycin-resistant Enterococcus faecium abdominal infection in a one-year-old, critically ill, and three times liver transplanted girl, which was successfully treated with intravenous injections (twice per day for 20 days) of a magistral preparation containing two Enterococcus phages. This correlated with a reduction in baseline C-reactive protein (CRP), successful weaning from mechanical ventilation and without associated clinical adverse events. Prior to clinical use, phage genome was sequenced to confirm the absence of genetic determinants conferring lysogeny, virulence or antibiotic resistance, and thus their safety. Using a phage neutralization assay, no neutralizing anti-phage antibodies in the patient's serum could be detected. Vancomycin-susceptible E. faecium isolates were identified in close relation to phage therapy and, by using whole-genome sequencing, it was demonstrated that vancomycin-susceptible E. faecium emerged from vancomycin-resistant progenitors. Covering a one year follow up, we provide further evidence for the feasibility of bacteriophage therapy that can serve as a basis for urgently needed controlled clinical trials.

Rhinoviruses (RVs) are the main cause of recurrent infections with rather mild symptoms characteristic of the common cold. Nevertheless, RVs give rise to enormous numbers of absences from work and school and may become life-threatening in particular settings. Vaccination is jeopardised by the large number of serotypes eliciting only poorly cross-neutralising antibodies. Conversely, antivirals developed over the years failed FDA approval because of a low efficacy and/or side effects. RV species A, B, and C are now included in the fifteen species of the genus Enteroviruses based upon the high similarity of their genome sequences. As a result of their comparably low pathogenicity, RVs have become a handy model for other, more dangerous members of this genus, e.g., poliovirus and enterovirus 71. We provide a short overview of viral proteins that are considered potential drug targets and their corresponding drug candidates. We briefly mention more recently identified cellular enzymes whose inhibition impacts on RVs and comment novel approaches to interfere with infection via aggregation, virus trapping, or preventing viral access to the cell receptor. Finally, we devote a large part of this article to adding the viral RNA genome to the list of potential drug targets by dwelling on its structure, folding, and the still debated way of its exit from the capsid. Finally, we discuss the recent finding that G-quadruplex stabilising compounds impact on RNA egress possibly via obfuscating the unravelling of stable secondary structural elements.

Since the 2000s, the distribution of bluetongue virus (BTV) has changed, leading to numerous epidemics and economic losses in Europe. Previously, we found a BTV-4 field strain with a higher infection rate of a Culicoides vector than a BTV-1 field strain has. We reverse-engineered parental BTV-1 and BTV-4 strains and created BTV-1/BTV-4 reassortants to elucidate the influence of individual BTV segments on BTV replication in both C. sonorensis midges and in KC cells. Substitution of segment 2 (Seg-2) with Seg-2 from the rBTV-4 significantly increased vector infection rate in reassortant BTV-14S2 (30.4%) in comparison to reverse-engineered rBTV-1 (1.0%). Replacement of Seg-2, Seg-6 and Seg-7 with those from rBTV-1 in reassortant BTV-41S2S6S7 (2.9%) decreased vector infection rate in comparison to rBTV-4 (30.2%). However, triple-reassorted BTV-14S2S6S7 only replicated to comparatively low levels (3.0%), despite containing Seg-2, Seg-6 and Seg-7 from rBTV-4, indicating that vector infection rate is influenced by interactions of multiple segments and/or host-mediated amino acid substitutions within segments. Overall, these results demonstrated that we could utilize reverse-engineered viruses to identify the genetic basis influencing BTV replication within Culicoides vectors. However, BTV replication dynamics in KC cells were not suitable for predicting the replication ability of these virus strains in Culicoides midges.

If viral strains are sufficiently similar in their immunodominant epitopes, then populations of cross-reactive T cells may be boosted by exposure to one strain and provide protection against infection by another at a later date. This type of pre-existing immunity may be important in the adaptive immune response to influenza and to coronaviruses. Patterns of recognition of epitopes by T cell clonotypes (a set of cells sharing the same T cell receptor) are represented as edges on a bipartite network. We describe different methods of constructing bipartite networks that exhibit cross-reactivity, and the dynamics of the T cell repertoire in conditions of homeostasis, infection and re-infection. Cross-reactivity may arise simply by chance, or because immunodominant epitopes of different strains are structurally similar. We introduce a circular space of epitopes, so that T cell cross-reactivity is a quantitative measure of the overlap between clonotypes that recognize similar (that is, close in epitope space) epitopes.

Congenital cytomegalovirus infection (cCMV) is the most common intrauterine infection with central nervous system (CNS) involvement. There is limited data on the associations between Single Nucleotide Polymorphisms (SNPs) in genes involving the first-line defense mechanism and the risk of CNS damage during cCMV. We investigated the associations between neuroimaging findings and SNPs in genes encoding the following cytokines and cytokine receptors in 92 infants with cCMV: interleukins (IL1B rs16944, IL12B rs3212227, IL28B rs12979860), C-C motif chemokine ligand 2 (CCL2 rs1024611), dendritic cell-specific intercellular adhesion grabbing non-integrin (DC-SIGN rs735240), Toll-like receptors (TLR2 rs5743708, TLR4 rs4986791, TLR9 rs352140). The SNP of IL1B rs16944 (G/A) was associated with a reduced risk of ventriculomegaly on MRI (OR = 0.46, 95% CI, 0.22-0.95; p = 0.03) and cUS (OR = 0.38, 95% CI, 0.0-0.93; p = 0.034). Infants carrying heterozygous (T/C) genotype at IL28B rs12979860 had an increased risk of cystic lesions on cUS (OR = 3.31, 95% CI, 1.37-8.01; p = 0.0064) and MRI (OR = 4.97, 95% CI, 1.84-13.43; p = 0.001), and an increased risk of ventriculomegaly on MRI (OR = 2.46, 95% CI, 1.03-5.90; p = 0.04). No other associations between genotyped SNPs and neuroimaging results were found. This is the first study demonstrating new associations between SNPs of IL1B and IL28B and abnormal neuroimaging in infants with cCMV.

Cell-free human cytomegalovirus (HCMV) can be inhibited by a soluble form of the cellular HCMV-receptor PDGFRα, resembling neutralization by antibodies. The cell-associated growth of recent HCMV isolates, however, is resistant against antibodies. We investigated whether PDGFRα-derivatives can inhibit this transmission mode. A protein containing the extracellular PDGFRα-domain and 40-mer peptides derived therefrom were tested regarding the inhibition of the cell-associated HCMV strain Merlin-pAL1502, hits were validated with recent isolates, and the most effective peptide was modified to increase its potency. The modified peptide was further analyzed regarding its mode of action on the virion level. While full-length PDGFRα failed to inhibit HCMV isolates, three peptides significantly reduced virus growth. A 30-mer version of the lead peptide (GD30) proved even more effective against the cell-free virus, and this effect was HCMV-specific and depended on the viral glycoprotein O. In cell-associated spread, GD30 reduced both the number of transferred particles and their penetration. This effect was reversible after peptide removal, which allowed the synchronized analysis of particle transfer, showing that two virions per hour were transferred to neighboring cells and one virion was sufficient for infection. In conclusion, PDGFRα-derived peptides are novel inhibitors of the cell-associated spread of HCMV and facilitate the investigation of this transmission mode.

Next generation influenza vaccines that target conserved epitopes are becoming a clinical reality but still have challenges to overcome. Universal next generation vaccines are considered a vital tool to combat future pandemic viruses and have the potential to vastly improve long-term protection against seasonal influenza viruses. Key vaccine strategies include HA-stem and T cell activating vaccines; however, they could have unintended effects for virus adaptation as they recognise the virus after cell entry and do not directly block infection. This may lead to immune pressure on residual viruses. The potential for immune escape is already evident, for both the HA stem and T cell epitopes, and mosaic approaches for pre-emptive immune priming may be needed to circumvent key variants. Live attenuated influenza vaccines have not been immunogenic enough to boost T cells in adults with established prior immunity. Therefore, viral vectors or peptide approaches are key to harnessing T cell responses. A plethora of viral vector vaccines and routes of administration may be needed for next generation vaccine strategies that require repeated long-term administration to overcome vector immunity and increase our arsenal against diverse influenza viruses.

A final diagnosis in a horse with clinical signs of encephalopathy can be challenging despite the use of extensive diagnostics. Clinical signs are often not pathognomonic and need to be interpreted in combination with (specific) laboratory results and epidemiological data of the geographical region of the origin of the case(s). Here we describe the diagnostic pathway of tick-borne encephalitis virus infection in two horses using established molecular diagnostic methods and a novel in situ hybridization technique to differentiate between regionally important/emerging diseases for central Europe: (i) hepatoencephalopathy, (ii) Borna disease virus, and (iii) West Nile virus infections.

A common viral replication strategy is characterized by the assembly of intracellular compartments that concentrate factors needed for viral replication and simultaneously conceal the viral genome from host-defense mechanisms. Recently, various membrane-less virus-induced compartments and cellular organelles have been shown to represent biomolecular condensates (BMCs) that assemble through liquid-liquid phase separation (LLPS). In the present work, we analyze biophysical properties of intranuclear replication compartments (RCs) induced during human adenovirus (HAdV) infection. The viral ssDNA-binding protein (DBP) is a major component of RCs that contains intrinsically disordered and low complexity proline-rich regions, features shared with proteins that drive phase transitions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse studies in living HAdV-infected cells, we show that DBP-positive RCs display properties of liquid BMCs, which can fuse and divide, and eventually form an intranuclear mesh with less fluid-like features. Moreover, the transient expression of DBP recapitulates the assembly and liquid-like properties of RCs in HAdV-infected cells. These results are of relevance as they indicate that DBP may be a scaffold protein for the assembly of HAdV-RCs and should contribute to future studies on the role of BMCs in virus-host cell interactions.

As a structural protein of the Foot-and-mouth disease virus (FMDV), VP3 plays a vital role in virus assembly and inhibiting the interferon (IFN) signal transduction to promote FMDV replication. Previous studies demonstrated that FMDV VP3 blocks the type-I IFN response by inhibiting the mRNA expression of the mitochondrial antiviral-signaling protein (MAVS); however, the underlying mechanism is poorly understood. Here, we describe the specificity of FMDV VP3 interaction with the transmembrane (TM) domain of MAVS as FMDV driven type-I IFN inhibitory mechanism for its effective replication. The TM domain of MAVS governs the mitochondria localization of MAVS, and it is a key factor in type-I IFN signaling transduction via MAVS aggregation. Thereby, the interaction of FMDV VP3 with the TM domain of MAVS leads to the inhibition of MAVS mitochondria localization, self-association, and aggregation, resulting in the suppression of type-I IFN response. Collectively, these results provide a clear understanding of a key molecular mechanism used by the FMDV VP3 for the suppression of IFN responses via targeting MAVS.

A reliable surveillance strategy of tick-borne encephalitis virus (TBEV) is necessary to ensure adequate disease control measures. However, current approaches assessing geographical TBEV circulation are ineffective or have significant limitations. In this study we investigated a total of 1363 goat and 312 sheep bulk tank milk samples for the presence of TBEV. Samples were collected from systematically selected farms in Lithuania every 4-5 days from April to November in 2018 and 2019. To validate results, we additionally tested 2685 questing ticks collected in the vicinity of milk collection sites. We found 4.25% (95% CI 3.25-5.47) and 4.48% (95% CI 2.47-7.41) goat and sheep milk samples to be positive for TBEV, respectively. Furthermore, geographical distribution of TBEV in milk samples coincided with the known TBE endemic zone and was correlated with incidence of TBE in humans in 2019. When sampling time coincides, TBEV detection in milk samples is as good a method as via flagged ticks, however bulk milk samples can be easier to obtain more frequently and regularly than tick samples. The minimal infectious rate (MIR) in ticks was 0.34% (CI 95% 0.15-0.64). Therefore, our results confirm that testing milk serves as a valuable tool to investigate the spatial distribution of TBEV at higher resolution and lower cost.

Antibodies that can neutralize diverse HIV-1 strains develop in ~10-20% of HIV-1 infected individuals, and their elicitation is a goal of vaccine design. Such antibodies can also serve as therapeutics for those who have already been infected with the virus. Structural characterizations of broadly reactive antibodies in complex with the HIV-1 spike indicate that there are a limited number of sites of vulnerability on the spike. Analysis of their structures can help reveal commonalities that would be useful in vaccine design and provide insights on combinations of antibodies that can be used to minimize the incidence of viral resistance mutations. In this review, we give an update on recent structures determined of the spike in complex with broadly neutralizing antibodies in the context of all epitopes on the HIV-1 spike identified to date.

Comment in Vet Q. 2021 Dec;41(1):321-322.

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

Infectious salmon anemia virus (ISAV) infection is currently detected by fish sampling for PCR and immunohistochemistry analysis. As an alternative to sampling fish, we evaluated two different membrane filters in combination with four buffers for elution, concentration, and detection of ISAV in seawater, during a bath challenge of Atlantic salmon (Salmo salar L.) post-smolts with high and low concentrations of ISAV. Transmission of ISAV in the bath challenge was confirmed by a high mortality, clinical signs associated with ISA disease, and detection of ISAV RNA in organ tissues and seawater samples. The electronegatively charged filter, combined with lysis buffer, gave significantly higher ISAV RNA detection by droplet digital PCR from seawater (5.6 × 104 ISAV RNA copies/L; p < 0.001). Viral shedding in seawater was first detected at two days post-challenge and peaked on day 11 post-challenge, one day before mortalities started in fish challenged with high dose ISAV, demonstrating that a large viral shedding event occurs before death. These data provide important information for ISAV shedding that is relevant for the development of improved surveillance tools based on water samples, transmission models, and management of ISA.

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

Pathogen discovery contributes to our knowledge of bat-borne viruses and is linked to the heightened interest globally in bats as recognised reservoirs of zoonotic agents. The transmission of lyssaviruses from bats-to-humans, domestic animals, or other wildlife species is uncommon, but interest in these pathogens remains due to their ability to cause an acute, progressive, invariably fatal encephalitis in humans. Consequently, the detection and characterisation of bat lyssaviruses continues to expand our knowledge of their phylogroup definition, viral diversity, host species association, geographical distribution, evolution, mechanisms for perpetuation, and the potential routes of transmission. Although the opportunity for lyssavirus cross-species transmission seems rare, adaptation in a new host and the possibility of onward transmission to humans requires continued investigation. Considering the limited efficacy of available rabies biologicals it is important to further our understanding of protective immunity to minimize the threat from these pathogens to public health. Hence, in addition to increased surveillance, the development of a niche pan-lyssavirus vaccine or therapeutic biologics for post-exposure prophylaxis for use against genetically divergent lyssaviruses should be an international priority as these emerging lyssaviruses remain a concern for global public health.

A significant increase in the number of viruses causing unexpected illnesses and epidemics among humans, wildlife and livestock has been observed in recent years. These new or re-emerging viruses have often caught the scientific community off-guard, without sufficient knowledge to combat them, as shown by the current coronavirus pandemic. The bunyaviruses, together with the flaviviruses and filoviruses, are the major etiological agents of viral hemorrhagic fever, and several of them have been listed as priority pathogens by the World Health Organization for which insufficient countermeasures exist. Based on new techniques allowing rapid analysis of the repertoire of protective antibodies induced during infection, combined with atomic-level structural information on viral surface proteins, structural vaccinology is now instrumental in the combat against newly emerging threats, as it allows rapid rational design of novel vaccine antigens. Here, we discuss the contribution of structural vaccinology and the current challenges that remain in the search for an efficient vaccine against some of the deadliest bunyaviruses.

The hepatitis B virus is responsible for most of the chronic liver disease and liver cancer worldwide. As actual therapeutic strategies have had little success in eradicating the virus from hepatocytes, and as lifelong treatment is often required, new drugs targeting the various phases of the hepatitis B virus (HBV) lifecycle are currently under investigation. In this review, we provide an overview of potential future treatments for HBV.

Measles virus (MeV) genotype B3 is one globally significant circulating genotype. Here, we present a systematic description of long-term evolutionary characterizations of the MeV genotype B3's hemagglutinin (H) gene in the elimination era. Our results show that the B3 H gene can be divided into two main sub-genotypes, and the highest intra-genotypic diversity was observed in 2004. MeV genotype B3's H gene diverged in 1976; its overall nucleotide substitution rate is estimated to be 5.697 × 10-4 substitutions/site/year, and is slowing down. The amino acid substitution rate of genotype B3's H gene is also decreasing, and the mean effective population size has been in a downward trend since 2000. Selection pressure analysis only recognized a few sites under positive selection, and the number of positive selection sites is getting smaller. All of these observations may reveal that genotype B3's H gene is not under strong selection pressure, and is becoming increasingly conservative. MeV H-gene or whole-genome sequencing should be routine, so as to better elucidate the molecular epidemiology of MeV in the future.

The applicability and safety of bacteriophage Delta as a potential anti-Pseudomonas aeruginosa agent belonging to genus Bruynoghevirus (family Podoviridae) was characterised. Phage Delta belongs to the species Pseudomonas virus PaP3, which has been described as a temperate, with cos sites at the end of the genome. The phage Delta possesses a genome of 45,970 bp that encodes tRNA for proline (Pro), aspartic acid (Asp) and asparagine (Asn) and does not encode any known protein involved in lysogeny formation or persistence. Analysis showed that phage Delta has 182 bp direct terminal repeats at the end of genome and lysogeny was confirmed, neither upon infection at low nor at high multiplicity of infection (MOI). The turbid plaques that appear on certain host lawns can result from bacteriophage insensitive mutants that occur with higher frequency (10-4). In silico analysis showed that the genome of Delta phage does not encode any known bacterial toxin or virulence factor, determinants of antibiotic resistance and known human allergens. Based on the broad host range and high lytic activity against planktonic and biofilm cells, phage Delta represents a promising candidate for phage therapy.

Update of medRxiv. 2021 Aug 10;:

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host's immune response in different virus-host combinations.

BACKGROUND: The immunological changes associated with COVID-19 are largely unknown. METHODS: Patients with COVID-19 showing moderate (n = 18; SpO2 > 93%, respiratory rate > 22 per minute, CRP > 10 mg/L) and severe (n = 23; SpO2 < 93%, respiratory rate >30 per minute, PaO2/FiO2 ≤ 300 mmHg, permanent oxygen therapy, qSOFA > 2) infection, and 37 healthy donors (HD) were enrolled. Circulating T- and B-cell subsets were analyzed by flow cytometry. RESULTS: CD4+Th cells were skewed toward Th2-like phenotypes within CD45RA+CD62L- (CM) and CD45RA-CD62L- (EM) cells in patients with severe COVID-19, while CM CCR6+ Th17-like cells were decreased if compared with HD. Within CM Th17-like cells "classical" Th17-like cells were increased and Th17.1-like cells were decreased in severe COVID-19 cases. Circulating CM follicular Th-like (Tfh) cells were decreased in all COVID-19 patients, and Tfh17-like cells represented the most predominant subset in severe COVID-19 cases. Both groups of patients showed increased levels of IgD-CD38++ B cells, while the levels of IgD+CD38- and IgD-CD38- were decreased. The frequency of IgD+CD27+ and IgD-CD27+ B cells was significantly reduced in severe COVID-19 cases. CONCLUSIONS: We showed an imbalance within almost all circulating memory Th subsets during acute COVID-19 and showed that altered Tfh polarization led to a dysregulated humoral immune response.

Several vaccines with varying efficacies have been developed and are currently administered globally to minimize the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite having an RNA-dependent RNA polymerase with a proofreading activity, new variants of SARS-CoV-2 are on the rise periodically. Some of the mutations in these variants, especially mutations on the spike protein, aid the virus in transmission, infectivity and host immune evasion. Further, these mutations also reduce the effectiveness of some of the current vaccines and monoclonal antibodies (mAbs). In the present study, using the available 984,769 SARS-CoV-2 nucleotide sequences on the NCBI database from the end of 2019 till 28 July 2021, we have estimated the global prevalence of so-called 'adaptive mutations' and 'mutations identified in the prolonged infections', in the receptor-binding domain (RBD) of the spike (S) protein. Irrespective of the geographical region, in the case of the adaptive mutations, N501Y (48.38%) was found to be the dominant mutation followed by L452R (17.52%), T478K (14.31%), E484K (4.69%), S477N (3.29%), K417T (1.64%), N439K (0.7%) and S494P (0.7%). Other mutations were found to be less prevalent (less than 0.7%). Since the last two months, there has been a massive increase of L452R and T478K mutations (delta variant) in certain areas. In the case of prolonged infections' mutations (long-term SARS-CoV-2 infections), V483A (0.009%) was found to be dominant followed by Q493R (0.009%), while other mutations were found in less than 0.007% of the studied sequences. The data obtained in this study will aid in the development of better infection control policies, thereby curbing the spread of this virus.

The genus Protoparvovirus (family Parvoviridae) includes several viruses of carnivores. We describe a novel fox protoparvovirus, which we named Newlavirus as it was discovered in samples from Newfoundland and Labrador, Canada. Analysis of the full non-structural protein (NS1) sequence indicates that this virus is a previously uncharacterized species. Newlavirus showed high prevalence in foxes from both the mainland (Labrador, 54/137, 39.4%) and the island of Newfoundland (22/50, 44%) but was not detected in samples from other carnivores, including coyotes (n = 92), lynx (n = 58), martens (n = 146), mink (n = 47), ermines (n = 17), dogs (n = 48), and ringed (n = 4), harp (n = 6), bearded (n = 6), and harbor (n = 2) seals. Newlavirus was found at similar rates in stool and spleen (24/80, 30% vs. 59/152, 38.8%, p = 0.2) but at lower rates in lymph nodes (2/37, 5.4%, p < 0.01). Sequencing a fragment of approximately 750 nt of the capsid protein gene from 53 samples showed a high frequency of co-infection by more than one strain (33.9%), high genetic diversity with 13 genotypes with low sequence identities (70.5-87.8%), and no geographic segregation of strains. Given the high prevalence, high diversity, and the lack of identification in other species, foxes are likely the natural reservoir of Newlavirus, and further studies should investigate its distribution.

Wnt/β-catenin signaling is a highly conserved pathway related to a variety of biological processes in different cells. The regulation of replication of various viruses by Wnt/β-catenin signaling pathway has been reported. However, the interaction between the Wnt/β-catenin pathway and avian leukosis virus is unknown. In the present study, we investigated the effect of modulating the Wnt/β-catenin pathway during avian leukosis virus subgroup J (ALV-J) infection. The activation of the Wnt/β-catenin pathway by GSK-3 inhibitor increased ALV-J mRNA, viral protein expression, and virus production in CEF cells. This increase was suppressed by iCRT14, one of the specific inhibitors of the Wnt/β-catenin signaling pathway. Moreover, treatment with iCRT14 reduced virus titer and viral gene expression significantly in CEF and LMH cells in a dose-dependent manner. Inhibition Wnt/β-catenin signaling pathway by knockdown of β-catenin reduced virus proliferation in CEF cells also. Collectively, these results suggested that the status of Wnt/β-catenin signaling pathway modulated ALV-J replication. These studies extend our understanding of the role of Wnt/β-catenin signaling pathway in ALV-J replication and make a new contribution to understanding the virus-host interactions of avian leukosis virus.

Cell-cell fusion is a fundamental and complex process that occurs during reproduction, organ and tissue growth, cancer metastasis, immune response, and infection. All enveloped viruses express one or more proteins that drive the fusion of the viral envelope with cellular membranes. The same proteins can mediate the fusion of the plasma membranes of adjacent cells, leading to the formation of multinucleated syncytia. While cell-cell fusion triggered by alpha- and gammaherpesviruses is well-studied, much less is known about the fusogenic potential of betaherpesviruses such as human cytomegalovirus (HCMV) and human herpesviruses 6 and 7 (HHV-6 and HHV-7). These are slow-growing viruses that are highly prevalent in the human population and associated with several diseases, particularly in individuals with an immature or impaired immune system such as fetuses and transplant recipients. While HHV-6 and HHV-7 are strictly lymphotropic, HCMV infects a very broad range of cell types including epithelial, endothelial, mesenchymal, and myeloid cells. Syncytia have been observed occasionally for all three betaherpesviruses, both during in vitro and in vivo infection. Since cell-cell fusion may allow efficient spread to neighboring cells without exposure to neutralizing antibodies and other host immune factors, viral-induced syncytia may be important for viral dissemination, long-term persistence, and pathogenicity. In this review, we provide an overview of the viral and cellular factors and mechanisms identified so far in the process of cell-cell fusion induced by betaherpesviruses and discuss the possible consequences for cellular dysfunction and pathogenesis.

Dengue is a mosquito-borne viral disease (arboviral) caused by the Dengue virus. It is one of the prominent public health problems in tropical and subtropical regions with no effective vaccines. Every year around 400 million people get infected by the Dengue virus, with a mortality rate of about 20% among the patients with severe dengue. The Dengue virus belongs to the Flaviviridae family, and it is an enveloped virus with positive-sense single-stranded RNA as the genetic material. Studies of the infection cycle of this virus revealed potential host targets important for the virus replication cycle. Here in this review article, we will be discussing different stages of the Dengue virus infection cycle inside mammalian host cells and how host proteins are exploited by the virus in the course of infection as well as how the host counteracts the virus by eliciting different antiviral responses.

In cells, the contributions of DEAD-box helicases (DDXs), without which cellular life is impossible, are of utmost importance. The extremely diverse roles of the nucleolar helicase DDX21, ranging from fundamental cellular processes such as cell growth, ribosome biogenesis, protein translation, protein-protein interaction, mediating and sensing transcription, and gene regulation to viral manipulation, drew our attention. We designed this project to study virus-host interactions and viral pathogenesis. A pulldown assay was used to investigate the association between foot-and-mouth disease virus (FMDV) and DDX21. Further insight into the DDX21-FMDV interaction was obtained through dual-luciferase, knockdown, overexpression, qPCR, and confocal microscopy assays. Our results highlight the antagonistic feature of DDX21 against FMDV, as it progressively inhibited FMDV internal ribosome entry site (IRES) -dependent translation through association with FMDV IRES domains 2, 3, and 4. To subvert this host helicase antagonism, FMDV degraded DDX21 through its non-structural proteins 2B, 2C, and 3C protease (3Cpro). Our results suggest that DDX21 is degraded during 2B and 2C overexpression and FMDV infection through the caspase pathway; however, DDX21 is degraded through the lysosomal pathway during 3Cpro overexpression. Further investigation showed that DDX21 enhanced interferon-beta and interleukin-8 production to restrict viral replication. Together, our results demonstrate that DDX21 is a novel FMDV IRES trans-acting factor, which negatively regulates FMDV IRES-dependent translation and replication.

Lassa virus (LASV)-a member of the family Arenaviridae-causes Lassa fever in humans and is endemic in West Africa. Currently, no approved drugs are available. We screened 2480 small compounds for their potential antiviral activity using pseudotyped vesicular stomatitis virus harboring the LASV glycoprotein (VSV-LASVGP) and a related prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). Follow-up studies confirmed that CP100356 hydrochloride (CP100356), a specific P-glycoprotein (P-gp) inhibitor, suppressed VSV-LASVGP, LCMV, and LASV infection with half maximal inhibitory concentrations of 0.52, 0.54, and 0.062 μM, respectively, without significant cytotoxicity. Although CP100356 did not block receptor binding at the cell surface, it inhibited low-pH-dependent membrane fusion mediated by arenavirus glycoproteins. P-gp downregulation did not cause a significant reduction in either VSV-LASVGP or LCMV infection, suggesting that P-gp itself is unlikely to be involved in arenavirus entry. Finally, our data also indicate that CP100356 inhibits the infection by other mammarenaviruses. Thus, our findings suggest that CP100356 can be considered as an effective virus entry inhibitor for LASV and other highly pathogenic mammarenaviruses.

Despite the probable zoonotic origin of SARS-CoV-2, only limited research efforts have been made to understand the role of companion animals in SARS-CoV-2 epidemiology. According to recent serological prevalence studies, human-to-companion animal transmission is quite frequent, which led us to consider that the risk of SARS-CoV-2 transmission from animal to human, albeit negligible in the present context, may have been underestimated. In this study, we provide the results of a prospective survey that was conducted to evaluate the SARS-CoV-2 isolation rate by qRT-PCR in dogs and cats with different exposure risks and clinical statuses. From April 2020 to April 2021, we analyzed 367 samples and investigated the presence of SARS-CoV-2 RNA using qRT-PCR. Only four animals tested positive, all of them being cats. Three cats were asymptomatic and one presented a coryza-like syndrome. We describe in detail the infection in two cats and the associated clinical characteristics. Importantly, we obtained SARS-CoV-2 genomes from one infected animal and characterized them as Alpha variants. This represents the first identification of the SARS-CoV-2 Alpha variant in an infected animal in France.

Surveillance of the evolving SARS-CoV-2 genome combined with epidemiological monitoring and emerging vaccination became paramount tasks to control the pandemic which is rapidly changing in time and space. Genomic surveillance must combine generation and sharing sequence data with appropriate bioinformatics monitoring and analysis methods. We applied molecular portrayal using self-organizing maps machine learning (SOM portrayal) to characterize the diversity of the virus genomes, their mutual relatedness and development since the beginning of the pandemic. The genetic landscape obtained visualizes the relevant mutations in a lineage-specific fashion and provides developmental paths in genetic state space from early lineages towards the variants of concern alpha, beta, gamma and delta. The different genes of the virus have specific footprints in the landscape reflecting their biological impact. SOM portrayal provides a novel option for 'bioinformatics surveillance' of the pandemic, with strong odds regarding visualization, intuitive perception and 'personalization' of the mutational patterns of the virus genomes.

While self-reported Coronavirus Disease 2019 (COVID-19) symptom checklists have been extensively used during the pandemic, they have not been sufficiently validated from a psychometric perspective. We, therefore, used advanced psychometric modelling to explore the construct validity and internal consistency of an online self-reported COVID-19 symptom checklist and suggested adaptations where necessary. Fit to the Rasch model was examined in a sample of 1638 Austrian citizens who completed the checklist on up to 20 days during a lockdown. The items' fatigue', 'headache' and 'sneezing' had the highest likelihood to be affirmed. The longitudinal application of the symptom checklist increased the fit to the Rasch model. The item 'cough' showed a significant misfit to the fundamental measurement model and an additional dependency to 'dry cough/no sputum production'. Several personal factors, such as gender, age group, educational status, COVID-19 test status, comorbidities, immunosuppressive medication, pregnancy and pollen allergy led to systematic differences in the patterns of how symptoms were affirmed. Raw scores' adjustments ranged from ±0.01 to ±0.25 on the metric scales (0 to 10). Except for some basic adaptations that increases the scale's construct validity and internal consistency, the present analysis supports the combination of items. More accurate item wordings co-created with laypersons would lead to a common understanding of what is meant by a specific symptom. Adjustments for personal factors and comorbidities would allow for better clinical interpretations of self-reported symptom data.

Cyprinid herpesvirus 2 (CyHV-2), a member of the Alloherpesviridae family belonging to the genus Cyprinivirus, is a fatal contagious aquatic pathogen that affects goldfish (Carassius auratus) and crucian carp (Carassius carassius). Although crucian carp and goldfish belong to the genus Carassius, it is unclear whether they are susceptible to the same CyHV-2 isolate. In addition, the origin of the crucian carp-derived CyHV-2 virus isolate remains unclear. CyHV-2 SH01 was isolated during herpesviral hematopoietic necrosis disease (HVHN) outbreaks in crucian carp at a local fish farm near Shanghai. CyHV-2 SH01 was confirmed by PCR and Western blot analysis of kidney, spleen, muscle, and blood tissue from the diseased crucian carp. Moreover, histopathological and ultra-pathological analyses revealed pathological changes characteristic of CyHV-2 SH01 infection in the tissues of the diseased crucian carp. In the present study, goldfish and crucian carp were challenged with CyHV-2 SH01 to elucidate viral virulence. We found that CyHV-2 SH01 could cause rapid and fatal disease progression in goldfish and crucian carp 24 h post-injection at 28 °C. Experimental infection of goldfish by injection indicated that the average virus titer in the kidney of the goldfish was 103.47 to 103.59 copies/mg. In addition, tissues exhibited the most prominent histopathological changes (cellular wrinkling and shrinkage, cytoplasmic vacuolation, fusion of the gill lamellae, and hepatic congestion) in CyHV-2 SH01-infected goldfish and crucian carp. Thus, crucian carp and goldfish showed a high sensitivity, with typical symptoms, to HVHN disease caused by CyHV-2 SH01.

Influenza A viruses (IAV) can infect a broad range of mammalian and avian species. However, the host innate immune system provides defenses that restrict IAV replication and infection. Likewise, IAV have evolved to develop efficient mechanisms to counteract host antiviral responses to efficiently replicate in their hosts. The IAV PA-X and NS1 non-structural proteins are key virulence factors that modulate innate immune responses and virus pathogenicity during infection. To study the determinants of IAV pathogenicity and their functional co-evolution, we evaluated amino acid differences in the PA-X and NS1 proteins of early (1996-1997) and more recent (since 2016) H5N1 IAV. H5N1 IAV have zoonotic and pandemic potential and represent an important challenge both in poultry farming and human health. The results indicate that amino acid changes occurred over time, affecting the ability of these two non-structural H5N1 IAV proteins to inhibit gene expression and affecting virus pathogenicity. These results highlight the importance to monitor the evolution of these two virulence factors of IAV, which could result in enhanced viral replication and virulence.

Oncolytic virus (OV) as a promising therapeutic agent can selectively infect and kill tumor cells with naturally inherited or engineered properties. Considering the limitations of OVs monotherapy, combination therapy has been widely explored. MEK inhibitor (MEKi) Trametinib is an FDA-approved kinase inhibitor indicated for the treatment of tumors with BRAF V600E or V600K mutations. In this study, the oncolytic activity in vitro and anti-tumor therapeutic efficacy in vivo when combined with oHSV and MEKi Trametinib were investigated. We found: (1) Treatment with MEKi Trametinib augmented oHSV oncolytic activity in BRAF V600E-mutated tumor cells. (2) Combination treatment with oHSV and MEKi Trametinib enhanced virus replication mediated by down-regulation of STAT1 and PKR expression or phosphorylation in BRAF V600E-mutated tumor cells as well as BRAF wt/KRAS-mutated tumor cells. (3) A remarkably synergistic therapeutic efficacy was shown in vivo for BRAF wt/KRAS-mutated tumor models, when a combination of oHSV including PD-1 blockade and MEK inhibition. Collectively, these data provide some new insights for clinical development of combination therapy with oncolytic virus, MEK inhibition, and checkpoint blockade for BRAF or KRAS-mutated tumors.

Respiratory Syncytial Virus (RSV) is the leading cause of acute lower respiratory infections in young children and infection has been linked to the development of persistent lung disease in the form of wheezing and asthma. Despite substantial research efforts, there are no RSV vaccines currently available and an effective monoclonal antibody targeting the RSV fusion protein (palivizumab) is of limited general use given the associated expense. Therefore, the development of novel approaches to prevent RSV infection is highly desirable to improve pediatric health globally. We have developed a method to generate alveolar-like macrophages (ALMs) from pluripotent stem cells. These ALMs have shown potential to promote airway innate immunity and tissue repair and so we hypothesized that ALMs could be used as a strategy to prevent RSV infection. Here, we demonstrate that ALMs are not productively infected by RSV and prevent the infection of epithelial cells. Prevention of epithelial infection was mediated by two different mechanisms: phagocytosis of RSV particles and release of an antiviral soluble factor different from type I interferon. Furthermore, intratracheal administration of ALMs protected mice from subsequent virus-induced weight loss and decreased lung viral titres and inflammation, indicating that ALMs can impair the pathogenesis of RSV infection. Our results support a prophylactic role for ALMs in the setting of RSV infection and warrant further studies on stem cell-derived ALMs as a novel cell-based therapy for pulmonary viral infections.

Ever since its brief introduction in the Brazilian territory in 1981, dengue virus serotype 4 (DENV-4) remained absent from the national epidemiological scenario for almost 25 years. The emergence of DENV-4 in 2010 resulted in epidemics in most Brazilian states. DENV-4, however, remains one of the least studied among the four DENV serotypes. Despite being known as a mild serotype, DENV-4 is associated with severe cases and deaths and deserves to be investigated; however, the lack of suitable experimental animal models is a limiting factor for pathogenesis studies. Here, we aimed to investigate the susceptibility and potential tropism of DENV-4 for liver, lung and heart of an immunocompetent mice model, and to evaluate and investigate the resulting morphological and ultrastructural alterations upon viral infection. BALB/c mice were inoculated intravenously with non-neuroadapted doses of DENV-4 isolated from a human case. The histopathological analysis of liver revealed typical alterations of DENV, such as microsteatosis, edema and vascular congestion, while in lung, widespread areas of hemorrhage and interstitial pneumonia were observed. While milder alterations were present in heart, characterized by limited hemorrhage and discrete presence of inflammatory infiltrate, the disorganization of the structure of the intercalated disc is of particular interest. DENV-4 RNA was detected in liver, lung, heart and serum of BALB/c mice through qRT-PCR, while the NS3 viral protein was observed in all of the aforementioned organs through immunohistochemistry. These findings indicate the susceptibility of the model to the serotype and further reinforce the usefulness of BALB/c mice in studying the many alterations caused by DENV.

Globally and in all age groups, noroviruses are a main cause of gastroenteritis. To assess their local epidemiology and genetic diversity, stool samples of 7509 inpatients with gastrointestinal complaints from all age groups were analyzed. After detection of norovirus genogroup I and II RNA by real-time RT-PCR, viral capsids were genotyped by partial nucleic acid sequencing. In the case of GII.2 strains, polymerase genotypes were also assessed. Between October 2013 and September 2017, presence of norovirus RNA was shown in 611 samples (8.1%), of which 610 (99.8%) were typed successfully. Norovirus positivity rate was higher in patients aged below five years (14.8%) than in older patients (5.7%). Among the 611 norovirus positive samples, GII.4 (56.6%) strains prevailed, followed by GII.6 (11.3%), GII.3 (11.0%) and GII.2 (9.5%). The most common genogroup I (GGI) genotype was GI.3 (3.6%). In addition, rare genotypes such as GII.13, GII.14 and GII.26 were detected. Interestingly, GII.3 infections were most common in children under the age of five years. Assessment of polymerase genotypes in GII.2 viruses showed a shift from P2 to P16, with higher diversity in P2 sequences. The varying distribution of norovirus genotypes depending on season, age and setting of infection highlights the importance of frequent genotyping as a basis for vaccine development and needful adjustments.

Campylobacter jejuni is a Gram-negative foodborne pathogen that causes diarrheal disease and is associated with severe post-infectious sequelae. Bacteriophages (phages) are a possible means of reducing Campylobacter colonization in poultry to prevent downstream human infections. However, the factors influencing phage-host interactions must be better understood before this strategy can be predictably employed. Most studies have focused on Campylobacter phage binding to the host surface, with all phages classified as either capsule- or flagella-specific. Here we describe the characterization of a C. jejuni phage that requires functional flagellar glycosylation and motor genes for infection, without needing the flagella for adsorption to the cell surface. Through phage infectivity studies of targeted C. jejuni mutants, transcriptomic analysis of phage-resistant mutants, and genotypic and phenotypic analysis of a spontaneous phage variant capable of simultaneously overcoming flagellar gene dependence and sensitivity to oxidative stress, we have uncovered a link between oxidative stress, flagellar motility, and phage infectivity. Taken together, our results underscore the importance of understanding phage-host interactions beyond the cell surface and point to host oxidative stress state as an important and underappreciated consideration for future phage-host interaction studies.

Ever since the immune regulatory strains of lymphocytic choriomeningitis virus (LCMV), such as Clone 13, were isolated, LCMV infection of mice has served as a valuable model for the mechanistic study of viral immune suppression and virus persistence. The exhaustion of virus-specific T cells was demonstrated during LCMV infection, and the underlying mechanisms have been extensively investigated using LCMV infection in mouse models. In particular, the mechanism for gradual CD8+ T cell exhaustion at molecular and transcriptional levels has been investigated. These studies revealed crucial roles for inhibitory receptors, surface markers, regulatory cytokines, and transcription factors, including PD-1, PSGL-1, CXCR5, and TOX in the regulation of T cells. However, the action mode for CD4+ T cell suppression is largely unknown. Recently, sphingosine kinase 2 was proven to specifically repress CD4+ T cell proliferation and lead to LCMV persistence. As CD4+ T cell regulation was also known to be important for viral persistence, research to uncover the mechanism for CD4+ T cell repression could help us better understand how viruses launch and prolong their persistence. This review summarizes discoveries derived from the study of LCMV in regard to the mechanisms for T cell suppression and approaches for the termination of viral persistence with special emphasis on CD8+ T cells.

Bacteriophages are viruses that specifically infect bacteria and are classified as either virulent phages or temperate phages. Despite virulent phages being promising antimicrobial agents due to their bactericidal effects, the implementation of phage therapy depends on the availability of virulent phages against target bacteria. Notably, virulent phages of Streptococcus gordonii, which resides in the oral cavity and is an opportunistic pathogen that can cause periodontitis and endocarditis have previously never been found. We thus attempted to isolate virulent phages against S. gordonii. In the present study, we report for the first time a virulent bacteriophage against S. gordonii, ΦSG005, discovered from drainage water. ΦSG005 is composed of a short, non-contractile tail and a long head, revealing Podoviridae characteristics via electron microscopic analysis. In turbidity reduction assays, ΦSG005 showed efficient bactericidal effects on S. gordonii. Whole-genome sequencing showed that the virus has a DNA genome of 16,127 bp with 21 coding sequences. We identified no prophage-related elements such as integrase in the ΦSG005 genome, demonstrating that the virus is a virulent phage. Phylogenetic analysis indicated that ΦSG005 forms a distinct clade among the streptococcus viruses and is positioned next to streptococcus virus C1. Molecular characterization revealed the presence of an anti-CRISPR (Acr) IIA5-like protein in the ΦSG005 genome. These findings facilitate our understanding of streptococcus viruses and advance the development of phage therapy against S. gordonii infection.

South Tyrol has implemented, in 2007, a mandatory eradication program against Caprine Arthritis Encephalitis Virus (CAEV), a virus known to cause economic losses related to decreases in milk production and milk quality in goats, along with poor animal welfare and premature death. After a great initial decrease in the seroprevalence, the program has reached a tailing phase with scattered positivities. Potential risk factors associated with the multispecies farming system, a traditional approach in South Tyrol, are evaluated in this study, in order to better understand some of the potential causes leading to the tailing phenomenon. A statistically significant number of farms was selected for the present study, based on the risk factors evaluated. Even though there is no statistically significant association between the practices evaluated and the incidence of infection, the authors believe that it is important to highlight potential risks that may threaten the outcome of this eradication program.

Recent advances in light and electron microscopy are uncovering viral lifecycle events with a level of detail never before seen [...].

Sequential infections of humans by the four different dengue serotypes (DENV-1-4) lead to neutralizing antibodies with group, cross, and type specificity. Virus neutralization of serotypes showed monotypic but mostly multitypic neutralization profiles due to multiple virus exposures. We have studied neutralization to heterologous, reference DENV serotypes using paired sera collected between days 6 and 37 after onset of fever. The DENV-primed neutralization profile of the first serum sample, which was monitored by a foci reduction neutralization test (FRNT), was boosted but the neutralization profile stayed unchanged in the second serum sample. In 45 of 47 paired serum samples, the predominant neutralization was directed against DENV serotypes distinct from the infecting serotype. Homologous neutralization studies using sera and viruses from the same area, 33 secondary sera from DENV-1 infected Cambodian patients and eight virus isolates from Cambodia, showed that the FRNT assay accurately predicted the lack of a predominant antibody response against the infecting DENV-1 serotype in contrast to FRNT results using the WHO set of DENV viruses. This report provides evidence that DENV-primed multitypic neutralizing antibody profiles were mainly boosted and stayed unchanged after secondary infection and that DENV neutralization was predominantly directed to heterologous DENV but not against the infecting homologous serotype.

To minimize immune responses against infected cells, HIV-1 has evolved different mechanisms to limit the surface expression of its envelope glycoproteins (Env). Recent observations suggest that the binding of certain broadly neutralizing antibodies (bNAbs) targeting the 'closed' conformation of Env induces its internalization. On the other hand, non-neutralizing antibodies (nNAbs) that preferentially target Env in its 'open' conformation, remain bound to Env on the cell surface for longer periods of time. In this study, we attempt to better understand the underlying mechanisms behind the differential rates of antibody-mediated Env internalization. We demonstrate that 'forcing' open Env using CD4 mimetics allows for nNAb binding and results in similar rates of Env internalization as those observed upon the bNAb binding. Moreover, we can identify distinct populations of Env that are differentially targeted by Abs that mediate faster rates of internalization, suggesting that the mechanism of antibody-induced Env internalization partially depends on the localization of Env on the cell surface.

Understanding how perturbations to trophic interactions influence virus-host dynamics is essential in the face of ongoing biodiversity loss and the continued emergence of RNA viruses and their associated zoonoses. Herein, we investigated the role of predator exclusion on rodent communities and the seroprevalence of hantaviruses within the Reserva Natural del Bosque Mbaracayú (RNBM), which is a protected area of the Interior Atlantic Forest (IAF). In the IAF, two sympatric rodent reservoirs, Akodon montensis and Oligoryzomys nigripes, harbor Jaborá and Juquitiba hantavirus (JABV, JUQV), respectively. In this study, we employed two complementary methods for predator exclusion: comprehensive fencing and trapping/removal. The goal of exclusion was to preclude the influence of predation on small mammals on the sampling grids and thereby potentially reduce rodent mortality. Following baseline sampling on three grid pairs with different habitats, we closed the grids and began predator removal. By sampling three habitat types, we controlled for habitat-specific effects, which is important for hantavirus-reservoir dynamics in neotropical ecosystems. Our six-month predator exclusion experiment revealed that the exclusion of terrestrial mammalian predators had little influence on the rodent community or the population dynamics of A. montensis and O. nigripes. Instead, fluctuations in species diversity and species abundances were influenced by sampling session and forest degradation. These results suggest that seasonality and landscape composition play dominant roles in the prevalence of hantaviruses in rodent reservoirs in the IAF ecosystem.

Bats have been identified as natural reservoirs of a variety of coronaviruses. They harbor at least 19 of the 33 defined species of alpha- and betacoronaviruses. Previously, the bat coronavirus HKU10 was found in two bat species of different suborders, Rousettus leschenaultia and Hipposideros pomona, in south China. However, its geographic distribution and evolution history are not fully investigated. Here, we screened this viral species by a nested reverse transcriptase PCR in our archived samples collected over 10 years from 25 provinces of China and one province of Laos. From 8004 bat fecal samples, 26 were found to be positive for bat coronavirus HKU10 (BtCoV HKU10). New habitats of BtCoV HKU10 were found in the Yunnan, Guangxi, and Hainan Provinces of China, and Louang Namtha Province in Laos. In addition to H. pomona, BtCoV HKU10 variants were found circulating in Aselliscus stoliczkanus and Hipposideros larvatus. We sequenced full-length genomes of 17 newly discovered BtCoV HKU10 strains and compared them with previously published sequences. Our results revealed a much higher genetic diversity of BtCoV HKU10, particularly in spike genes and accessory genes. Besides the two previously reported lineages, we found six novel lineages in their new habitats, three of which were located in Yunnan province. The genotypes of these viruses are closely related to sampling locations based on polyproteins, and correlated to bat species based on spike genes. Combining phylogenetic analysis, selective pressure, and molecular-clock calculation, we demonstrated that Yunnan bats harbor a gene pool of BtCoV HKU10, with H. pomona as a natural reservoir. The cell tropism test using spike-pseudotyped lentivirus system showed that BtCoV HKU10 could enter cells from human and bat, suggesting a potential interspecies spillover. Continuous studies on these bat coronaviruses will expand our understanding of the evolution and genetic diversity of coronaviruses, and provide a prewarning of potential zoonotic diseases from bats.

The spatiotemporal patterns of spread of influenza A(H1N1)pdm09 viruses on a countrywide scale are unclear in many tropical/subtropical regions mainly because spatiotemporally representative sequence data are lacking. We isolated, sequenced, and analyzed 383 A(H1N1)pdm09 viral genomes from hospitalized patients between 2009 and 2018 from seven locations across Kenya. Using these genomes and contemporaneously sampled global sequences, we characterized the spread of the virus in Kenya over several seasons using phylodynamic methods. The transmission dynamics of A(H1N1)pdm09 virus in Kenya were characterized by (i) multiple virus introductions into Kenya over the study period, although only a few of those introductions instigated local seasonal epidemics that then established local transmission clusters, (ii) persistence of transmission clusters over several epidemic seasons across the country, (iii) seasonal fluctuations in effective reproduction number (Re) associated with lower number of infections and seasonal fluctuations in relative genetic diversity after an initial rapid increase during the early pandemic phase, which broadly corresponded to epidemic peaks in the northern and southern hemispheres, (iv) high virus genetic diversity with greater frequency of seasonal fluctuations in 2009-2011 and 2018 and low virus genetic diversity with relatively weaker seasonal fluctuations in 2012-2017, and (v) virus spread across Kenya. Considerable influenza virus diversity circulated within Kenya, including persistent viral lineages that were unique to the country, which may have been capable of dissemination to other continents through a globally migrating virus population. Further knowledge of the viral lineages that circulate within understudied low-to-middle-income tropical and subtropical regions is required to understand the full diversity and global ecology of influenza viruses in humans and to inform vaccination strategies within these regions.

Cydia pomonella granulovirus (CpGV) is a widely used biological control agent of the codling moth. Recently, however, the codling moth has developed different types of field resistance against CpGV isolates. Whereas type I resistance is Z chromosomal inherited and targeted at the viral gene pe38 of isolate CpGV-M, type II resistance is autosomal inherited and targeted against isolates CpGV-M and CpGV-S. Here, we report that mixtures of CpGV-M and CpGV-S fail to break type II resistance and is expressed at all larval stages. Budded virus (BV) injection experiments circumventing initial midgut infection provided evidence that resistance against CpGV-S is midgut-related, though fluorescence dequenching assay using rhodamine-18 labeled occlusion derived viruses (ODV) could not fully elucidate whether the receptor binding or an intracellular midgut factor is involved. From our peroral and intra-hemocoel infection experiments, we conclude that two different (but genetically linked) resistance mechanisms are responsible for type II resistance in the codling moth: resistance against CpGV-M is systemic whereas a second and/or additional resistance mechanism against CpGV-S is located in the midgut of CpR5M larvae.

Camelpox virus (CMLV) is the causative agent of camelpox, which frequently occurs in the Old World camelids-rearing countries except for Australia. It has also been described in experimentally inoculated New World camelids. Camelpox outbreaks are often experienced shortly after the rainy season, which occurs twice a year on the Arabian Peninsula because of the increased density of the insect population, particularly mosquitos. A systemic form of camelpox outbreak in seven dromedary camels was diagnosed by histology, virus isolation, and PCR. A phylogenetic analysis using full length CMLV genomes of the isolated CMLV strains showed a single phylogenetic unit without any distinctive differences between them. The United Arab Emirates (UAE) isolate sequences showed phylogenetical relatedness with CMLV isolates from Israel with only minor sequence differences. Although the sequences of viruses from both countries were closely related, the disease manifestation was vastly different. Our study shows that the virulence is not only determined by genetic features of CMLV alone but may also depend on other factors such as unknown aspects of the host (e.g., age, overall fitness), management, and the environment.

Human papillomavirus (HPV) type 31 (HPV31) is closely related to the most carcinogenic type, HPV16, but only accounts for 4% of cervical cancer cases worldwide. Viral genetic and epigenetic variations have been associated with carcinogenesis for other high-risk HPV types, but little is known about HPV31. We sequenced 2093 HPV31 viral whole genomes from two large studies, one from the U.S. and one international. In addition, we investigated CpG methylation in a subset of 175 samples. We evaluated the association of HPV31 lineages/sublineages, single nucleotide polymorphisms (SNPs) and viral methylation with cervical carcinogenesis. HPV31 A/B clade was >1.8-fold more associated with cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) compared to the most common C lineage. Lineage/sublineage distribution varied by race/ethnicity and geographic region. A viral genome-wide association analysis identified SNPs within the A/B clade associated with CIN3+, including H23Y (C626T) (odds ratio = 1.60, confidence intervals = 1.17-2.19) located in the pRb CR2 binding-site within the E7 oncogene. Viral CpG methylation was higher in lineage B, compared to the other lineages, and was most elevated in CIN3+. In conclusion, these data support the increased oncogenicity of the A/B lineages and suggest variation of E7 as a contributing risk factor.

Immature HIV virions harbor a lattice of Gag molecules with significant ordering in CA-NTD, CA-CTD and SP1 regions. This ordering plays a major role during HIV maturation. To test the condition in which the Gag lattice forms in vivo, we assembled virus like particles (VLPs) by expressing only HIV Gag in mammalian cells. Here we show that these VLPs incorporate a similar number of Gag molecules compared to immature HIV virions. However, within these VLPs, Gag molecules diffuse with a pseudo-diffusion rate of 10 nm2/s, this pseudo-diffusion is abrogated in the presence of melittin and is sensitive to mutations within the SP1 region. Using cryotomography, we show that unlike immature HIV virions, in the Gag lattice of VLPs the CA-CTD and SP1 regions are significantly less ordered. Our observations suggest that within immature HIV virions, other viral factors in addition to Gag, contribute to ordering in the CA-CTD and SP1 regions.

Advances in antiretroviral treatment improved the life expectancy of perinatally HIV-infected children. However, growing up with HIV provides challenges in daily functioning. This cross-sectional cohort study investigated the neuropsychological and psychosocial functioning of a group of perinatally HIV-infected children in the Netherlands and compared their outcomes with Dutch normative data and outcomes of a control group of uninfected siblings. The children's functioning was assessed with internationally well-known and standardized questionnaires, using a multi-informant approach, including the perspectives of caregivers, teachers, and school-aged children. In addition, we explored the associations of socio-demographic and medical characteristics of the HIV-infected children with their neuropsychological and psychosocial functioning. Caregivers reported compromised functioning when compared to Dutch normative data for HIV-infected children in the areas of attention, sensory processing, social-emotional functioning, and health-related quality of life. Teachers reported in addition compromised executive functioning for HIV-infected children. A comparison with siblings revealed differences in executive functioning, problems with peers, and general health. The concurrent resemblance between HIV-infected children and siblings regarding problems in other domains implies that social and contextual factors may be of influence. A family-focused approach with special attention to the child's socio-environmental context and additional attention for siblings is recommended.

Since SARS-CoV-2 appeared in late 2019, many studies on the immune response to COVID-19 have been conducted, but the asymptomatic or light symptom cases were somewhat understudied as respective individuals often did not seek medical help. Here, we analyze the production of the IgG antibodies to viral nucleocapsid (N) protein and receptor-binding domain (RBD) of the spike protein and assess the serum neutralization capabilities in a cohort of patients with different levels of disease severity. In half of light or asymptomatic cases the antibodies to the nucleocapsid protein, which serve as the main target in many modern test systems, were not detected. They were detected in all cases of moderate or severe symptoms, and severe lung lesions correlated with respective higher signals. Antibodies to RBD were present in the absolute majority of samples, with levels being sometimes higher in light symptom cases. We thus suggest that the anti-RBD/anti-N antibody ratio may serve as an indicator of the disease severity. Anti-RBD IgG remained detectable after a year or more since the infection, even with a slight tendency to raise over time, and the respective signal correlated with the serum capacity to inhibit the RBD interaction with the ACE-2 receptor.

The recently discovered porcine circovirus 3 (PCV3) belongs to the Circovirus genus of the Circoviridae family together with the other three PCVs, PCV1, PCV2, and PCV4. As reported, PCV3 can infect pig, wild boar, and several other intermediate hosts, resulting in single or multiple infections in the affected animal. The PCV3 infection can lead to respiratory diseases, digestive disorders, reproductive disorders, multisystemic inflammation, and immune responses. Up to now, PCV3 infection, as well as the disease caused by PCV3, has been reported in many swine farms worldwide with high positive rates, which indicates that the virus may be another important pathogen in the swine industry. Therefore, we reviewed the current progress on epidemiology and pathobiology of PCV3, which may provide the latest knowledge of the virus and PCV3-related diseases.

Nipah virus (NiV) and respiratory syncytial virus (RSV) possess two surface glycoproteins involved in cellular attachment and membrane fusion, both of which are potential targets for vaccines. The majority of vaccine development is focused on the attachment (G) protein of NiV, which is the immunodominant target. In contrast, the fusion (F) protein of RSV is the main target in vaccine development. Despite this, neutralising epitopes have been described in NiV F and RSV G, making them alternate targets for vaccine design. Through rational design, we have developed a vaccine strategy applicable to phylogenetically divergent NiV and RSV that comprises both the F and G proteins (FxG). In a mouse immunization model, we found that NiV FxG elicited an improved immune response capable of neutralising pseudotyped NiV and a NiV mutant that is able to escape neutralisation by two known F-specific antibodies. RSV FxG elicited an immune response against both F and G and was able to neutralise RSV; however, this was inferior to the immune response of F alone. Despite this, RSV FxG elicited a response against a known protective epitope within G that is conserved across RSV A and B subgroups, which may provide additional protection in vivo. We conclude that inclusion of F and G antigens within a single design provides a streamlined subunit vaccine strategy against both emerging and established pathogens, with the potential for broader protection against NiV.

Numerous species of RNA viruses pathogenic for humans are present worldwide [...].

Neurological diseases in cattle can be caused by several infectious agents. Astroviruses are increasingly recognized as the causative agent of encephalitis in various animals, including humans. In this study, a neuroinvasive astrovirus (BoAstV 20B05) was discovered in the brain tissues of an 81-month-old Korean native cattle with neurological symptoms. Lymphocyte infiltration and multifocal perivascular cuffing were observed in the cerebrum and brain stem, and viral antigens were also detected in the meninges. In particular, the concentration of the astroviral genome was high in the brain tissues. Korean BoAstV 20B05 was classified into the CH13/NeuroS1 clade and was closely related to the Neuro-Uy and KagoshimaSR28-462 strains. Our evolutionary analysis showed that Korean BoAstV 20B05 belongs to the sub-lineage NeuroS1 and evolved independently of BoAstV KagoshimaSR28-462. These results suggest that neuroinvasive astroviruses were first introduced in Korea. However, analysis is limited by the lack of reference astrovirus sequences reported in various countries within Asia, and further analysis should be performed using more strains. In this study, we identified a neuroinvasive astrovirus infection with neurological symptoms for the first time in South Korea and confirmed that BoAstV 20B05 may have been introduced in South Korea a long time ago.

Most endolysins of dsDNA phages are exported by a holin-dependent mechanism, while in some cases endolysins are exported via a holin-independent mechanism. However, it is still unclear whether the same endolysins can be exported by both holin-dependent and holin-independent mechanisms. This study investigated the lysis system of OP2-like phage X2 infecting Xanthomonas oryzae pv. oryzae, causing devastating bacterial leaf blight disease in rice. Based on bioinformatics and protein biochemistry methods, we show that phage X2 employs the classic "holin-endolysin" lysis system. The endolysin acts on the cell envelope and exhibits antibacterial effects in vitro, while the holin facilitates the release of the protein into the periplasm. We also characterized the role of the transmembrane domain (TMD) in the translocation of the endolysin across the inner membrane. We found that the TMD facilitated the translocation of the endolysin via the Sec secretion system. The holin increases the efficiency of protein release, leading to faster and more efficient lysis. Interestingly, in E. coli, the expression of either holin or endolysin with TMDs resulted in the formation of long rod shaped cells. We conclude that the TMD of X2-Lys plays a dual role: One is the transmembrane transport while the other is the inhibition of cell division, resulting in larger cells and thus in a higher number of released viruses per cell.

To evaluate whether uterine injury caused by hepatitis E virus (HEV) infection is responsible for adverse pregnancy outcomes. HEV-infected female BALB/c mice were coupled with healthy male BALB/c mice at 0, 7, 14, 21, and 91 dpi to explore the uterine injury caused by HEV infection. Mice were euthanized after 10 days of copulation, and uteruses were collected for HEV RNA and antigen detection and histopathological analysis. Inflammatory responses; apoptosis; and estrogen receptor ɑ (ER-ɑ), endomethal antibody (ERAb), cytokeratin-7 (CK7), vimentin (VIM), and vascular endothelial growth factor (VEGF) expression levels were evaluated. After 10 days of copulation, miscarriage and nonpregnancy, as well as enlarged uteruses filled with inflammatory cytokines, were found in HEV-infected mice. HEV RNA and antigens were detected in the sera and uteruses of HEV-infected mice. Significant endometrial thickness (EMT) thinning, severe inflammatory responses, and aggravated apoptosis in the uteruses of HEV-infected mice that experienced miscarriage might contribute to adverse pregnancy outcomes. Furthermore, significantly suppressed ER-ɑ expression and increased ERAb, CK7, VIM, and VEGF expression levels were found in the uteruses of HEV-infected mice that had miscarried. However, uterine damage recovered after complete HEV clearance, and impaired fertility was improved. EMT injury, severe inflammatory responses, and aggravated apoptosis in the uterus caused by HEV infection are responsible for poor pregnancy outcomes.

High throughput sequencing (HTS) has revolutionised virus detection and discovery, allowing for the untargeted characterisation of whole viromes. Viral metagenomics studies have demonstrated the ubiquity of virus infection - often in the absence of disease symptoms - and tend to discover many novel viruses, highlighting the small fraction of virus biodiversity described to date. The majority of the studies using high-throughput sequencing to characterise plant viromes have focused on economically important crops, and only a small number of studies have considered weeds and wild plants. Characterising the viromes of wild plants is highly relevant, as these plants can affect disease dynamics in crops, often by acting as viral reservoirs. Moreover, the viruses in unmanaged systems may also have important effects on wild plant populations and communities. Here, we review metagenomic studies on weeds and wild plants to show the benefits and limitations of this approach and identify knowledge gaps. We consider key genomics developments that are likely to benefit the field in the near future. Although only a small number of HTS studies have been performed on weeds and wild plants, these studies have already discovered many novel viruses, demonstrated unexpected trends in virus distributions, and highlighted the potential of metagenomics as an approach.

BACKGROUND: The prevalence and prognostic implications of metabolic syndrome (MetS) in patients infected by the SARS-CoV-2 remain unclear. We performed a systematic review and meta-analysis of prevalence and mortality risk in COVID-19 patients with MetS. METHODS: Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed in abstracting data and assessing validity. We searched MEDLINE and Scopus to locate every article published up to 1 September 2021, reporting data on MetS among COVID-19 patients. The pooled prevalence of MetS was calculated using a random effects model and presented using the related 95% confidence interval (CI), while the mortality risk was estimated using the Mantel-Haenszel random effects models with odds ratio (OR) and related 95% CI. Statistical heterogeneity was measured using the Higgins I2 statistic. RESULTS: Six studies, enrolling 209.569 COVID-19 patients [mean age 57.2 years, 114.188 males (54.4%)] met the inclusion criteria and were included in the final analysis. The pooled prevalence of dyslipidaemia was 20.5% of cases (95% CI: 6.7-47.8%, p = 0.03), with high heterogeneity (I2 = 98.9%). Pre-existing MetS was significantly associated with higher risk of short-term mortality (OR: 2.30, 95% CI: 1.52-3.45, p < 0.001), with high heterogeneity (I2 = 89.4%). Meta-regression showed a direct correlation with male gender (p = 0.03), hypertension (p < 0.001), DM (p = 0.01) and hyperlipidaemia (p = 0.04), but no effect when considering age (p = 0.75) and chronic pulmonary disease (p = 0.86) as moderators. CONCLUSIONS: MetS represents a major comorbidity in about 20% of COVID-19 patients and it is associated with a 230% increased risk of short-term mortality.

The novel coronavirus pneumonia (COVID-19) pandemic is a great threat to human society and now is still spreading. Although several vaccines have been authorized for emergency use, only one recombinant subunit vaccine has been permitted for widespread use. More subunit vaccines for COVID-19 should be developed in the future. The receptor binding domain (RBD), located at the S protein of SARS-CoV-2, contains most of the neutralizing epitopes. However, the immunogenicity of RBD monomers is not strong enough. In this study, we fused the RBD-monomer with a modified Fc fragment of human IgG1 to form an RBD-Fc fusion protein. The recombinant vaccine candidate based on the RBD-Fc protein could induce high levels of IgG and neutralizing antibody in mice, and these could last for at least three months. The secretion of IFN-γ, IL-2 and IL-10 in the RBD-stimulated splenocytes of immunized mice also increased significantly. Our results first showed that the RBD-Fc vaccine could induce both humoral and cellular immune responses and might be an optional strategy to control COVID-19.

Nascent HIV-1 particles incorporate the viral envelope glycoprotein and multiple host transmembrane proteins during assembly at the plasma membrane. At least some of these host transmembrane proteins on the surface of virions are reported as pro-viral factors that enhance virus attachment to target cells or facilitate trans-infection of CD4+ T cells via interactions with non-T cells. In addition to the pro-viral factors, anti-viral transmembrane proteins are incorporated into progeny virions. These virion-incorporated transmembrane proteins inhibit HIV-1 entry at the point of attachment and fusion. In infected polarized CD4+ T cells, HIV-1 Gag localizes to a rear-end protrusion known as the uropod. Regardless of cell polarization, Gag colocalizes with and promotes the virion incorporation of a subset of uropod-directed host transmembrane proteins, including CD162, CD43, and CD44. Until recently, the functions of these virion-incorporated proteins had not been clear. Here, we review the recent findings about the roles played by virion-incorporated CD162, CD43, and CD44 in HIV-1 spread to CD4+ T cells.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

Polyomavirus JC (JCPyV) causes the demyelinating disease progressive multifocal leukoencephalopathy (PML). JCPyV infection is very common in childhood and, under conditions of severe immunosuppression, JCPyV may reactivate to cause PML. JC viral proteins expression is regulated by the JCPyV non-coding control region (NCCR), which contains binding sites for cellular transcriptional factors which regulate JCPyV transcription. Our earlier studies suggest that JCPyV reactivation occurs within glial cells due to cytokines such as TNF-α which stimulate viral gene expression. In this study, we examined interferon-α (IFNα) or β (IFNβ) which have a negative effect on JCPyV transcriptional regulation. We also showed that these interferons induce the endogenous liver inhibitory protein (LIP), an isoform of CAAT/enhancer binding protein beta (C/EBPβ). Treatment of glial cell line with interferons increases the endogenous level of C/EBPβ-LIP. Furthermore, we showed that the negative regulatory role of the interferons in JCPyV early and late transcription and viral replication is more pronounced in the presence of C/EBPβ-LIP. Knockdown of C/EBPβ-LIP by shRNA reverse the inhibitory effect on JCPyV viral replication. Therefore, IFNα and IFNβ negatively regulate JCPyV through induction of C/EBPβ-LIP, which together with other cellular transcriptional factors may control the balance between JCPyV latency and activation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent of coronavirus disease 2019 (COVID-19), is responsible for the worst pandemic of the 21st century. Like all human coronaviruses, SARS-CoV-2 originated in a wildlife reservoir, most likely from bats. As SARS-CoV-2 has spread across the globe in humans, it has spilled over to infect a variety of non-human animal species in domestic, farm, and zoo settings. Additionally, a broad range of species, including one neotropical monkey, have proven to be susceptible to experimental infection with SARS-CoV-2. Together, these findings raise the specter of establishment of novel enzootic cycles of SARS-CoV-2. To assess the potential exposure of free-living non-human primates to SARS-CoV-2, we sampled 60 neotropical monkeys living in proximity to Manaus and São José do Rio Preto, two hotspots for COVID-19 in Brazil. Our molecular and serological tests detected no evidence of SAR-CoV-2 infection among these populations. While this result is reassuring, sustained surveillance efforts of wildlife living in close association with human populations is warranted, given the stochastic nature of spillover events and the enormous implications of SARS-CoV-2 spillover for human health.

Pepper mottle virus (PepMoV) is a destructive pathogen that infects various solanaceous plants, including pepper, bell pepper, potato, and tomato. In this review, we summarize what is known about the molecular characteristics of PepMoV and its interactions with host plants. Comparisons of symptom variations caused by PepMoV isolates in plant hosts indicates a possible relationship between symptom development and genetic variation. Researchers have investigated the PepMoV-plant pathosystem to identify effective and durable genes that confer resistance to the pathogen. As a result, several recessive pvr or dominant Pvr resistance genes that confer resistance to PepMoV in pepper have been characterized. On the other hand, the molecular mechanisms underlying the interaction between these resistance genes and PepMoV-encoded genes remain largely unknown. Our understanding of the molecular interactions between PepMoV and host plants should be increased by reverse genetic approaches and comprehensive transcriptomic analyses of both the virus and the host genes.

Modern bacteriophage encapsulation methods based on polymers such as alginate have been developed recently for their use in phage therapy for veterinary purposes. In birds, it has been proven that using this delivery system allows the release of the bacteriophage in the small intestine, the site of infection by Salmonella spp. This work designed an approach for phage therapy using encapsulation by ionotropic gelation of the lytic bacteriophage S1 for Salmonella&nbsp;enterica in 2% w/v alginate beads using 2% w/v calcium chloride as crosslinking agent. This formulation resulted in beads with an average size of 3.73 ± 0.04 mm and an encapsulation efficiency of 70%. In vitro, the beads protected the bacteriophages from pH 3 and released them at higher pH. To confirm that this would protect the bacteriophages from gastrointestinal pH changes, we tested the phage infectivity in vivo assay. Using a model chicken (Gallus gallus domesticus) infected with Salmonella Enteritidis, we confirmed that after 3 h of the beads delivery, infective phages were present in the chicken's duodenal and caecal sections. This study demonstrates that our phage formulation is an effective system for release and delivery of bacteriophage S1 against Salmonella Enteritidis with potential use in the poultry sector.

To further classify the oomycete viruses that have been discovered in recent years, we investigated virus infection in the plant-parasitic oomycete Globisporangium ultimum in Japan. Double-stranded RNA detection, high-throughput sequencing, and RT-PCR revealed that the G. ultimum isolate UOP226 contained two viruses related to fusarivirus and totivirus, named Pythium ultimum RNA virus 1 (PuRV1) and Pythium ultimum RNA virus 2 (PuRV2), respectively. Phylogenetic analysis of the deduced amino acid sequence of the RNA-dependent RNA polymerase (RdRp) showed that fusari-like PuRV1 belonged to a different phylogenetic group than Plasmopara viticola lesion-associated fusari virus (PvlaFV) 1-3 from oomycete Plasmopara viticola. Codon usage bias of the PuRV1 RdRp gene was more similar to those of fungi than Globisporangium and Phytophthora, suggesting that the PuRV1 ancestor horizontally transmitted to G. ultimum ancestor from fungi. Phylogenetic analysis of the deduced amino acid sequence of the RdRp of toti-like PuRV2 showed a monophyletic group with the other toti-like oomycete viruses from Globisporangium, Phytophthora, and Pl. viticola. However, the nucleotide sequences of toti-like oomycete viruses were not so homologous, suggesting the possibility of convergent evolution of toti-like oomycete viruses.

Herpes simplex virus type 2 (HSV-2) infection affects 24 million births annually and is associated with adverse pregnancy outcomes, including neonatal herpes; however, the mechanisms underlying in utero transmission of HSV-2 are largely unknown. We examined the effects of primary HSV-2 infection during early pregnancy on gestational outcomes in a novel, clinically relevant mouse model. Pregnant C57BL/6 mice were infected intravaginally with 102-105 pfu/mL HSV-2 on gestation day (gd) 4.5. Controls were infected, nonpregnant, diestrus-staged mice and pregnant, uninfected mice. Compared to nonpregnant mice, pregnant mice were 100-fold more susceptible to HSV-2 infection. Three days post-inoculation (gd7.5), viral DNA was present in implantation sites, but pregnancy outcomes were largely unaffected by infection. Eight days post-inoculation (gd12.5), HSV-2 DNA persisted in placental tissues, resulting in inflammation and hemorrhage. Fetal and placental weights were reduced and fetal loss was observed with high viral doses. HSV-2 DNA and increased expression of pro-inflammatory mediators were detected in fetal tissues at gd12.5, signifying viral transmission and fetal infection, even with low viral doses. This mouse model shows a dose-dependent effect of primary HSV-2 infection on pregnancy outcomes and suggests that fetal loss may occur due to placental inflammation, thus providing valuable insight into in utero transmission of HSV-2.

Oral health care workers (OHCW) are exposed to pathogenic microorganisms during dental aerosol-generating procedures. Technologies aimed at the reduction of aerosol, droplets and splatter are essential. This in vivo study assessed aerosol, droplet and splatter contamination in a simulated clinical scenario. The coolant of the high-speed air turbine was colored with red concentrate. The red aerosol, droplets and splatter contamination on the wrists of the OHCW and chests of the OHCW/volunteer protective gowns, were assessed and quantified in cm2. The efficacy of various evacuation strategies was assessed: low-volume saliva ejector (LV) alone, high-volume evacuator (HV) plus LV and an extra-oral dental aerosol suction device (DASD) plus LV. The Kruskal-Wallis rank-sum test for multiple independent samples with a post-hoc test was used. No significant difference between the LV alone compared to the HV plus LV was demonstrated (p = 0.372059). The DASD combined with LV resulted in a 62% reduction of contamination of the OHCW. The HV plus LV reduced contamination by 53% compared to LV alone (p = 0.019945). The DASD demonstrated a 50% reduction in the contamination of the OHCWs wrists and a 30% reduction in chest contamination compared to HV plus LV. The DASD in conjunction with LV was more effective in reducing aerosol, droplets and splatter than HV plus LV.

Recently, two cases of complete remission of classical Hodgkin lymphoma (cHL) and follicular lymphoma (FL) after SARS-CoV-2 infection were reported. However, the precise molecular mechanism of this rare event is yet to be understood. Here, we hypothesize a potential anti-tumor immune response of SARS-CoV-2 and based on a computational approach show that: (i) SARS-CoV-2 Spike-RBD may bind to the extracellular domains of CD15, CD27, CD45, and CD152 receptors of cHL or FL and may directly inhibit cell proliferation. (ii) Alternately, upon internalization after binding to these CD molecules, the SARS-CoV-2 membrane (M) protein and ORF3a may bind to gamma-tubulin complex component 3 (GCP3) at its tubulin gamma-1 chain (TUBG1) binding site. (iii) The M protein may also interact with TUBG1, blocking its binding to GCP3. (iv) Both the M and ORF3a proteins may render the GCP2-GCP3 lateral binding where the M protein possibly interacts with GCP2 at its GCP3 binding site and the ORF3a protein to GCP3 at its GCP2 interacting residues. (v) Interactions of the M and ORF3a proteins with these gamma-tubulin ring complex components potentially block the initial process of microtubule nucleation, leading to cell-cycle arrest and apoptosis. (vi) The Spike-RBD may also interact with and block PD-1 signaling similar to pembrolizumab and nivolumab- like monoclonal antibodies and may induce B-cell apoptosis and remission. (vii) Finally, the TRADD interacting "PVQLSY" motif of Epstein-Barr virus LMP-1, that is responsible for NF-kB mediated oncogenesis, potentially interacts with SARS-CoV-2 Mpro, NSP7, NSP10, and spike (S) proteins, and may inhibit the LMP-1 mediated cell proliferation. Taken together, our results suggest a possible therapeutic potential of SARS-CoV-2 in lymphoproliferative disorders.

Coronavirus disease 2019 (COVID-19) is a multisystem disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), that primarily causes respiratory symptoms. However, an increasing number of cutaneous manifestations associated with this disease have been reported. The aim of this study is to analyze the scientific literature on cutaneous manifestations associated with SARS-CoV-2 by means of a narrative literature review until June 2021. The search was conducted in the following electronic databases: Medline (PubMed), SciELO, and Cochrane Library Plus. The most common cutaneous manifestations in patients with COVID-19 are vesicular eruptions, petechial/purpuric rashes, acral lesions, liveoid lesions, urticarial rash, and maculopapular-erythematous rash. These manifestations may be the first presenting symptoms of SARS-CoV-2 infection, as is the case with acral lesions, vesicular eruptions, and urticaria. In relation to severity, the presence of liveoid lesions may be associated with a more severe course of the disease. Treatment used for dermatological lesions includes therapy with anticoagulants, corticosteroids, and antihistamines. Knowledge of the dermatologic manifestations associated with SARS-CoV-2 contributes to the diagnosis of COVID-19 in patients with skin lesions associated with respiratory symptoms or in asymptomatic patients. In addition, understanding the dermatologic lesions associated with COVID-19 could be useful to establish a personalized care plan.

In the present study, we evaluated the effect of spray-drying formulations and operating parameters of a laboratory-scale spray-dryer on the characteristics of spray-dried powders containing two Pseudomonas aeruginosa bacteriophages exhibiting different morphotypes: a podovirus (LUZ19) and a myovirus (14-1). We optimized the production process for bacteriophage-loaded powders, with an emphasis on long-term storage under ICH (international conference on harmonization) conditions. D-trehalose-/L-isoleucine-containing bacteriophage mixtures were spray-dried from aqueous solutions using a Büchi Mini Spray-dryer B-290 (Flawil, Switzerland). A response surface methodology was used for the optimization of the spray-drying process, with the following as-evaluated parameters: Inlet temperature, spray gas flow rate, and the D-trehalose/L-isoleucine ratio. The dried powders were characterized in terms of yield, residual moisture content, and bacteriophage lytic activity. L-isoleucine has demonstrated a positive impact on the activity of LUZ19, but a negative impact on 14-1. We observed a negligible impact of the inlet temperature and a positive correlation of the spray gas flow rate with bacteriophage activity. After optimization, we were able to obtain dry powder preparations of both bacteriophages, which were stable for a minimum of one year under different ICH storage conditions (up to and including 40 °C and 75% relative humidity).

Erratum in Viruses. 2022 Jun 28;14(7):

The nucleocapsid (NC) protein of human immunodeficiency (HIV) is a small, highly basic protein containing two CCHC zinc-finger motifs, which is cleaved from the NC domain of the Gag polyprotein during virus maturation. We previously reported that recombinant HIV-1 Gag and NCp7 overexpressed in an E. coli host contains two and one zinc ions, respectively, and Gag exhibited much higher selectivity for packaging signal (Psi) and affinity for the stem-loop (SL)-3 of Psi than NCp7. In this study, we prepared NCp7 containing 0 (0NCp7), 1 (NCp7) or 2 (2NCp7) zinc ions, and compared their secondary structure, Psi-selectivity and SL3-affinity. Along with the decrease of the zinc content, less ordered conformations were detected. Compared to NCp7, 2NCp7 exhibited a much higher Psi-selectivity and SL3-affinity, similar to Gag, whereas 0NCp7 exhibited a lower Psi-selectivity and SL3-affinity, similar to the H23&H44K double mutant of NCp7, indicating that the different RNA-binding property of Gag NC domain and the mature NCp7 may be resulted, at least partially, from their different zinc content. This study will be helpful to elucidate the critical roles that zinc played in the viral life cycle, and benefit further investigations of the functional switch from the NC domain of Gag to the mature NCp7.

The coxsackievirus B3 strain PD-0 has been proposed as a new oncolytic virus for the treatment of colorectal carcinoma. Here, we generated a cDNA clone of PD-0 and analyzed the virus PD-H, newly generated from this cDNA, in xenografted and syngenic models of colorectal cancer. Replication and cytotoxic assays revealed that PD-H replicated and lysed colorectal carcinoma cell lines in vitro as well as PD-0. Intratumoral injection of PD-H into subcutaneous DLD-1 tumors in nude mice resulted in strong inhibition of tumor growth and significantly prolonged the survival of the animals, but virus-induced systemic infection was observed in one of the six animals. In a syngenic mouse model of subcutaneously growing Colon-26 tumors, intratumoral administration of PD-H led to a significant reduction of tumor growth, the prolongation of animal survival, the prevention of tumor-induced cachexia, and the elevation of CD3+ and dendritic cells in the tumor microenvironment. No virus-induced side effects were observed. After intraperitoneal application, PD-H induced weak pancreatitis and myocarditis in immunocompetent mice. By equipping the virus with target sites of miR-375, which is specifically expressed in the pancreas, organ infections were prevented. Moreover, employment of this virus in a syngenic mouse model of CT-26 peritoneal carcinomatosis resulted in a significant reduction in tumor growth and an increase in animal survival. The results demonstrate that the immune status of the host, the route of virus application, and the engineering of the virus with target sites of suitable microRNAs are crucial for the use of PD-H as an oncolytic virus.

The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR-Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR-Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR-Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.

Viruses are an underappreciated cause of heart failure. Indeed, several types of viral infections carry cardiovascular risks. Understanding shared and unique mechanisms by which each virus compromises heart function is critical to inform on therapeutic interventions. This review describes how the key viruses known to lead to cardiac dysfunction operate. Both direct host-damaging mechanisms and indirect actions on the immune systems are discussed. As viral myocarditis is a key pathologic driver of heart failure in infected individuals, this review also highlights the role of cytokine storms and inflammation in virus-induced cardiomyopathy.

Influenza A viruses (IAV) are widespread viruses affecting avian and mammalian species worldwide. IAVs from avian species can be transmitted to mammals including humans and, thus, they are of inherent pandemic concern. Most of the efforts to understand the pathogenicity and transmission of avian origin IAVs have been focused on H5 and H7 subtypes due to their highly pathogenic phenotype in poultry. However, IAV of the H9 subtype, which circulate endemically in poultry flocks in some regions of the world, have also been associated with cases of zoonotic infections. In this review, we discuss the mammalian transmission of H9N2 and the molecular factors that are thought relevant for this spillover, focusing on the HA segment. Additionally, we discuss factors that have been associated with the ability of these viruses to transmit through the respiratory route in mammalian species. The summarized information shows that minimal amino acid changes in the HA and/or the combination of H9N2 surface genes with internal genes of human influenza viruses are enough for the generation of H9N2 viruses with the ability to transmit via aerosol.

Equine parvovirus-hepatitis (EqPV-H) is a newly identified etiologic agent of Theiler's disease (TD). We present a case of EqPV-H-related fulminant hepatitis in a 14-year-old thoroughbred mare in Korea. The mare had acute hepatopathy and gastrointestinal symptoms, with abnormal liver-related blood parameters. The horse was born in the USA and imported to Korea in 2017, with no history of administration of equine biological products after entry into Korea. The horse was diagnosed with EqPV-H-associated hepatitis after abdominal ultrasonography, laparotomy, and nested polymerase chain reaction (PCR) and in situ hybridization (ISH) assays. The serum, nasal swab, oral swab, and liver biopsy were positive for EqPV-H according to the PCR assay. Genetic analysis of the partial NS1 gene of EqPV-H showed a unique nucleotide substitution, distinct from that in previously deposited strains. EqPV-H DNA was found not only in hepatocytes but also in bile duct epithelium and Kupffer cells, particularly via ISH. To the best of our knowledge, this is the first case of EqPV-H-associated TD in Asia, providing the first clinical evidence for viral shedding from the mouth and nose, and identification of EqPV-H in the liver. This study contributes to a better understanding of the pathological features of EqPV-H-associated TD.

SARS-CoV-2 vaccine clinical trials assess efficacy against disease (VEDIS), the ability to block symptomatic COVID-19. They only partially discriminate whether VEDIS is mediated by preventing infection completely, which is defined as detection of virus in the airways (VESUSC), or by preventing symptoms despite infection (VESYMP). Vaccine efficacy against transmissibility given infection (VEINF), the decrease in secondary transmissions from infected vaccine recipients, is also not measured. Using mathematical modeling of data from King County Washington, we demonstrate that if the Moderna (mRNA-1273QS) and Pfizer-BioNTech (BNT162b2) vaccines, which demonstrated VEDIS > 90% in clinical trials, mediate VEDIS by VESUSC, then a limited fourth epidemic wave of infections with the highly infectious B.1.1.7 variant would have been predicted in spring 2021 assuming rapid vaccine roll out. If high VEDIS is explained by VESYMP, then high VEINF would have also been necessary to limit the extent of this fourth wave. Vaccines which completely protect against infection or secondary transmission also substantially lower the number of people who must be vaccinated before the herd immunity threshold is reached. The limited extent of the fourth wave suggests that the vaccines have either high VESUSC or both high VESYMP and high VEINF against B.1.1.7. Finally, using a separate intra-host mathematical model of viral kinetics, we demonstrate that a 0.6 log vaccine-mediated reduction in average peak viral load might be sufficient to achieve 50% VEINF, which suggests that human challenge studies with a relatively low number of infected participants could be employed to estimate all three vaccine efficacy metrics.

Chronic hepatitis B virus (HBV) infection is a global health problem that can lead to liver dysfunction, including liver cirrhosis and hepatocellular carcinoma (HCC). Current antiviral therapies can control viral replication in patients with chronic HBV infection; however, there is a risk of HCC development. HBV-related proteins may be produced in hepatocytes regardless of antiviral therapies and influence intracellular metabolism and signaling pathways, resulting in liver carcinogenesis. To understand the mechanisms of liver carcinogenesis, the effect of HBV infection in human hepatocytes should be analyzed. HBV infects human hepatocytes through transfer to the sodium taurocholate co-transporting polypeptide (NTCP). Although the NTCP is expressed on the hepatocyte surface in several animals, including mice, HBV infection is limited to human primates. Due to this species-specific liver tropism, suitable animal models for analyzing HBV replication and developing antivirals have been lacking since the discovery of the virus. Recently, a humanized mouse model carrying human hepatocytes in the liver was developed based on several immunodeficient mice; this is useful for analyzing the HBV life cycle, antiviral effects of existing/novel antivirals, and intracellular signaling pathways under HBV infection. Herein, the usefulness of human hepatocyte chimeric mouse models in the analysis of HBV-associated hepatocarcinogenesis is discussed.

Cercospora leaf spot (CLS) caused by Cercospora beticola is a devastating foliar disease of sugar beet (Beta vulgaris), resulting in high yield losses worldwide. Mycoviruses are widespread fungi viruses and can be used as a potential biocontrol agent for fugal disease management. To determine the presence of mycoviruses in C. beticola, high-throughput sequencing analysis was used to determine the diversity of mycoviruses in 139 C. beticola isolates collected from major sugar beet production areas in China. The high-throughput sequencing reads were assembled and searched against the NCBI database using BLASTn and BLASTx. The results showed that the obtained 93 contigs were derived from eight novel mycoviruses, which were grouped into 3 distinct lineages, belonging to the families Hypoviridae, Narnaviridae and Botourmiaviridae, as well as some unclassified (-)ssRNA viruses in the order Bunyavirales and Mononegavirales. To the best of our knowledge, this is the first identification of highly diverse mycoviruses in C. beticola. The novel mycoviruses explored in this study will provide new viral materials to biocontrol Cercospora diseases. Future studies of these mycoviruses will aim to assess the roles of each mycovirus in biological function of C. beticola in the future.

Human herpesviruses, classified into three subfamilies, are double-stranded DNA viruses that establish lifelong latent infections within most of the world's population and can cause severe disease, especially in immunocompromised people. There is no cure, and current preventative and therapeutic options are limited. Therefore, understanding the biology of these viruses is essential for finding new ways to stop them. Capsids play a central role in herpesvirus biology. They are sophisticated vehicles that shelter the pressurized double-stranded-DNA genomes while ensuring their delivery to defined cellular destinations on the way in and out of the host cell. Moreover, the importance of capsids for multiple key steps in the replication cycle makes their assembly an attractive therapeutic target. Recent cryo-electron microscopy reconstructions of capsids from all three subfamilies of human herpesviruses revealed not only conserved features but also remarkable structural differences. Furthermore, capsid assembly studies have suggested subfamily-specific roles of viral capsid protein homologs. In this review, we compare capsid structures, assembly mechanisms, and capsid protein functions across human herpesvirus subfamilies, highlighting the differences.

At the end of 2019 a newly emerged betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of an outbreak of severe pneumonia, subsequently termed COVID-19, in a number of patients in Wuhan, China. Subsequently, SARS-CoV-2 rapidly spread globally, resulting in a pandemic that has to date infected over 200 million individuals and resulted in more than 4.3 million deaths. While SARS-CoV-2 results in severe disease in 13.8%, with increasing frequency of severe disease with age, over 80% of infections are asymptomatic or mild. The immune response is an important determinant of outcome following SARS-CoV-2 infection. While B cell and T cell responses are associated with control of infection and protection against subsequent challenge with SARS-CoV-2, failure to control viral replication and the resulting hyperinflammation are associated with severe COVID-19. Towards the end of 2020, several variants of concern emerged that demonstrate increased transmissibility and/or evasion of immune responses from prior SARS-CoV-2 infection. This article reviews what is known about the humoral and cellular immune responses to SARS-CoV-2 and how mutation and structural/functional changes in the emerging variants of concern impact upon the immune protection from prior infection or vaccination.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) commonly complicates with coagulopathy. A syndrome called Long-COVID-19 is emerging recently in COVID-19 survivors, characterized, in addition to the persistence of symptoms typical of the acute phase, by alterations in inflammatory and coagulation parameters due to endothelial damage. The related disseminated intravascular coagulation (DIC) can be associated with high death rates in COVID-19 patients. It is possible to find a prothrombotic state also in Long-COVID-19. Early administration of anticoagulants in COVID-19 was suggested in order to improve patient outcomes, although exact criteria for their application were not well-established. Low-molecular-weight heparin (LMWH) was commonly adopted for counteracting DIC and venous thromboembolism (VTE), due to its pharmacodynamics and anti-inflammatory properties. However, the efficacy of anticoagulant therapy for COVID-19-associated DIC is still a matter of debate. Thrombin and Factor Xa (FXa) are well-known components of the coagulation cascade. The FXa is known to strongly promote inflammation as the consequence of increased cytokine expression. Endothelial cells and mononuclear leucocytes release cytokines, growth factors, and adhesion molecules due to thrombin activation. On the other hand, cytokines can activate coagulation. The cross-talk between coagulation and inflammation is mediated via protease-activated receptors (PARs). These receptors might become potential targets to be considered for counteracting the clinical expressions of COVID-19. SARS-CoV-2 is effectively able to activate local and circulating coagulation factors, thus inducing the generation of disseminated coagula. LMWH may be considered as the new frontier in the treatment of COVID-19 and Long-COVID-19. Indeed, direct oral anticoagulants (DOACs) may be an alternative option for both early and later treatment of COVID-19 patients due to their ability to inhibit PARs. The aim of this report was to evaluate the role of anticoagulants-and DOACs in particular in COVID-19 and Long-COVID-19 patients. We report the case of a COVID-19 patient who, after administration of enoxaparin developed DIC secondary to virosis and positivity for platelet factor 4 (PF4) and a case of Long-COVID with high residual cardiovascular risk and persistence of blood chemistry of inflammation and procoagulative state.

Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). The availability of effective and well-tolerated antiviral drugs for the treatment of COVID-19 patients is still very limited. Traditional herbal medicines elicit antiviral activity against various viruses and might therefore represent a promising option for the complementary treatment of COVID-19 patients. The application of turmeric root in herbal medicine has a very long history. Its bioactive ingredient curcumin shows a broad-spectrum antimicrobial activity. In the present study, we investigated the antiviral activity of aqueous turmeric root extract, the dissolved content of a curcumin-containing nutritional supplement capsule, and pure curcumin against SARS-CoV-2. Turmeric root extract, dissolved turmeric capsule content, and pure curcumin effectively neutralized SARS-CoV-2 at subtoxic concentrations in Vero E6 and human Calu-3 cells. Furthermore, curcumin treatment significantly reduced SARS-CoV-2 RNA levels in cell culture supernatants. Our data uncover curcumin as a promising compound for complementary COVID-19 treatment. Curcumin concentrations contained in turmeric root or capsules used as nutritional supplements completely neutralized SARS-CoV-2 in vitro. Our data argue in favor of appropriate and carefully monitored clinical studies that vigorously test the effectiveness of complementary treatment of COVID-19 patients with curcumin-containing products.

Hepatitis D is the smallest virus known to infect humans, the most aggressive, causing the most severe disease. It is considered a satellite or defective virus requiring the hepatitis B surface antigen (HBsAg) for its replication with approximately 10-70 million persons infected. Elimination of hepatitis D is, therefore, closely tied to hepatitis B elimination. There is a paucity of quality data in many resource-poor areas. Despite its aggressive natural history, treatment options for hepatitis D to date have been limited and, in many places, inaccessible. For decades, Pegylated interferon alpha (Peg IFN α) offered limited response rates (20%) where available. Developments in understanding viral replication pathways has meant that, for the first time in over three decades, specific therapy has been licensed for use in Europe. Bulevirtide (Hepcludex®) is an entry inhibitor approved for use in patients with confirmed viraemia and compensated disease. It can be combined with Peg IFN α and/or nucleos(t)ide analogue for hepatitis B. Early reports suggest response rates of over 50% with good tolerability profile. Additional agents showing promise include the prenylation inhibitor lonafarnib, inhibitors of viral release (nucleic acid polymers) and better tolerated Peg IFN lambda (λ). These agents remain out of reach for most resource limited areas where access to new therapies are delayed by decades. strategies to facilitate access to care for the most vulnerable should be actively sought by all stakeholders.

Dengue is the most significant arbovirus worldwide and a public health threat to non-endemic areas in which Aedes vectors are present. Autochthonous dengue transmission has been reported in several European countries in the last decade. Infected travelers from endemic regions arriving to areas colonized by Aedes albopictus in Europe need to be monitored in surveillance and control programs. We aimed to perform molecular characterization of RT-PCR-positive dengue cases detected in Catalonia, northeastern Spain, from 2013 to 2018. The basic demographic information and the geographical regions of importation were also analyzed. One-hundred four dengue cases were studied (103 imported infections and the first autochthonous case in our region). The dengue virus strains detected were serotyped and genotyped using molecular methods, and phylogenetic analyses were conducted. All four dengue serotypes were detected in travelers, including up to 10 different genotypes, reflecting the global circulation of dengue in endemic areas. The primary travel-related case of the 2018 autochthonous transmission was not identified, but the molecular analysis revealed dengue serotype 1, genotype I of Asian origin. Our results highlight the diversity of imported dengue virus strains and the role of molecular epidemiology in supporting arbovirus surveillance programs.

Comment in Viruses. 2021 Nov 27;13(12):

Viruses are extraordinary biological entities that can only thrive as obligate intracellular parasites, exploiting other living cellular components in order to reproduce [...].

Influenza A virus nonstructural protein 1 (NS1) plays an important role in evading host innate immunity. NS1 inhibits interferon (IFN) responses via multiple mechanisms, including sequestering dsRNA and suppressing retinoic acid-inducible gene I (RIG-I) signaling by interacting with RIG-I and tripartite motif-containing protein 25 (TRIM25). In the current study, we demonstrated the mitochondrial localization of NS1 at the early stage of influenza virus infection. Since NS1 does not contain mitochondria-targeting signals, we suspected that there is an association between the NS1 and mitochondrial proteins. This hypothesis was tested by demonstrating the interaction of NS1 with mitochondrial antiviral-signaling protein (MAVS) in a RIG-I-independent manner. Importantly, the association with MAVS facilitated the mitochondrial localization of NS1 and thereby significantly impeded MAVS-mediated Type I IFN production.

Diverse coronavirus (CoV) strains can infect both humans and animals and produce various diseases. CoVs have caused three epidemics and pandemics in the last two decades, and caused a severe impact on public health and the global economy. Therefore, it is of utmost importance to understand the emergence and evolution of endemic and emerging CoV diversity in humans and animals. For diverse bird species, the Infectious Bronchitis Virus is a significant one, whereas feline enteric and canine coronavirus, recombined to produce feline infectious peritonitis virus, infects wild cats. Bovine and canine CoVs have ancestral relationships, while porcine CoVs, especially SADS-CoV, can cross species barriers. Bats are considered as the natural host of diverse strains of alpha and beta coronaviruses. Though MERS-CoV is significant for both camels and humans, humans are nonetheless affected more severely. MERS-CoV cases have been reported mainly in the Arabic peninsula since 2012. To date, seven CoV strains have infected humans, all descended from animals. The severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) are presumed to be originated in Rhinolopoid bats that severely infect humans with spillover to multiple domestic and wild animals. Emerging alpha and delta variants of SARS-CoV-2 were detected in pets and wild animals. Still, the intermediate hosts and all susceptible animal species remain unknown. SARS-CoV-2 might not be the last CoV to cross the species barrier. Hence, we recommend developing a universal CoV vaccine for humans so that any future outbreak can be prevented effectively. Furthermore, a One Health approach coronavirus surveillance should be implemented at human-animal interfaces to detect novel coronaviruses before emerging to humans and to prevent future epidemics and pandemics.

Calf diarrhea is one of the common diseases involved in the process of calf feeding. In this study, a sample of calf diarrhea that tested positive for bovine coronavirus and bovine astrovirus was subjected to high-throughput sequencing. The reassembly revealed the complete genomes of bovine norovirus, bovine astrovirus, bovine kobuvirus, and the S gene of bovine coronavirus. Phylogenetic analysis showed that the ORF2 region of bovine astrovirus had the lowest similarity with other strains and gathered in the Mamastrovirus unclassified genogroup, suggesting a new serotype/genotype could appear. Compared with the most closely related strain, there are six amino acid mutation sites in the S gene of bovine coronavirus, most of which are located in the S1 subunit region. The bovine norovirus identified in our study was BNoV-GIII 2, based on the VP1 sequences. The bovine kobuvirus is distributed in the Aichi virus B genus; the P1 gene shows as highly variable, while the 3D gene is highly conserved. These findings enriched our knowledge of the viruses in the role of calf diarrhea, and help to develop an effective strategy for disease prevention and control.

Rotavirus is the most significant cause of severe acute gastroenteritis among children under 5 years of age, worldwide. Sub-Saharan Africa particularly bears the brunt of the diarrheal deaths. A meta-analysis was conducted on 43 eligible studies published between 1982 and 2020 to estimate the pooled prevalence of rotavirus infection and changes in the main rotavirus strains circulating before and after vaccine introduction among under-five children in South Africa. The pooled national prevalence of rotavirus infection was estimated at 24% (95% CI: 21-27%) for the pre-vaccination period and decreased to 23% (95% CI: 21-25%) in the post-vaccination period. However, an increased number of cases was observed in the KwaZulu-Natal (21-28%) and Western Cape (18-24%) regions post-vaccination. The most dominant genotype combinations in the pre-vaccine era was G1P[8], followed by G2P[4], G3P[8], and G1P[6]. After vaccine introduction, a greater genotype diversity was observed, with G9P[8] emerging as the predominant genotype combination, followed by G2P[4], G12P[8], and G1P[8]. The introduction of the rotavirus vaccine was associated with a reduction in the burden of rotavirus-associated diarrhea in South Africa, although not without regional fluctuation. The observed changing patterns of genotype distribution highlights the need for ongoing surveillance to monitor the disease trend and to identify any potential effects associated with the dynamics of genotype changes on vaccine pressure/failure.

During the 2020-2021 winter season, an outbreak of clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) virus occurred in South Korea. Here, we evaluated the pathogenicity and transmissibility of A/mandarin duck/Korea/H242/2020 (H5N8) (H242/20(H5N8)) first isolated from this outbreak in specific pathogen-free (SPF) chickens and commercial ducks in comparison with those of A/duck/Korea/HD1/2017(H5N6) (HD1/17(H5N6)) from a previous HPAI outbreak in 2017-2018. In chickens, the 50% chicken lethal dose and mean death time of H242/20(H5N8) group were 104.5 EID50 and 4.3 days, respectively, which indicate less virulent than those of HD1/17(H5N6) (103.6 EID50 and 2.2 days). Whereas, chickens inoculated with H242/20(H5N8) survived longer and had a higher titer of viral shedding than those inoculated with HD1/17(H5N6), which may increase the risk of viral contamination on farms. All ducks infected with either HPAI virus survived without clinical symptoms. In addition, they exhibited a longer virus shedding period and a higher transmission rate, indicating that ducks may play an important role as a silent carrier of both HPAI viruses. These results suggest that the pathogenic characteristics of HPAI viruses in chickens and ducks need to be considered to effectively control HPAI outbreaks in the field.

In this retrospective descriptive study we focus on cases of three patients who underwent phage therapy procedures at Eliava Phage Therapy Center (EPTC) in Tbilisi, Georgia. Patients with chronic infectious diseases related to Pseudomonas aeruginosa (two patients, lower respiratory tract infection (LRTI)) and Klebsiella pneumoniae (one patient, urinary tract infection (UTI)) are among those very few EPTC patients whose pathogens persisted through phage therapy. By looking at bacterial strains and personalized phages used against them we tried to point towards possible adaptation strategies that are employed by these pathogens. Genome restriction-based Pulsed Field Gel Electrophoresis (PFGE) profiling of strains isolated before and after phage therapy hints towards two strategies of adaptation. In one patient case (Pseudomonas aeruginosa related lung infection) bacterial strains before and after phage therapy were indistinguishable according to their PFGE profiles, but differed in their phage susceptibility properties. On the other hand, in two other patient cases (Pseudomonas aeruginosa related LRTI and Klebsiella pneumoniae related UTI) bacterial adaptation strategy seemed to have resulted in diversification of infecting strains of the same species. With this work we want to attract more attention to phage resistance in general as well as to its role in phage therapy.

The hepatitis A virus (HAV) is a leading cause of acute viral hepatitis worldwide. It is transmitted mainly by direct contact with patients who have been infected or by ingesting contaminated water or food. The virus is endemic in low-income countries where sanitary and sociodemographic conditions are poor. Paradoxically, improving sanitary conditions in these countries, which reduces the incidence of HAV infections, can lead to more severe disease in susceptible adults. The populations of developed countries are highly susceptible to HAV, and large outbreaks can occur when the virus is spread by globalization and by increased travel and movement of foodstuffs. Most of these outbreaks occur among high-risk groups: travellers, men who have sex with men, people who use substances, and people facing homelessness. Hepatitis A infections can be prevented by vaccination; safe and effective vaccines have been available for decades. Several countries have successfully introduced universal mass vaccination for children, but high-risk groups in high-income countries remain insufficiently protected. The development of HAV antivirals may be important to control HAV outbreaks in developed countries where a universal vaccination programme is not recommended.

Phage therapy (PT) shows promising potential in managing biofilm infections, which include refractory orthopedic infections. We report the case of a 13-year-old girl who developed chronic polymicrobial biofilm infection of a pelvic bone allograft after Ewing's sarcoma resection surgery. Chronic infection by Clostridium hathewayi, Proteus mirabilis and Finegoldia magna was worsened by methicillin-susceptible Staphylococcus aureus exhibiting an inducible Macrolides-Lincosamides-Streptogramin B resistance phenotype (iMLSB). After failure of conventional conservative treatment, combination of in situ anti-S. aureus PT with surgical debridement and intravenous antibiotic therapy led to marked clinical and microbiological improvement, yet failed to prevent a recurrence of infection on the midterm. This eventually led to surgical graft replacement. Multiple factors can explain this midterm failure, among which incomplete coverage of the polymicrobial infection by PT. Indeed, no phage therapy against C. hathewayi, P. mirabilis or F. magna could be administered. Phage-antibiotic interactions were investigated using OmniLog® technology. Our results suggest that phage-antibiotic interactions should not be considered "unconditionally synergistic", and should be assessed on a case-by-case basis. Specific pharmacodynamics of phages and antibiotics might explain these differences. More than two years after final graft replacement, the patient remains cured of her sarcoma and no further infections occurred.

West Nile virus (WNV, Flaviviridae, Flavivirus) is a mosquito-borne flavivirus introduced to North America in 1999. Since 1999, the Earth's average temperature has increased by 0.6 °C. Mosquitoes are ectothermic organisms, reliant on environmental heat sources. Temperature impacts vector-virus interactions which directly influence arbovirus transmission. RNA viral replication is highly error-prone and increasing temperature could further increase replication rates, mutation frequencies, and evolutionary rates. The impact of temperature on arbovirus evolutionary trajectories and fitness landscapes has yet to be sufficiently studied. To investigate how temperature impacts the rate and extent of WNV evolution in mosquito cells, WNV was experimentally passaged 12 times in Culex tarsalis cells, at 25 °C and 30 °C. Full-genome deep sequencing was used to compare genetic signatures during passage, and replicative fitness was evaluated before and after passage at each temperature. Our results suggest adaptive potential at both temperatures, with unique temperature-dependent and lineage-specific genetic signatures. Further, higher temperature passage was associated with significantly increased replicative fitness at both temperatures and increases in nonsynonymous mutations. Together, these data indicate that if similar selective pressures exist in natural systems, increases in temperature could accelerate emergence of high-fitness strains with greater phenotypic plasticity.

People with human immunodeficiency virus (HIV) (PWH) have reduced gut barrier integrity ("leaky gut") that permits diffusion of microbial antigens (microbial translocation) such as lipopolysaccharide (LPS) into the circulation, stimulating inflammation. A potential source of this disturbance, in addition to gut lymphoid tissue CD4+ T-cell depletion, is the interaction between the gut barrier and gut microbes themselves. We evaluated the relationship of gut barrier integrity, as indexed by plasma occludin levels (higher levels corresponding to greater loss of occludin from the gut barrier), to gut microbial diversity. PWH and people without HIV (PWoH) participants were recruited from community sources and provided stool, and 16S rRNA amplicon sequencing was used to characterize the gut microbiome. Microbial diversity was indexed by Faith's phylogenetic diversity (PD). Participants were 50 PWH and 52 PWoH individuals, mean ± SD age 45.6 ± 14.5 years, 28 (27.5%) women, 50 (49.0%) non-white race/ethnicity. PWH had higher gut microbial diversity (Faith's PD 14.2 ± 4.06 versus 11.7 ± 3.27; p = 0.0007), but occludin levels were not different (1.84 ± 0.311 versus 1.85 ± 0.274; p = 0.843). Lower gut microbial diversity was associated with higher plasma occludin levels in PWH (r = -0.251; p = 0.0111), but not in PWoH. A multivariable model demonstrated an interaction (p = 0.0459) such that the correlation between Faith's PD and plasma occludin held only for PWH (r = -0.434; p = 0.0017), but not for PWoH individuals (r = -0.0227; p = 0.873). The pattern was similar for Shannon alpha diversity. Antiretroviral treatment and viral suppression status were not associated with gut microbial diversity (ps > 0.10). Plasma occludin levels were not significantly related to age, sex or ethnicity, nor to current or nadir CD4 or plasma viral load. Higher occludin levels were associated with higher plasma sCD14 and LPS, both markers of microbial translocation. Together, the findings suggest that damage to the gut epithelial barrier is an important mediator of microbial translocation and inflammation in PWH, and that reduced gut microbiome diversity may have an important role.

Nearly 40 years have passed since the initial cases of infection with the human mmunodeficiency virus (HIV) were identified as a new disease entity and the cause of acquired immunodeficiency disease (AIDS). This virus, unlike any other, is capable of causing severe suppression of our adaptive immune defense mechanisms by directly infecting and destroying helper T cells leading to increased susceptibility to a wide variety of microbial pathogens, especially those considered to be intracellular or opportunistic. After T cells are infected, HIV reproduces itself via a somewhat unique mechanism involving various metabolic steps, which includes the use of a reverse transcriptase enzyme that enables the viral RNA to produce copies of its complementary DNA. Subsequent physiologic steps lead to the production of new virus progeny and the eventual death of the invaded T cell. Fortunately, both serologic and molecular tests (such as PCR) can be used to confirm the diagnosis of an HIV infection. In the wake of the current COVID-19 pandemic, it appears that people living with HIV/AIDS are equally or slightly more susceptible to the etiologic agent, SARS-CoV-2, than the general population having intact immune systems, but they may have more serious outcomes. Limited clinical trials have also shown that the currently available COVID-19 vaccines are both safe and effective in affording protection to HIV/AIDS patients. In this review, we further explore the unique dynamic of HIV/AIDS in the context of the worldwide COVID-19 pandemic and the implementation of vaccines as a protective measure against COVID-19, as well as what immune parameters and safeguards should be monitored in this immunocompromised group following vaccination.

Influenza B viruses (IBVs) are causing an increasing burden of morbidity and mortality, yet the prevalence of culture-adapted mutations in human seasonal IBVs are unclear. We collected 368 clinical samples from patients with influenza-like illness in Missouri during the 2019-2020 influenza season and recovered 146 influenza isolates including 38 IBV isolates. Of MDCK-CCL34, MDCK-Siat1, and humanized MDCK (hCK), hCK showed the highest virus recovery efficiency. All Missourian IBVs belonged to the Victoria V1A.3 lineage, all of which contained a three-amino acid deletion on the HA protein and were antigenically distant from the Victoria lineage IBV vaccine strain used during that season. By comparing genomic sequences of these IBVs in 31 paired samples, eight cell-adapted nonsynonymous mutations were identified, with the majority in the RNA polymerase. Analyses of IBV clinical sample-isolate pairs from public databases further showed that cell- and egg-adapted mutations occurred more widely in viral proteins, including the receptor and antibody binding sites on HA. Our study suggests that hCK is an effective platform for IBV isolation and that culture-adapted mutations may occur during IBV isolation. As culture-adapted mutations may affect subsequent virus studies and vaccine development, the knowledge from this study may help optimize strategies for influenza surveillance, vaccine strain selection, and vaccine development.

Cytomegalovirus (CMV) infections acquired by very-low-birthweight (VLBW) infants are incompletely characterized. To examine CMV transmission in VLBW infants, we evaluated maternal DNAlactia, infant DNAemia, and presence of clinical disease in a blinded study in VLBW infants in our newborn intensive care unit (NICU). To examine these issues, 200 VLBW infants were enrolled in a surveillance study, with weekly breast milk and infant whole blood samples collected, as available. Virologic (breast milk and infant whole blood real time PCR) and immunologic (IgG, IgM, and IgG avidity) correlates were evaluated. A chart review examined whether infants had symptoms compatible with CMV disease. DNAlactia was identified in 65/150 (43%) of lactating mothers. Nine CMV infections were identified in 9/75 CMV-exposed infants (12% of exposed infants). A higher median breast milk viral load (DNAlactia) correlated with an increased likelihood of DNAemia (p = 0.05). Despite potential symptoms compatible with CMV infection, clinicians had not considered the diagnosis of CMV in 6/9 cases (66%). All of these infants had chronic lung disease at discharge. There was no correlation between IgG antibody titer or IgG avidity index and the likelihood of transmission or CMV disease. In conclusion, in VLBW infants receiving milk from seropositive mothers, CMV infections are commonly acquired, and are frequently unrecognized. Future studies are needed to determine whether routine surveillance for CMV of either breast milk or infant plasma is beneficial in preventing or recognizing infection.

Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (>3000 EU/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding.

Infection with HPV starts with the access of the viral particles to basal cells in the epidermis, potentially via microtraumas to the skin. The basal cells are able to keep away these pathogens in normal circumstances through a robust immune response from the host, as HPV infections are, in general, cleared within 2 to 3 weeks. However, the rare instances of persistent infection and/or in cases where the host immune system is compromised are major risk factors for the development of lesions potentially leading to malignancy. Evolutionarily, obligatory pathogens such as HPVs would not be expected to risk exposing the host to lethal cancer, as this would entail challenging their own life cycle, but infection with these viruses is highly correlated with cancer and malignancy-as in cancer of the cervix, which is almost always associated with these viruses. Despite this key associative cause and the availability of very effective vaccines against these viruses, therapeutic interventions against HPV-induced cancers are still a challenge, indicating the need for focused translational research. In this review, we will consider the key roles that the viral proteins play in driving the host cells to carcinogenesis, mainly focusing on events orchestrated by early proteins E5, E6 and E7-the not-so-good, the bad and the ugly-and discuss and summarize the major events that lead to these viruses mechanistically corrupting cellular homeostasis, giving rise to cancer and malignancy.

The mechanisms generating variability in viruses are diverse. Variability allows baculoviruses to evolve with their host and with changes in their environment. We examined the role of one genetic variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and its contribution to the variability of the virus under laboratory conditions. A mixture of natural isolates (ChinNPV-Mex1) contained two genetic variants that dominated over other variants in individual larvae that consumed high (ChinNPV-K) and low (ChinNPV-E) concentrations of inoculum. Studies on the ChinNPV-K variant indicated that it was capable of generating novel variation in a concentration-dependent manner. In cell culture, cells inoculated with high concentrations of ChinNPV-K produced OBs with the ChinNPV-K REN profile, whereas a high diversity of ChinNPV variants was recovered following plaque purification of low concentrations of ChinNPV-K virion inoculum. Interestingly, the ChinNPV-K variant could not be recovered from plaques derived from low concentration inocula originating from budded virions or occlusion-derived virions of ChinNPV-K. Genome sequencing revealed marked differences between ChinNPV-K and ChinNPV-E, with high variation in the ChinNPV-K genome, mostly due to single nucleotide polymorphisms. We conclude that ChinNPV-K is an unstable genetic variant that is responsible for generating much of the detected variability in the natural ChinNPV isolates used in this study.

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.

RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.

The increase of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) causes a threat to human health. LA-MRSA can be transmitted from animals to animal caretakers, which may further spread MRSA to communities and health care facilities. The objective of this work was to study the efficacy of phage treatment in the eradication of LA-MRSA from healthy carrier pigs. A total of 19 MRSA -positive weanling pigs were assigned to a test (n = 10) and a control group (n = 9). A phage cocktail containing three Staphylococcus phages, or a control buffer was administered to the nares and skin of the pigs three times every two days, after which the phage and MRSA levels in nasal and skin swab samples were monitored for a three-week period. The sensitivity of the strains isolated during the follow-up period to the phage cocktail and each phage individually was analyzed and the pig sera were tested for antibodies against the phages used in the cocktail. The phage treatment did not cause any side effects to the pigs. Phages were found in the skin and nasal samples on the days following the phage applications, but there was no reduction in the MRSA levels in the sampled animals. Phage-resistant strains or phage-specific antibodies were not detected during the experiment. The MRSA load in these healthy carrier animals was only 10-100 CFU/swab or nasal sample, which was likely below the replication threshold of phages. The effectiveness of phage treatment to eradicate MRSA from the pigs could thus not be (reliably) determined.

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals with serious economic consequences. FMD is endemic in Southeast Asia (SEA) and East Asia (EA) with the circulation of multiple serotypes, posing a threat to Australia and other FMD-free countries. Although vaccination is one of the most important control measures to prevent FMD outbreaks, the available vaccines may not be able to provide enough cross-protection against the FMD viruses (FMDVs) circulating in these countries due to the incursion of new lineages and sub-lineages as experienced in South Korea during 2010, a FMD-free country, when a new lineage of serotype O FMDV (Mya-98) spread to the country, resulting in devastating economic consequences. In this study, a total of 62 serotype O (2013-2018) viruses selected from SEA and EA countries were antigenically characterized by virus neutralization tests using three existing (O/HKN/6/83, O/IND/R2/75 and O/PanAsia-2) and one putative (O/MYA/2009) vaccine strains and full capsid sequencing. The Capsid sequence analysis revealed three topotypes, Cathay, SEA and Middle East-South Asia (ME-SA) of FMDVs circulating in the region. The vaccines used in this study showed a good match with the SEA and ME-SA viruses. However, none of the recently circulating Cathay topotype viruses were protected by any of the vaccine strains, including the existing Cathay topotype vaccine (O/HKN/6/83), indicating an antigenic drift and, also the urgency to monitor this topotype in the region and develop a new vaccine strain if necessary, although currently the presence of this topotype is mainly restricted to China, Hong Kong, Taiwan and Vietnam. Further, the capsid sequences of these viruses were analyzed that identified several capsid amino acid substitutions involving neutralizing antigenic sites 1, 2 and 5, which either individually or together could underpin the observed antigenic drift.

Human Adenoviruses (HAdV) are known to be potentially associated with strong inflammatory responses and morbidity in pediatric patients. Although most of the primary infections are self-limiting, the severity of clinical presentation, the elevation of the white blood cell count and inflammatory markers often mimic a bacterial infection and lead to an inappropriate use of antibiotics. In infections caused by HAdV, rapid antigen detection kits are advisable but not employed routinely; costs and feasibility of rapid syndromic molecular diagnosis may limit its use in the in-hospital setting; lymphocyte cultures and two-sampled serology are time consuming and impractical when considering the use of antibiotics. In this review, we aim to describe the principal diagnostic tools and the immune response in HAdV infections and evaluate whether markers based on the response of the host may help early recognition of HAdV and avoid inappropriate antimicrobial prescriptions in acute airway infections.

Evidence is emerging that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect various organs of the body, including cardiomyocytes and cardiac endothelial cells in the heart. This review focuses on the effects of SARS-CoV-2 in the heart after direct infection that can lead to myocarditis and an outline of potential treatment options. The main points are: (1) Viral entry: SARS-CoV-2 uses specific receptors and proteases for docking and priming in cardiac cells. Thus, different receptors or protease inhibitors might be effective in SARS-CoV-2-infected cardiac cells. (2) Viral replication: SARS-CoV-2 uses RNA-dependent RNA polymerase for replication. Drugs acting against ssRNA(+) viral replication for cardiac cells can be effective. (3) Autophagy and double-membrane vesicles: SARS-CoV-2 manipulates autophagy to inhibit viral clearance and promote SARS-CoV-2 replication by creating double-membrane vesicles as replication sites. (4) Immune response: Host immune response is manipulated to evade host cell attacks against SARS-CoV-2 and increased inflammation by dysregulating immune cells. Efficiency of immunosuppressive therapy must be elucidated. (5) Programmed cell death: SARS-CoV-2 inhibits programmed cell death in early stages and induces apoptosis, necroptosis, and pyroptosis in later stages. (6) Energy metabolism: SARS-CoV-2 infection leads to disturbed energy metabolism that in turn leads to a decrease in ATP production and ROS production. (7) Viroporins: SARS-CoV-2 creates viroporins that lead to an imbalance of ion homeostasis. This causes apoptosis, altered action potential, and arrhythmia.

Viruses are obligate intracellular parasites that are dependent on host factors for their replication. One such host protein, p97 or the valosin-containing protein (VCP), is a highly conserved AAA ATPase that facilitates replication of diverse RNA- and DNA-containing viruses. The wide range of cellular functions attributed to this ATPase is consistent with its participation in multiple steps of the virus life cycle from entry and uncoating to viral egress. Studies of VCP/p97 interactions with viruses will provide important information about host processes and cell biology, but also viral strategies that take advantage of these host functions. The critical role of p97 in viral replication might be exploited as a target for development of pan-antiviral drugs that exceed the capability of virus-specific vaccines or therapeutics.

(1) This study aimed to evaluate characteristics, perinatal outcomes, and placental pathology of pregnant women with or without SARS-CoV-2 infection in the context of maternal PCR cycle threshold (CT) values. (2) This was a retrospective case-control study in a third-level health center in Mexico City with universal screening by RT-qPCR. The association of COVID-19 manifestations, preeclampsia, and preterm birth with maternal variables and CT values were assessed by logistic regression models and decision trees. (3) Accordingly, 828 and 298 women had a negative and positive test, respectively. Of those positive, only 2.6% of them presented mild to moderate symptoms. Clinical characteristics between both groups of women were similar. No associations between CT values were found for maternal features, such as pre-gestational BMI, age, and symptomatology. A significantly higher percentage of placental fibrinoid was seen with women with low CTs (<25; p < 0.01). Regarding perinatal outcomes, preeclampsia was found to be significantly associated with symptomatology but not with risk factors or CT values (p < 0.01, aOR = 14.72). Moreover, 88.9% of women diagnosed with COVID-19 at <35 gestational weeks and symptomatic developed preeclampsia. (4) The data support strong guidance for pregnancies with SARS-CoV-2 infection, in particular preeclampsia and placental pathology, which need further investigation.

The neurovascular units (NVU) are the minimal functional units of the blood-brain barrier (BBB), composed of endothelial cells, pericytes, astrocytes, microglia, neurons, and the basement membrane. The BBB serves as an important interface for immune communication between the brain and peripheral circulation. Disruption of the NVU by the human immunodeficiency virus-1 (HIV-1) induces dysfunction of the BBB and triggers inflammatory responses, which can lead to the development of neurocognitive impairments collectively known as HIV-1-associated neurocognitive disorders (HAND). Methamphetamine (METH) use disorder is a frequent comorbidity among individuals infected with HIV-1. METH use may be associated not only with rapid HIV-1 disease progression but also with accelerated onset and increased severity of HAND. However, the molecular mechanisms of METH-induced neuronal injury and cognitive impairment in the context of HIV-1 infection are poorly understood. In this review, we summarize recent progress in the signaling pathways mediating synergistic impairment of the BBB and neuronal injury induced by METH and HIV-1, potentially accelerating the onset or severity of HAND in HIV-1-positive METH abusers. We also discuss potential therapies to limit neuroinflammation and NVU damage in HIV-1-infected METH abusers.

The error rate displayed during template copying to produce viral RNA progeny is a biologically relevant parameter of the replication complexes of viruses. It has consequences for virus-host interactions, and it represents the first step in the diversification of viruses in nature. Measurements during infections and with purified viral polymerases indicate that mutation rates for RNA viruses are in the range of 10-3 to 10-6 copying errors per nucleotide incorporated into the nascent RNA product. Although viruses are thought to exploit high error rates for adaptation to changing environments, some of them possess misincorporation correcting activities. One of them is a proofreading-repair 3' to 5' exonuclease present in coronaviruses that may decrease the error rate during replication. Here we review experimental evidence and models of information maintenance that explain why elevated mutation rates have been preserved during the evolution of RNA (and some DNA) viruses. The models also offer an interpretation of why error correction mechanisms have evolved to maintain the stability of genetic information carried out by large viral RNA genomes such as the coronaviruses.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has expanded into a global pandemic, with more than 220 million affected persons and almost 4.6 million deaths by 8 September 2021. In particular, Europe and the Americas have been heavily affected by high infection and death rates. In contrast, much lower infection rates and mortality have been reported generally in Africa, particularly in the sub-Saharan region (with the exception of the Southern Africa region). There are different hypotheses for this African paradox, including less testing, the young age of the population, genetic disposition, and behavioral and epidemiological factors. In the present review, we address different immunological factors and their correlation with genetic factors, pre-existing immune status, and differences in cytokine induction patterns. We also focus on epidemiological factors, such as specific medication coverage, helminth distribution, and malaria endemics in the sub-Saharan region. An analysis combining different factors is presented that highlights the central role of the NF-κB signaling pathway in the African paradox. Importantly, insights into the interplay of different factors with the underlying immune pathological mechanisms for COVID-19 can provide a better understanding of the disease and the development of new targets for more efficient treatment strategies.

The COVID-19 pandemic has hugely impacted global public health and economy. The COVID-19 has also shown potential impacts on maternal perinatal and neonatal outcomes. This systematic review aimed to summarize the evidence from existing systematic reviews about the effects of SARS-CoV-2 infections on maternal perinatal and neonatal outcomes. We searched PubMed, MEDLINE, Embase, and Web of Science in accordance with PRISMA guidelines, from 1 December 2019 to 7 July 2021, for published review studies that included case reports, primary studies, clinical practice guidelines, overviews, case-control studies, and observational studies. Systematic reviews that reported the plausibility of mother-to-child transmission of COVID-19 (also known as vertical transmission), maternal perinatal and neonatal outcomes, and review studies that addressed the effect of SARS-CoV-2 infection during pregnancy were also included. We identified 947 citations, of which 69 studies were included for further analysis. Most (>70%) of the mother-to-child infection was likely due to environmental exposure, although a significant proportion (about 20%) was attributable to potential vertical transmission of SARS-CoV-2. Further results of the review indicated that the mode of delivery of pregnant women infected with SARS-CoV-2 could not increase or decrease the risk of infection for the newborns (outcomes), thereby emphasizing the significance of breastfeeding. The issue of maternal perinatal and neonatal outcomes with SARS-CoV-2 infection continues to worsen during the ongoing COVID-19 pandemic, increasing maternal and neonatal mortality, stillbirth, ruptured ectopic pregnancies, and maternal depression. Based on this study, we observed increasing rates of cesarean delivery from mothers with SARS-CoV-2 infection. We also found that SARS-CoV-2 could be potentially transmitted vertically during the gestation period. However, more data are needed to further investigate and follow-up, especially with reports of newborns infected with SARS-CoV-2, in order to examine a possible long-term adverse effect.

Since its outbreak in December 2019, the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to an enormous rise in scientific response with an excess of COVID-19-related studies on the pathogenesis and potential therapeutic approaches. Solid organ transplant (SOT) recipients are a heterogeneous population with long-lasting immunosuppression as a joining element. Immunocompromised patients are a vulnerable population with a high risk of severe infections and an increased infection-related mortality rate. It was postulated that the hyperinflammatory state due to cytokine release syndrome during severe COVID-19 could be alleviated by immunosuppressive therapy in SOT patients. On the other hand, it was previously established that T cell-mediated immunity, which is significantly weakened in SOT recipients, is the main component of antiviral immune responses. In this paper, we present the current state of science on COVID-19 immunology in relation to solid organ transplantation with prospective therapeutic and vaccination strategies in this population.

Viral infections can give rise to a systemic decrease in the total number of lymphocytes in the blood, referred to as lymphopenia. Lymphopenia may affect the host adaptive immune responses and impact the clinical course of acute viral infections. Detailed knowledge on how viruses induce lymphopenia would provide valuable information into the pathogenesis of viral infections and potential therapeutic targeting. In this review, the current progress of viruses-induced lymphopenia is summarized and the potential mechanisms and factors involved are discussed.

Infants exposed to Zika virus (ZIKV) prenatally may develop birth defects, developmental deficits, or remain asymptomatic. It is unclear why some infants are more affected than others, although enhancement of maternal ZIKV infection via immunity to an antigenically similar virus, dengue virus (DENV), may play a role. We hypothesized that DENV immunity may worsen prenatal ZIKV infection and developmental deficits in offspring. We utilized a translational macaque model to examine how maternal DENV immunity influences ZIKV-exposed infant macaque neurodevelopment in the first month of life. We inoculated eight macaques with prior DENV infection with ZIKV, five macaques with ZIKV, and four macaques with saline. DENV/ZIKV-exposed infants had significantly worse visual orientation skills than ZIKV-exposed infants whose mothers were DENV-naive, with no differences in motor, sensory or state control development. ZIKV infection characteristics and pregnancy outcomes did not individually differ between dams with and without DENV immunity, but when multiple factors were combined in a multivariate model, maternal DENV immunity combined with ZIKV infection characteristics and pregnancy parameters predicted select developmental outcomes. We demonstrate that maternal DENV immunity exacerbates visual orientation and tracking deficits in ZIKV-exposed infant macaques, suggesting that human studies should evaluate how maternal DENV immunity impacts long-term neurodevelopment.

Syncytium formation, i.e., cell-cell fusion resulting in the formation of multinucleated cells, is a hallmark of infection by paramyxoviruses and other pathogenic viruses. This natural mechanism has historically been a diagnostic marker for paramyxovirus infection in vivo and is now widely used for the study of virus-induced membrane fusion in vitro. However, the role of syncytium formation in within-host dissemination and pathogenicity of viruses remains poorly understood. The diversity of henipaviruses and their wide host range and tissue tropism make them particularly appropriate models with which to characterize the drivers of syncytium formation and the implications for virus fitness and pathogenicity. Based on the henipavirus literature, we summarized current knowledge on the mechanisms driving syncytium formation, mostly acquired from in vitro studies, and on the in vivo distribution of syncytia. While these data suggest that syncytium formation widely occurs across henipaviruses, hosts, and tissues, we identified important data gaps that undermined our understanding of the role of syncytium formation in virus pathogenesis. Based on these observations, we propose solutions of varying complexity to fill these data gaps, from better practices in data archiving and publication for in vivo studies, to experimental approaches in vitro.

Human T-lymphotropic virus 1 and 2 (HTLV-1/2) belong to the delta group of retroviruses which may cause a life-long infection in humans, HTLV-1 leading to adult T-cell leukemia/lymphoma and other diseases. Different transmission modes have been described, such as breastfeeding, and, as for other blood-borne pathogens, unsafe sexual activity, intravenous drug usage, and blood transfusion and transplantation. The present systematic review was conducted to identify all peer-reviewed studies concerning the work-related infection by HTLV-1/2. A literature search was conducted from January to May 2021, according to the PRISMA methodology, selecting 29 studies: seven related to health care workers (HCWs), five to non-HCWs, and 17 to sex workers (SWs). The findings showed no clear evidence as to the possibility of HTLV-1/2 occupational transmission in HCWs, according to the limited number and quality of the papers. Moreover, non-HCWs showed a higher prevalence in jobs consistent with a lower socioeconomic status or that could represent a familial cluster, and an increased risk of zoonotic transmission from STLV-1-infected non-human primates has been observed in African hunters. Finally, a general increase of HTLV-1 infection was observed in SWs, whereas only one paper described an increased prevalence for HTLV-2, supporting the urgent need for prevention and control measures, including screening, diagnosis, and treatment of HTLV-1/2, to be offered routinely as part of a comprehensive approach to decrease the impact of sexually transmitted diseases in SWs.

SARS-CoV-2 uses ACE2 and TMPRSS2 to gain entry into the cell. However, recent studies have shown that SARS-CoV-2 may use additional host factors that are required for the viral lifecycle. Here we used publicly available datasets, CoV-associated genes, and machine learning algorithms to explore the SARS-CoV-2 interaction landscape in different tissues. We found that in general a small fraction of cells express ACE2 in the different tissues, including nasal, bronchi, and lungs. We show that a small fraction of immune cells (including T cells, macrophages, dendritic cells) found in tissues also express ACE2. We show that healthy circulating immune cells do not express ACE2 and TMPRSS2. However, a small fraction of circulating immune cells (including dendritic cells, monocytes, T cells) in the PBMC of COVID-19 patients express ACE2 and TMPRSS2. Additionally, we found that a large spectrum of cells (in tissues and circulation) in both healthy and COVID-19-positive patients were significantly enriched for SARS-CoV-2 factors, such as those associated with RHOA and RAB GTPases, mRNA translation proteins, COPI- and COPII-mediated transport, and integrins. Thus, we propose that further research is needed to explore if SARS-CoV-2 can directly infect tissue and circulating immune cells to better understand the virus' mechanism of action.

Influenza D virus (IDV) was first isolated in 2011 in Oklahoma, USA from pigs presenting with influenza-like symptoms. IDV is known to mainly circulate in ruminants, especially cattle. In Africa, there is limited information on the epidemiology of IDV, although the virus has likely circulated in the region since 2012. In the present study, we investigated the seropositivity of IDV among domestic ruminants and swine in West and East Africa from 2017 to 2020. Serum samples were analyzed using the hemagglutination inhibition (HI) assay. Our study demonstrated that IDV is still circulating in Africa, with variations in seropositivity among countries and species. The highest seropositivity was detected in cattle (3.9 to 20.9%). Our data highlights a need for extensive surveillance of IDV in Africa in order to better understand the epidemiology of the virus in the region.

Wild-type or engineered bacteriophages have been reported as therapeutic agents in the treatment of several types of diseases, including cancer. They might be used either as naked phages or as carriers of antitumor molecules. Here, we evaluate the role of bacteriophages M13 and T4 in modulating the expression of genes related to cell adhesion, growth, and survival in the androgen-responsive LNCaP prostatic adenocarcinoma-derived epithelial cell line. LNCaP cells were exposed to either bacteriophage M13 or T4 at a concentration of 1 × 105 pfu/mL, 1 × 106 pfu/mL, and 1 × 107 pfu/mL for 24, 48, and 72 h. After exposure, cells were processed for general morphology, cell viability assay, and gene expression analyses. Neither M13 nor T4 exposure altered cellular morphology, but both decreased the MTT reduction capacity of LNCaP cells at different times of treatment. In addition, genes AKT, ITGA5, ITGB1, ITGB3, ITGB5, MAPK3, and PI3K were significantly up-regulated, whilst the genes AR, HSPB1, ITGAV, and PGC1A were down-regulated. Our results show that bacteriophage M13 and T4 interact with LNCaP cells and effectively promote gene expression changes related to anchorage-dependent survival and androgen signaling. In conclusion, phage therapy may increase the response of PCa treatment with PI3K/AKT pathway inhibitors.

The Michael addition reaction is a spontaneous and quick chemical reaction that is widely applied in various fields. This reaction is performed by conjugating an addition of nucleophiles with α, β-unsaturated carbonyl compounds, resulting in the bond formation of C-N, C-S, C-O, and so on. In the development of molecular materials, the Michael addition is not only used to synthesize chemical compounds but is also involved in the mechanism of drug action. Several covalent drugs that bond via Michael addition are regarded as anticarcinogens and anti-inflammatory drugs. Although drug development is mainly focused on pharmaceutical drug discovery, target-based discovery can provide a different perspective for drug usage. However, considerable time and labor are required to define a molecular target through molecular biological experiments. In this review, we systematically examine the chemical structures of current FDA-approved antiviral drugs for potential Michael addition moieties with α, β-unsaturated carbonyl groups, which may exert an unidentified broad-spectrum inhibitory mechanism to target viral or host factors. We thus propose that profiling the targets of antiviral agents, such as Michael addition products, can be achieved by employing a high-throughput LC-MS approach to comprehensively analyze the interaction between drugs and targets, and the subsequent drug responses in the cellular environment to facilitate drug repurposing and/or identify potential adverse effects, with a particular emphasis on the pros and cons of this shotgun proteomic approach.

3D-printed alternatives to standard flocked swabs were rapidly developed to provide a response to the unprecedented and sudden need for an exponentially growing amount of diagnostic tools to fight the COVID-19 pandemic. In light of the anticipated shortage, a hospital-based 3D-printing platform was implemented in our institution for the production of swabs for nasopharyngeal and oropharyngeal sampling based on the freely available, open-source design provided to the community by University of South Florida's Health Radiology and Northwell Health System teams as a replacement for locally used commercial swabs. Validation of our 3D-printed swabs was performed with a head-to-head diagnostic accuracy study of the 3D-printed "Northwell model" with the cobas PCR Media® swab sample kit. We observed an excellent concordance (total agreement 96.8%, Kappa 0.936) in results obtained with the 3D-printed and flocked swabs, indicating that the in-house 3D-printed swab could be used reliably in the context of a shortage of flocked swabs. To our knowledge, this is the first study to report on autonomous hospital-based production and clinical validation of 3D-printed swabs.

Antiretroviral therapy (ART) suppresses HIV-1 replication but does not eradicate the virus. Persistence of HIV-1 latent reservoirs in ART-treated individuals is considered the main obstacle to achieving an HIV-1 cure. However, these HIV-1 reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individuals. HIV-1 latency is regulated at the transcriptional and at multiple post-transcriptional levels. Here, we review recent insights into the possible contribution of viral RNA processing to the persistence of HIV-1 reservoirs, and discuss the clinical implications of persistence of viral RNA species in ART-treated individuals.

IL-15, a proinflammatory cytokine critical for the generation, maintenance, and homeostasis of T cell responses, is produced naturally in response to HIV/SIV infection, but has also demonstrated therapeutic potential. IL-15 can boost CD4+ and CD8+ T cell and NK cell proliferation, activation, and function. However, IL-15 treatment may cause aberrant immune activation and accelerated disease progression in certain circumstances. Moreover, the relationship between the timing of IL-15 administration and disease progression remains unclear. The IL-15 superagonist N-803 was developed to expand the therapeutic potential of IL-15 by maximizing its tissue distribution and half-life. N-803 has garnered enthusiasm recently as a way to enhance the innate and cellular immune responses to HIV/SIV by improving CD8+ T cell recognition and killing of virus-infected cells and directing immune cells to mucosal sites and lymph nodes, the primary sites of virus replication. N-803 has also been evaluated in "shock and kill" strategies due to its potential to reverse latency (shock) and enhance antiviral immunity (kill). This review examines the current literature about the effects of IL-15 and N-803 on innate and cellular immunity, viral burden, and latency reversal in the context of HIV/SIV, and their therapeutic potential both alone and combined with additional interventions such as antiretroviral therapy (ART) and vaccination.

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its emergence of variants needs rapid and point-of-care testing methods for a broad diagnosis. The regular RT-qPCR is time-consuming and limited in central laboratories, so a broad and large-scale screening requirement calls for rapid and in situ methods. In this regard, a reverse transcription recombinase-aided amplification (RT-RAA) is proposed here for the rapid and point-of-care detection of SARS-CoV-2. A set of highly conserved primers and probes targeting more than 98% of SARS-CoV-2 strains, including currently circulating variants (four variants of concerns (VOCs) and three variants of interest (VOIs)), was used in this study. With the preferred primers, the RT-RAA assay showed a 100% specificity to SARS-CoV-2 from eight other respiratory RNA viruses. Moreover, the assay here is of a high sensitivity and 0.48 copies/μL can be detected within 25 min at a constant temperature (42 °C), which can be realized on portable equipment. Furthermore, the RT-RAA assay demonstrated its high agreement for the detection of SARS-CoV-2 in clinical specimens compared with RT-qPCR. The rapid, simple and point-of-care RT-RAA method is expected to be an appealing detection tool to detect SARS-CoV-2, including variants, in clinical diagnostic applications.

HIV persists via integration of the viral DNA into the human genome. The HIV DNA pool within an infected individual is a complex population that comprises both intact and defective viral genomes, each with a distinct integration site, in addition to a unique repertoire of viral quasi-species. Obtaining an accurate profile of the viral DNA pool is critical to understanding viral persistence and resolving interhost differences. Recent advances in next-generation deep sequencing (NGS) technologies have enabled the development of two sequencing assays to capture viral near-full- genome sequences at single molecule resolution (FLIP-seq) or to co-capture full-length viral genome sequences in conjunction with its associated viral integration site (MIP-seq). This commentary aims to provide an overview on both FLIP-seq and MIP-seq, discuss their strengths and limitations, and outline specific chemistry and bioinformatics concerns when using these assays to study HIV persistence.

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in addition to the autochthonous tick-borne encephalitis virus (TBEV) in Europe causes rising concern for public and animal health. The first equine case of West Nile neuroinvasive disease in Austria was diagnosed in 2016. As a consequence, a cross-sectional seroprevalence study was conducted in 2017, including 348 equids from eastern Austria. Serum samples reactive by ELISA for either flavivirus immunoglobulin G or M were further analyzed with the plaque reduction neutralization test (PRNT-80) to identify the specific etiologic agent. Neutralizing antibody prevalences excluding vaccinated equids were found to be 5.3% for WNV, 15.5% for TBEV, 0% for USUV, and 1.2% for WNV from autochthonous origin. Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect WNV nucleic acid in horse sera and was found to be negative in all cases. Risk factor analysis did not identify any factors significantly associated with seropositivity.

Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism.

Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Over the past decade, tremendous progress has been made in systems biology-based approaches to studying immunity to viral infections and responses to vaccines. These approaches that integrate multiple facets of the immune response, including transcriptomics, serology and immune functions, are now being applied to understand correlates of protective immunity against hepatitis C virus (HCV) infection and to inform vaccine development. This review focuses on recent progress in understanding immunity to HCV using systems biology, specifically transcriptomic and epigenetic studies. It also examines proposed strategies moving forward towards an integrated systems immunology approach for predicting and evaluating the efficacy of the next generation of HCV vaccines.

The causal connection between serum biomarkers and COVID-19 severity or pathogenicity in children is unclear. The aim of this study was to describe clinical and immunological features of children affected by COVID-19. The secondary aim was to evaluate whether these cytokines could predict severity of COVID-19. All children (aged 0-18) admitted to the Pediatric Emergency Department and tested with nasopharyngeal swab for SARS-CoV-2 were recruited and assigned to three groups: COVID-19, other infections, control group. Clinical and laboratory data of these patients, including circulating cytokine levels, were analyzed in three groups. Fever was the most frequent symptom in COVID-19 (67.3%). Neutropenia was found in the COVID-19 group (p < 0.05); no difference was observed for lymphocyte counts in the three groups. Higher levels of IL-6 and TNF-alpha were found in the COVID-19 group compared to other infections and control groups (p = 0.014 and p = 0.001, respectively). Whereas, in the COVID-19 group, no difference was observed as for the same cytokines among sub-groups of different disease severity (p = 0.7 and p = 0.8). Serum levels of IL-6 and TNF-alpha were higher in COVID-19 children than in children with other infectious diseases, but those levels did not correlate with disease severity. Clinical studies in a large pediatric population are necessary to better define the role of the immune-mediated response in SARS-CoV-2 infections in children.

Minipigs play an important role in biomedical research and they have also been used as donor animals for preclinical xenotransplantations. Since zoonotic microorganisms including viruses can be transmitted when pig cells, tissues or organs are transplanted, virus safety is an important feature in xenotransplantation. Whereas most porcine viruses can be eliminated from pig herds by different strategies, this is not possible for porcine endogenous retroviruses (PERVs). PERVs are integrated in the genome of pigs and some of them release infectious particles able to infect human cells. Whereas PERV-A and PERV-B are present in all pigs and can infect cells from humans and other species, PERV-C is present in most, but not all pigs and infects only pig cells. Recombinant viruses between PERV-A and PERV-C have been found in some pigs; these recombinants infect human cells and are characterized by high replication rates. PERV-A/C recombinants have been found mainly in minipigs of different origin. The possible reasons of this high prevalence of PERV-A/C in minipigs, including inbreeding and higher numbers and expression of replication-competent PERV-C in these animals, are discussed in this review. Based on these data, it is highly recommended to use only pig donors in clinical xenotransplantation that are negative for PERV-C.

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

The classical laboratory mouse strains are genetic mosaics of three Mus musculus subspecies that occupy distinct regions of Eurasia. These strains and subspecies carry infectious and endogenous mouse leukemia viruses (MLVs) that can be pathogenic and mutagenic. MLVs evolved in concert with restrictive host factors with some under positive selection, including the XPR1 receptor for xenotropic/polytropic MLVs (X/P-MLVs) and the post-entry restriction factor Fv1. Since positive selection marks host-pathogen genetic conflicts, we examined MLVs for counter-adaptations at sites that interact with XPR1, Fv1, and the CAT1 receptor for ecotropic MLVs (E-MLVs). Results describe different co-adaptive evolutionary paths within the ranges occupied by these virus-infected subspecies. The interface of CAT1, and the otherwise variable E-MLV envelopes, is highly conserved; antiviral protection is afforded by the Fv4 restriction factor. XPR1 and X/P-MLVs variants show coordinate geographic distributions, with receptor critical sites in envelope, under positive selection but with little variation in envelope and XPR1 in mice carrying P-ERVs. The major Fv1 target in the viral capsid is under positive selection, and the distribution of Fv1 alleles is subspecies-correlated. These data document adaptive, spatial and temporal, co-evolutionary trajectories at the critical interfaces of MLVs and the host factors that restrict their replication.

The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. One of the strategies developed for this purpose is the use of nucleotide analogs such as bromodeoxyuridine (BrdU, analog to thymine) or bromouridine (BrU, analog of uridine), which are incorporated into the nucleic acids during replication or transcription. In adenovirus infections, BrdU has been used to localize newly synthesized viral genomes in the nucleus, where it is key to distinguish between host and viral DNA. Here, we describe our experience with methodological variations of BrdU labeling to localize adenovirus genomes in fluorescence and electron microscopy. We illustrate the need to define conditions in which most of the newly synthesized DNA corresponds to the virus and not the host, and the amount of BrdU provided is enough to incorporate to the new DNA molecules without hampering the cell metabolism. We hope that our discussion of problems encountered and solutions implemented will help other researches interested in viral genome localization in infected cells.

Rotarix® vaccine was implemented nationwide in Zambia in 2013. In this study, four unusual strains collected in the post-vaccine period were subjected to whole genome sequencing and analysis. The four strains possessed atypical genotype constellations, with at least one reassortant genome segment within the constellation. One of the strains (UFS-NGS-MRC-DPRU4749) was genetically and phylogenetically distinct in the VP4 and VP1 gene segments. Pairwise analyses demonstrated several amino acid disparities in the VP4 antigenic sites of this strain compared to that of Rotarix®. Although the impact of these amino acid disparities remains to be determined, this study adds to our understanding of the whole genomes of reassortant strains circulating in Zambia following Rotarix® vaccine introduction.

It has been a year since Stefan Kunz, a Full Professor since 2017 at the Faculty of Biology and Medicine of the University of Lausanne, Switzerland, passed away [...].

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74-100% (94.79-100%), 91.9-100% (96.3-100%) and 90.71-100% (93.51-100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5'UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3'UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3' termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses.

Human leukocyte antigen (HLA) is a group of molecules involved in inflammatory and infective responses. We evaluated blood sHLA-E and sHLA-G levels in hospitalized COVID-19 patients with respiratory failure and their relationship with clinical evolution, changes in endothelial activation biomarker profile, and neutrophil adhesion. sHLA-E, sHLA-G, and endothelial activation biomarkers were quantified by ELISA assay in plasma samples. Neutrophil adhesion to endothelium was assessed in the presence/absence of patients' plasma samples. At admission, plasma levels of sHLA-G and sHLA-E were significantly higher in COVID-19 patients with respiratory failure compared to controls. COVID-19 clinical improvement was associated with increased sHLA-G plasma levels. In COVID-19, but not in control patients, an inverse correlation was found between serum sICAM-1 and E-selectin levels and plasma sHLA-G values. The in vitro analysis of activated endothelial cells confirmed the ability of HLA-G molecules to control sICAM-1 and sE-selectin expression via CD160 interaction and FGF2 induction and consequently neutrophil adhesion. We suggest a potential role for sHLA-G in improving COVID-19 patients' clinical condition related to the control of neutrophil adhesion to activated endothelium.

Bovine herpesvirus-1 (BoHV-1) is a major cause of rhinotracheitis and vulvovaginitis in cattle. VP8, the major tegument protein of BoHV-1, is essential for viral replication in the host. VP8 is phosphorylated by the viral kinase US3, mediating its translocation to the cytoplasm. VP8 remains nuclear when not phosphorylated. Interestingly, VP8 has a significant presence in mature BoHV-1YmVP8, in which the VP8 phosphorylation sites are mutated. This suggests that VP8 might be packaged during primary envelopment of BoHV-1. This was investigated by mass spectrometry and Western blotting, which showed VP8, as well as VP22, to be constituents of the primary enveloped virions. VP8 and VP22 were shown to interact via co-immunoprecipitation experiments, in both BoHV-1-infected and VP8-transfected cells. VP8 and VP22 also co-localised with one another and with nuclear lamin-associated protein 2 in BoHV-1-infected cells, suggesting an interaction between VP8 and VP22 in the perinuclear region. In cells infected with VP22-deleted BoHV-1 (BoHV-1ΔUL49), VP8 was absent from the primary enveloped virions, implying that VP22 might be critical for the early packaging of VP8. In conclusion, a novel VP22-dependent mechanism for packaging of VP8 was identified, which may be responsible for a significant amount of VP8 in the viral particle.

More than 50% of the HIV-1 latent reservoir is maintained by clonal expansion. The clonally expanded HIV-1-infected cells can contribute to persistent nonsuppressible low-level viremia and viral rebound. HIV-1 integration site and proviral genome landscape profiling reveals the clonal expansion dynamics of HIV-1-infected cells. In individuals under long-term suppressive antiretroviral therapy (ART), HIV-1 integration sites are enriched in specific locations in certain cancer-related genes in the same orientation as the host transcription unit. Single-cell transcriptome analysis revealed that HIV-1 drives aberrant cancer-related gene expression through HIV-1-to-host RNA splicing. Furthermore, the HIV-1 promoter dominates over the host gene promoter and drives high levels of cancer-related gene expression. When HIV-1 integrates into cancer-related genes and causes gain of function of oncogenes or loss of function of tumor suppressor genes, HIV-1 insertional mutagenesis drives the proliferation of HIV-1-infected cells and may cause cancer in rare cases. HIV-1-driven aberrant cancer-related gene expression at the integration site can be suppressed by CRISPR-mediated inhibition of the HIV-1 promoter or by HIV-1 suppressing agents. Given that ART does not suppress HIV-1 promoter activity, therapeutic agents that suppress HIV-1 transcription and halt the clonal expansion of HIV-1-infected cells should be explored to block the clonal expansion of the HIV-1 latent reservoir.

Herpes stromal keratitis (HSK) is a disease that commonly affects the cornea and external eye and is caused by Herpes Simplex Virus type 1 (HSV-1). This virus infects approximately 66% of people worldwide; however, only a small portion of these people will develop symptoms in their lifetime. There is no cure or vaccine available for HSV-1; however, there are treatments available that aim to control the inflammation caused by the virus and prevent its recurrence. While these treatments are beneficial to those suffering with HSK, there is a need for more effective treatments to minimise the need for topical steroids, which can have harmful effects, and to prevent bouts of disease reactivation, which can lead to progressive corneal scarring and visual impairment. This review details the current understanding of HSV-1 infection and discusses potential novel treatment options including microRNAs, TLRs, mAbs, and aptamers.

The small cellular molecule inositol hexakisphosphate (IP6) has been known for ~20 years to promote the in vitro assembly of HIV-1 into immature virus-like particles. However, the molecular details underlying this effect have been determined only recently, with the identification of the IP6 binding site in the immature Gag lattice. IP6 also promotes formation of the mature capsid protein (CA) lattice via a second IP6 binding site, and enhances core stability, creating a favorable environment for reverse transcription. IP6 also enhances assembly of other retroviruses, from both the Lentivirus and the Alpharetrovirus genera. These findings suggest that IP6 may have a conserved function throughout the family Retroviridae. Here, we discuss the different steps in the viral life cycle that are influenced by IP6, and describe in detail how IP6 interacts with the immature and mature lattices of different retroviruses.

An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is a recently defined tumor subtype with apparent favorable clinical outcome despite aggressive histomorphology. However, in recent years, additional numbers of cases, with more variable features and at locations outside the sinonasal region, have complicated the definition of HMSC. Here, we have performed a systematic review of all cases described so far in order to accumulate more knowledge. We identified 127 articles published between 2013 and 2021, of which 21 presented unique cases. In total, 79 unique patient cases were identified and their clinical and micromorphological nature are herein summarized. In our opinion, better clinical follow-up data and a more detailed tumor characterization are preferably needed before HMSC can finally be justified as its own tumor entity.

Hepatitis B virus (HBV) is a major pathogen that causes acute/chronic hepatitis. Continuous HBV infection can lead to the development of hepatocellular carcinoma (HCC). Although several different anti-HBV treatments are available for chronic hepatitis B patients, discontinuing these medications is difficult. Patients with chronic hepatitis B at high risk for HCC therefore require close observation. However, no suitable biomarkers for detecting high-risk groups for HCC exist, except for serum HBV-DNA, but a number of HCC biomarkers are used clinically, such as alpha-fetoprotein (AFP) and protein induced by vitamin K absence-II (PIVKA-II). Glycosylation is an important post-translational protein modification involved in many human pathologic conditions. HBV surface proteins contain various oligosaccharides, and several reports have described their biological functions. Inhibition of HBV glycosylation represents a potential novel anti-HBV therapy. It is thought that glycosylation of hepatocytes/hepatoma cells is also important for HBV infection, as it prevents HBV from infecting cells other than hepatocytes, even if the cells express the HBV receptor. In this review, we summarize considerable research regarding the relationship between HBV and glycosylation as it relates to the development of novel diagnostic tests and therapies for HBV.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting annually in ~219,000 deaths and a societal cost of ~USD 60 billion, and no antivirals or vaccines are available. Here, we assess the anti-norovirus activity of new peptidomimetic aldehydes related to the protease inhibitor rupintrivir. The early hit compound 4 inhibited the replication of murine norovirus (MNV) and the HuNoV GI.1 replicon in vitro (EC50 ~1 µM) and swiftly cleared the HuNoV GI.1 replicon from the cells. Compound 4 still inhibits the proteolytic activity. We selected a resistant GI.1 replicon, with a mutation (I109V) in a highly conserved region of the viral protease, conferring a low yield of resistance against compound 4 and rupintrivir. After testing new derivatives, compound 10d was the most potent (EC50 nanomolar range). Molecular docking indicated that the aldehyde group of compounds 4 and 10d bind with Cys139 in the HuNoV 3CL protease by a covalent linkage. Finally, compound 10d inhibited the replication of HuNoV GII.4 in infected zebrafish larvae, and PK studies in mice showed an adequate profile.

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.

Many viruses that cause serious diseases in humans and animals, including the betacoronaviruses (beta-CoVs), such as SARS-CoV, MERS-CoV, and the recently identified SARS-CoV-2, have natural reservoirs in bats. Because these viruses rely entirely on the host cellular machinery for survival, their evolution is likely to be guided by the link between the codon usage of the virus and that of its host. As a result, specific cellular microenvironments of the diverse hosts and/or host tissues imprint peculiar molecular signatures in virus genomes. Our study is aimed at deciphering some of these signatures. Using a variety of genetic methods we demonstrated that trends in codon usage across chiroptera-hosted CoVs are collaboratively driven by geographically different host-species and temporal-spatial distribution. We not only found that chiroptera-hosted CoVs are the ancestors of SARS-CoV-2, but we also revealed that SARS-CoV-2 has the codon usage characteristics similar to those seen in CoVs infecting the Rhinolophus sp. Surprisingly, the envelope gene of beta-CoVs infecting Rhinolophus sp., including SARS-CoV-2, had extremely high CpG levels, which appears to be an evolutionarily conserved trait. The dissection of the furin cleavage site of various CoVs infecting hosts revealed host-specific preferences for arginine codons; however, arginine is encoded by a wider variety of synonymous codons in the murine CoV (MHV-A59) furin cleavage site. Our findings also highlight the latent diversity of CoVs in mammals that has yet to be fully explored.

An outbreak caused by H7N3 low pathogenicity avian influenza virus (LPAIV) occurred in commercial turkey farms in the states of North Carolina (NC) and South Carolina (SC), United States in March of 2020. Subsequently, H7N3 high pathogenicity avian influenza virus (HPAIV) was detected on a turkey farm in SC. The infectivity, transmissibility, and pathogenicity of the H7N3 HPAIV and two LPAIV isolates, including one with a deletion in the neuraminidase (NA) protein stalk, were studied in turkeys and chickens. High infectivity [<2 log10 50% bird infectious dose (BID50)] and transmission to birds exposed by direct contact were observed with the HPAIV in turkeys. In contrast, the HPAIV dose to infect chickens was higher than for turkeys (3.7 log10 BID50), and no transmission was observed. Similarly, higher infectivity (<2-2.5 log10 BID50) and transmissibility were observed with the H7N3 LPAIVs in turkeys compared to chickens, which required higher virus doses to become infected (5.4-5.7 log10 BID50). The LPAIV with the NA stalk deletion was more infectious in turkeys but did not have enhanced infectivity in chickens. These results show clear differences in the pathobiology of AIVs in turkeys and chickens and corroborate the high susceptibility of turkeys to both LPAIV and HPAIV infections.

Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are prototypical alphaherpesviruses that are characterized by their unique properties to infect trigeminal and dorsal root ganglionic neurons, respectively, and establish life-long latent infections. These viruses initially infect mucosal epithelial tissues and subsequently spread to neurons. They are associated with a significant disease spectrum, including orofacial and ocular infections for HSV-1 and genital and neonatal infections for HSV-2. Viral glycoproteins within the virion envelope bind to specific cellular receptors to mediate virus entry into cells. This is achieved by the fusion of the viral envelope with the plasma membrane. Similarly, viral glycoproteins expressed on cell surfaces mediate cell-to-cell fusion and facilitate virus spread. An interactive complex of viral glycoproteins gB, gD/gH/gL, and gK and other proteins mediate these membrane fusion phenomena with glycoprotein B (gB), the principal membrane fusogen. The requirement for the virion to enter neuronal axons suggests that the heterodimeric protein complex of gK and membrane protein UL20, found only in alphaherpesviruses, constitute a critical determinant for neuronal entry. This hypothesis was substantiated by the observation that a small deletion in the amino terminus of gK prevents entry into neuronal axons while allowing entry into other cells via endocytosis. Cellular receptors and receptor-mediated signaling synergize with the viral membrane fusion machinery to facilitate virus entry and intercellular spread. Unraveling the underlying interactions among viral glycoproteins, envelope proteins, and cellular receptors will provide new innovative approaches for antiviral therapy against herpesviruses and other neurotropic viruses.

Papillomaviruses cause persistent, and usually self-limiting, infections in the mucosal and cutaneous surfaces of the host epithelium. However, in some cases, infection with an oncogenic HPV can lead to cancer. The viral genome is a small, double-stranded circular DNA molecule that is assembled into nucleosomes at all stages of infection. The viral minichromosome replicates at a low copy number in the nucleus of persistently infected cells using the cellular replication machinery. When the infected cells differentiate, the virus hijacks the host DNA damage and repair pathways to replicate viral DNA to a high copy number to generate progeny virions. This strategy is highly effective and requires a close association between viral and host chromatin, as well as cellular processes associated with DNA replication, repair, and transcription. However, this association can lead to accidental integration of the viral genome into host DNA, and under certain circumstances integration can promote oncogenesis. Here we describe the fate of viral DNA at each stage of the viral life cycle and how this might facilitate accidental integration and subsequent carcinogenesis.

Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13-mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct, non-overlapping sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4+ T-cells and suppression of HIV-1 reactivated from latently infected cells using the CRISPR/Cas13d system. Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV.

In plants, HEN1-facilitated methylation at 3' end ribose is a critical step of small-RNA (sRNA) biogenesis. A mutant of well-studied Arabidopsis HEN1 (AtHEN1), hen1-1, showed a defective developmental phenotype, indicating the importance of sRNA methylation. Moreover, Marchantia polymorpha has been identified to have a HEN1 ortholog gene (MpHEN1); however, its function remained unfathomed. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex microRNA (miRNA) remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution of the HEN1 family in plants. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and inhibits HEN1 methylation of sRNAs. Here, we report that the HC-Pro physically binds with AtHEN1 through FRNK motif, inhibiting HEN1's methylation activity. Moreover, the in vitro EMSA data indicates GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, the HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner, suggesting the possibility of interaction between HC-Pro and MpHEN1 as well. Further investigations on understanding interaction mechanisms of HEN1 and various HC-Pros can advance the knowledge of viral suppressors.

The use of dengue virus (DENV) vaccines has been hindered by the complexities of antibody dependent enhancement (ADE). Current late-stage vaccine candidates utilize attenuated and chimeric DENVs that produce particles of varying maturities. Antibodies that are elicited by preferentially exposed epitopes on immature virions have been linked to increased ADE. We aimed to further understand the humoral immunity promoted by DENV particles of varying maturities in an AG129 mouse model using a chimeric insect specific vaccine candidate, bDENV-2. We immunized mice with mature, partially mature, and immature bDENV-2 and found that immunization with partially mature bDENV-2 produced more robust and cross-neutralizing immune responses than immunization with immature or mature bDENV-2. Upon challenge with mouse adapted DENV-2 (D220), we observed 80% protection for mature bDENV-2 vaccinated mice and 100% for immature and partially mature vaccinated mice, suggesting that protection to homotypic challenge is not dependent on maturation. Finally, we found reduced in vitro ADE at subneutralising serum concentrations for mice immunized with mature bDENV-2. These results suggest that both immature and mature DENV particles play a role in homotypic protection; however, the increased risk of in vitro ADE from immature particles indicates potential safety benefits from mature DENV-based vaccines.

Understanding how viruses with multi-segmented genomes incorporate one copy of each segment into their capsids remains an intriguing question. Here, we review our recent progress and describe the advancements made in understanding the genome packaging mechanism of a model nonenveloped virus, Bluetongue virus (BTV), with a 10-segment (S1-S10) double-strand RNA (dsRNA) genome. BTV (multiple serotypes), a member of the Orbivirus genus in the Reoviridae family, is a notable pathogen for livestock and is responsible for significant economic losses worldwide. This has enabled the creation of an extensive set of reagents and assays, including reverse genetics, cell-free RNA packaging, and bespoke bioinformatics approaches, which can be directed to address the packaging question. Our studies have shown that (i) UTRs enable the conformation of each segment necessary for the next level of RNA-RNA interaction; (ii) a specific order of intersegment interactions leads to a complex RNA network containing all the active components in sorting and packaging; (iii) networked segments are recruited into nascent assembling capsids; and (iv) select capsid proteins might be involved in the packaging process. The key features of genome packaging mechanisms for BTV and related dsRNA viruses are novel and open up new avenues of potential intervention.

COVID-19 is an ongoing pandemic with high morbidity and mortality. Despite meticulous research, only dexamethasone has shown consistent mortality reduction. Convalescent plasma (CP) infusion might also develop into a safe and effective treatment modality on the basis of recent studies and meta-analyses; however, little is known regarding the kinetics of antibodies in CP recipients. To evaluate the kinetics, we followed 31 CP recipients longitudinally enrolled at a median of 3 days post symptom onset for changes in binding and neutralizing antibody titers and viral loads. Antibodies against the complete trimeric Spike protein and the receptor-binding domain (Spike-RBD), as well as against the complete Nucleocapsid protein and the RNA binding domain (N-RBD) were determined at baseline and weekly following CP infusion. Neutralizing antibody (pseudotype NAb) titers were determined at the same time points. Viral loads were determined semi-quantitatively by SARS-CoV-2 PCR. Patients with low humoral responses at entry showed a robust increase of antibodies to all SARS-CoV-2 proteins and Nab, reaching peak levels within 2 weeks. The rapid increase in binding and neutralizing antibodies was paralleled by a concomitant clearance of the virus within the same timeframe. Patients with high humoral responses at entry demonstrated low or no further increases; however, virus clearance followed the same trajectory as in patients with low antibody response at baseline. Together, the sequential immunological and virological analysis of this well-defined cohort of patients early in infection shows the presence of high levels of binding and neutralizing antibodies and potent clearance of the virus.

Nonstructural protein 1 (NS1) of influenza virus (IFV) is essential for evading interferon (IFN)-mediated antiviral responses, thereby contributing to the pathogenesis of influenza. Mitophagy is a type of autophagy that selectively removes damaged mitochondria. The role of NS1 in IFV-mediated mitophagy is currently unknown. Herein, we showed that overexpression of NS1 protein led to enhancement of mitophagy. Mitophagy induction via carbonyl cyanide 3-chlorophenylhydrazone treatment in IFV-infected A549 cells led to increased viral replication efficiency, whereas the knockdown of PTEN-induced kinase 1 (PINK1) led to the opposite effect on viral replication. Overexpression of NS1 protein led to changes in mitochondrial dynamics, including depolarization of mitochondrial membrane potential. In contrast, infection with NS1-deficient virus resulted in impaired mitochondrial fragmentation, subsequent mitolysosomal formation, and mitophagy induction, suggesting an important role of NS1 in mitophagy. Meanwhile, NS1 protein increased the phosphorylation of Unc-51-like autophagy activating kinase 1 (ULK1) and the mitochondrial expression of BCL2- interacting protein 3 (BNIP3), both of which were found to be important for IFV-mediated mitophagy. Overall, these data highlight the importance of IFV NS1, ULK1, and BNIP3 during mitophagy activation.

Recent years have witnessed the discovery of several new viruses belonging to the family Arteriviridae, expanding the known diversity and host range of this group of complex RNA viruses. Although the pathological relevance of these new viruses is not always clear, several well-studied members of the family Arteriviridae are known to be important animal pathogens. Here, we report the complete genome sequences of four new arterivirus variants, belonging to two putative novel species. These new arteriviruses were discovered in African rodents and were given the names Lopma virus and Praja virus. Their genomes follow the characteristic genome organization of all known arteriviruses, even though they are only distantly related to currently known rodent-borne arteriviruses. Phylogenetic analysis shows that Lopma virus clusters in the subfamily Variarterivirinae, while Praja virus clusters near members of the subfamily Heroarterivirinae: the yet undescribed forest pouched giant rat arterivirus and hedgehog arterivirus 1. A co-divergence analysis of rodent-borne arteriviruses confirms that they share similar phylogenetic patterns with their hosts, with only very few cases of host shifting events throughout their evolutionary history. Overall, the genomes described here and their unique clustering with other arteriviruses further illustrate the existence of multiple rodent-borne arterivirus lineages, expanding our knowledge of the evolutionary origin of these viruses.

We do not always remember the exact moment in which we first met our friends [...].

Type I Interferons (IFN-I) are a family of potent antiviral cytokines that act through the direct restriction of viral replication and by enhancing antiviral immunity. However, these powerful cytokines are a caged lion, as excessive and sustained IFN-I production can drive immunopathology during infection, and aberrant IFN-I production is a feature of several types of autoimmunity. As specialized producers of IFN-I plasmacytoid (p), dendritic cells (DCs) can secrete superb quantities and a wide breadth of IFN-I isoforms immediately after infection or stimulation, and are the focus of this review. Notably, a few days after viral infection pDCs tune down their capacity for IFN-I production, producing less cytokines in response to both the ongoing infection and unrelated secondary stimulations. This process, hereby referred to as "pDC exhaustion", favors viral persistence and associates with reduced innate responses and increased susceptibility to secondary opportunistic infections. On the other hand, pDC exhaustion may be a compromise to avoid IFN-I driven immunopathology. In this review we reflect on the mechanisms that initially induce IFN-I and subsequently silence their production by pDCs during a viral infection. While these processes have been long studied across numerous viral infection models, the 2019 coronavirus disease (COVID-19) pandemic has brought their discussion back to the fore, and so we also discuss emerging results related to pDC-IFN-I production in the context of COVID-19.

Viral hepatitis remains one of the most significant health issues globally, directly responsible for over 1 million deaths each year and affecting almost 300 million people around the world. Scientific research in recent decades has brought about improvements in the lives of people living with chronic viral hepatitis. On the 29 July 2021, the Australian Centre for Hepatitis Virology (ACHV) for the first time held a public educational forum for the general public. The main aim of this event was to inform the affected community about the importance of scientific research and give an overview of upcoming developments in the field. Here, we provide a detailed report of the panel discussion (including its organisation, execution, and lessons learned to incorporate into future events) and provide strategies that can be used by other scientific societies to hold similar events in their own communities.

Liver transplantation for hepatitis C virus (HCV)-related disease has the lowest five-year graft survival among all liver transplant recipients. Graft failure due to accelerated fibrosis from unrestrained HCV replication is common. Optimal timing of HCV treatment with direct-acting antiviral agents remains unknown. We compared HCV liver transplant recipients successfully treated for HCV before transplant to those treated within 1 year of transplant to determine if graft fibrosis, measured by Fib-4 scores, differs with timing of treatment. Fib-4 scores less than or equal to 1.45 defined minimal fibrosis and greater than 1.45 defined greater than minimal fibrosis. We identified 117 liver transplant recipients: 52 treated before transplantation and 65 treated within 1 year of transplantation. Overall, 34% of recipients had minimal fibrosis, and the likelihood of having minimal fibrosis following treatment and liver transplantation did not differ by timing of treatment. The odds ratio of having greater than minimal fibrosis was 0.65 (95% CI 0.30, 1.42) among those treated within 1 year after transplantation compared to those treated before transplantation (p-value 0.28). Importantly, nearly 2/3 of these patients had evidence of fibrosis progression one year after sustained virologic response, supporting recommendations for early antiviral-based treatment to prevent accumulation of HCV-related disease.

Human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as those of the oropharynx. HPV proteins activate host DNA damage repair factors to promote their viral life cycle in stratified epithelia. Activation of both the ATR pathway and the ATM pathway are essential for viral replication and differentiation-dependent genome amplification. These pathways are also important for maintaining host genomic integrity and their dysregulation or mutation is often seen in human cancers. The APOBEC3 family of cytidine deaminases are innate immune factors that are increased in HPV positive cells leading to the accumulation of TpC mutations in cellular DNAs that contribute to malignant progression. The activation of DNA damage repair factors may corelate with expression of APOBEC3 in HPV positive cells. These pathways may actively drive tumor development implicating/suggesting DNA damage repair factors and APOBEC3 as possible therapeutic targets.

Aleutian mink disease virus (AMDV) is known to cause the most significant disease in the mink industry. It is globally widespread and manifested as a deadly plasmacytosis and hyperglobulinemia. So far, measures to control the viral spread have been limited to manual serological testing for AMDV-positive mink. Further, due to the persistent nature of this virus, attempts to eradicate Aleutian disease (AD) have largely failed. Therefore, effective strategies to control the viral spread are of crucial importance for wildlife protection. One potentially key tool in the fight against this disease is by the immunization of mink against AMDV. Throughout many years, several researchers have tried to develop AMDV vaccines and demonstrated varying degrees of protection in mink by those vaccines. Despite these attempts, there are currently no vaccines available against AMDV, allowing the continuation of the spread of Aleutian disease. Herein, we summarize previous AMDV immunization attempts in mink as well as other preventative measures with the purpose to shed light on future studies designing such a potentially crucial preventative tool against Aleutian disease.

As one of the most infectious livestock diseases in the world, foot and mouth disease (FMD) presents a constant global threat to animal trade and national economies. FMD remains a severe constraint on development and poverty reduction throughout the developing world due to many reasons, including the cost of control measures, closure of access to valuable global FMD-free markets for livestock products, production losses through reduced milk yield, reduced live weight gain, and the inability of infected livestock to perform traction. FMD virus infects a variety of cloven-hoofed animals, including cattle, sheep, goats, swine, all wild ruminants, and suidae, with high morbidity in adult animals. High mortality can occur in young animals due to myocarditis. FMD is endemic in Africa, most of Asia, the Middle East, and parts of South America. The global clustering of FMD viruses has been divided into seven virus pools, where multiple serotypes occur but within which are topotypes that remain mostly confined to that pool. Three pools cover Europe, the Middle East, and Asia; three pools cover Africa; and one pool covers the Americas. The highly infectious nature of FMDV, the existence of numerous continually circulating serotypes and associated topotypes, the potential for wildlife reservoirs, and the frequent emergence of new strains that are poorly matched to existing vaccines all serve to compound the difficulties faced by the governments of endemic countries to effectively control and reduce the burden of the disease at the national and regional levels. This clustering of viruses suggests that if vaccination is to be a major tool for control, each pool could benefit from the use of tailored or more specific vaccines relevant to the topotypes present in that pool, rather than a continued reliance on the currently more widely available vaccines. It should also be noted that, currently, there are varying degrees of effort to identify improved vaccines in different regions. There are relatively few targeted for use in Africa, while the developed world's vaccine banks have a good stock of vaccines destined for emergency outbreak use in FMDV-free countries. The AgResults Foot and Mouth Disease (FMD) Vaccine Challenge Project (the "Project") is an eight-year, US $17.68 million prize competition that supports the development and uptake of high-quality quadrivalent FMD vaccines tailored to meet the needs of Eastern Africa (EA). The Project targets the following Pool Four countries: Burundi, Ethiopia, Kenya, Rwanda, Tanzania and Uganda. The Project is being run in two phases: a development phase, which will encourage the production of regionally relevant vaccines, and a cost-share phase, designed to help to reduce the price of these vaccines in the marketplace to the end users, which is hoped will encourage broader uptake. Manufacturers can submit quadrivalent FMD vaccines containing serotypes A, O, SAT1, and SAT2, which will be assessed as relevant for use in the region through a unique component of the Project requiring the screening of vaccines against the Eastern Africa Foot and Mouth Disease Virus Reference Antigen Panel assembled by the World Reference Laboratory for FMD (WRLFMD), at the Pirbright Institute, UK, in collaboration with the OIE/FAO FMD Reference Laboratory Network. To be eligible for the Project, sera from vaccinated cattle will be used to evaluate serological responses of FMD vaccines for their suitability for use in Eastern African countries. If they pass a determined cut-off threshold, they will be confirmed as relevant for use in the region and will be entered into the Project's cost-share phase.

The host's immune status may affect virus evolution. Little is known about how developing fetal and placental immune milieus affect virus heterogeneity. This knowledge will help us better understand intra-host virus evolution and how new virus variants emerge. The goal of our study was to find out whether the isolated in utero environment-an environment with specialized placental immunity and developing fetal immunity-supports the emergence of RNA and DNA virus variants. We used well-established porcine models for isolated Zika virus (RNA virus) and porcine circovirus 2 (DNA virus) fetal infections. We found that the isolated in utero environment was conducive to the emergence of RNA and DNA virus variants. Next-generation sequencing of nearly whole virus genomes and validated bioinformatics pipelines identified both unique and convergent single nucleotide variations in virus genomes isolated from different fetuses. Zika virus and PCV2 in utero evolution also resulted in single nucleotide variations previously reported in the human and porcine field samples. These findings should encourage further studies on virus evolution in placenta and fetuses, to better understand how virus variants emerge and how in utero viral evolution affects congenital virus transmission and pathogenicity.

Presently, the use of convalescent plasma and hyperimmunoglobulin obtained from individuals who have recovered from coronavirus disease 2019 (COVID-19) has proved to potentially provide passive antibody-based immunity, thereby leading to several clinical trials to develop an immune-based COVID-19 treatment. However, the therapeutic efficacy of hyperimmunoglobulin in critically ill patients with COVID-19 remains unknown. On 23 October 2020, we first administered GC5131 in a compassionate-use program to critically ill patients at the Kyungpook National University, Chilgok Hospital, Korea. Since then, five more critically ill patients were treated with GC5131 in this compassionate-use program in our hospital up until 17 December 2020. We retrospectively reviewed the clinical responses of six critically ill patients diagnosed with COVID-19 who received the hyperimmunoglobulin concentrate, GC5131, which was produced by the Green Cross Corporation. After the administration of GC5131, five patients died due to an exacerbation of COVID-19 pneumonia. GC5131 was ineffective when administered to critically ill patients with COVID-19. Nevertheless, we propose that to expect a therapeutic effect from GC5131, it should be administered as early as possible to avoid the excessive inflammatory response phase in patients with severe and advanced COVID-19 infection. This step was difficult to achieve in the real world due to the time required for decision making and the process of the compassionate-use program.

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

Congenital Zika Syndrome (CZS) is caused by vertical transmission of Zika virus (ZIKV) to the gestating human fetus. A subset of CZS microcephalic infants present with reduced otoacoustic emissions; this test screens for hearing loss originating in the cochlea. This observation leads to the question of whether mammalian cochlear tissues are susceptible to infection by ZIKV during development. To address this question using a mouse model, the sensory cochlea was explanted at proliferative, newly post-mitotic or maturing stages. ZIKV was added for the first 24 h and organs cultured for up to 6 days to allow for cell differentiation. Results showed that ZIKV can robustly infect proliferating sensory progenitors, as well as post-mitotic hair cells and supporting cells. Virus neutralization using ZIKV-117 antibody blocked cochlear infection. AXL is a cell surface molecule known to enhance the attachment of flavivirus to host cells. While Axl mRNA is widely expressed in embryonic cochlear tissues susceptible to ZIKV infection, it is selectively downregulated in the post-mitotic sensory organ by E15.5, even though these cells remain infectible. These findings may offer insights into which target cells could potentially contribute to hearing loss resulting from fetal exposure to ZIKV in humans.

Coxsackievirus B3 (CVB3) belongs to the enteroviruses, which are a well-known cause of acute and chronic myocarditis, primarily infecting cardiac myocytes. As primary human cardiomyocytes are difficult to obtain, viral myocarditis is quite frequently studied in vitro in different non-cardiac and cardiac-like cell lines. Recently, cardiomyocytes that have been differentiated from human-induced pluripotent stem cells have been described as a new model system to study CVB3 infection. Here, we compared iCell® Cardiomyocytes with other cell lines that are commonly used to study CVB3 infection regarding their susceptibility and patterns of infection and the mode of cell death. iCell® Cardiomyocytes, HeLa cells, HL-1 cells and H9c2 cells were infected with CVB3 (Nancy strain). The viral load, CVB3 RNA genome localization, VP1 expression (including the intracellular localization), cellular morphology and the expression of cell death markers were compared. The various cell lines clearly differed in their permissiveness to CVB3 infection, patterns of infection, viral load, and mode of cell death. When studying the mode of cell death of CVB3-infected iCell® Cardiomyocytes in more detail, especially regarding the necroptosis key players RIPK1 and RIPK3, we found that RIPK1 is cleaved during CVB3 infection. iCell® Cardiomyocytes represent well the natural host of CVB3 in the heart and are thus the most appropriate model system to study molecular mechanisms of CVB3-induced myocarditis in vitro. Doubts are raised about the suitability of commonly used cell lines such as HeLa cells, HL-1 cells and H9c2 cells to evaluate molecular pathways and processes occurring in vivo in enteroviral myocarditis.

This study aimed to characterize the HCV genetic subtypes variability and the presence of natural occurring resistance-associated substitutions (RASs) in Saudi Arabia patients. A total of 17 GT patients were analyzed. Sequence analysis of NS3, NS5A, and NS5B regions was performed by direct sequencing, and phylogenetic analyses were used to determine genetic subtypes, RAS, and polymorphisms. Nine patients were infected by GT 4a, two with GT 4o and three with GT 4d. Two patients were infected with apparent recombinant virus (4a/4o/4a in NS3/NS5A/NS5B), and one patient was infected with a previously unknown, unclassifiable, virus of GT 4. Natural RASs were found in six patients (35%), including three infected by GT 4a, two by GT 4a/GT 4o/GT 4a, and one patient infected by an unknown, unclassifiable, virus of GT 4. In particular, NS3-RAS V170I was demonstrated in three patients, while NS5A-RASs (L28M, L30R, L28M + M31L) were detected in the remaining three patients. All patients were treated with sofosbuvir plus daclatasvir; three patients were lost to follow-up, whereas 14 patients completed the treatment. A sustained virological response (SVR) was obtained in all but one patient carrying NS3-RAS V170I who later relapsed. GT 4a is the most common subtype in this small cohort of Saudi Arabia patients infected with hepatitis C infection. Natural RASs were observed in about one-third of patients, but only one of them showed a treatment failure.

Porcine reproductive and respiratory syndrome virus (PRRSV) modulates host innate immunity which plays a key role against PRRSV infection. As a RNA virus, PRRSV is mainly sensed by innate immune RNA receptors, whereas the role of innate immune DNA sensors in the PRRSV infection has not been elucidated. Here, we investigated the roles of DNA sensing cGAS-STING pathway in both PRRSV infected Marc-145 cells and porcine macrophages. The results show that in Marc-145 cells, the stable expression of STING with or without stimulations exhibited anti-PRRSV activity, and STING knockout heightened PRRSV infection. In CD163-3D4/21 porcine macrophages, either expression of STING or stimulation of cGAS-STING signaling obviously suppressed PRRSV infection, whereas in STING knockdown macrophages, the PRRSV infection was upregulated. Our results clearly demonstrate that the host cGAS-STING signal exerts an important antiviral role in PRRSV infection.

Temperature plays a significant role in the vector competence, extrinsic incubation period, and intensity of infection of arboviruses within mosquito vectors. Most laboratory infection studies use static incubation temperatures that may not accurately reflect daily temperature ranges (DTR) to which mosquitoes are exposed. This could potentially compromise the application of results to real world scenarios. We evaluated the effect of fluctuating DTR versus static temperature treatments on the infection, dissemination, and transmission rates and viral titers of Culex tarsalis and Culex quinquefasciatus mosquitoes for West Nile virus. Two DTR regimens were tested including an 11 and 15 °C range, both fluctuating around an average temperature of 28 °C. Overall, no significant differences were found between DTR and static treatments for infection, dissemination, or transmission rates for either species. However, significant treatment differences were identified for both Cx. tarsalis and Cx. quinquefasciatus viral titers. These effects were species-specific and most prominent later in the infection. These results indicate that future studies on WNV infections in Culex mosquitoes should consider employing realistic DTRs to reflect interactions most accurately between the virus, vector, and environment.

During lytic infection, herpes simplex virus (HSV) 1 induces a rapid shutoff of host RNA synthesis while redirecting transcriptional machinery to viral genes. In addition to being a major human pathogen, there is burgeoning clinical interest in HSV as a vector in gene delivery and oncolytic therapies, necessitating research into transcriptional control. This review summarizes the array of impacts that HSV has on RNA Polymerase (Pol) II, which transcribes all mRNA in infected cells. We discuss alterations in Pol II holoenzymes, post-translational modifications, and how viral proteins regulate specific activities such as promoter-proximal pausing, splicing, histone repositioning, and termination with respect to host genes. Recent technological innovations that have reshaped our understanding of previous observations are summarized in detail, along with specific research directions and technical considerations for future studies.

The Open Reading Frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific, immediate-early, tegument protein required for efficient viral replication and virion production. We have previously shown that ORF45 interacts with the conserved herpesviral protein ORF33 through the highly conserved C-terminal 19 amino acids (C19) of ORF45. Because the deletion of C19 abolished ORF33 accumulation and viral production, we reasoned that this interaction could be critical for viral production and explored as an antiviral target for gammaherpesviruses. In work described in this article, we characterize this interaction in further detail, first by revealing that this interaction is conserved among gammaherpesviruses, then by identifying residues in C19 critical for its interaction with and stabilization of ORF33. More importantly, we show that disruption of the interaction, either by mutating key residues (W403A or W405A) in C19 or by using competing cell penetration peptide TAT-C19, dramatically reduce the yield of KSHV progeny viruses. Our results not only reveal critical roles of this interaction to viral production but also provide a proof of concept for targeting the ORF33-ORF45 interaction as a novel antiviral strategy against KSHV and other gammaherpesviruses.

The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

JC polyomavirus (JCPyV) is a neuroinvasive pathogen causing a fatal, demyelinating disease of the central nervous system (CNS) known as progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types: oligodendrocytes and astrocytes. The underlying mechanisms of astrocytic infection are poorly understood, yet recent findings suggest critical differences in JCPyV infection of primary astrocytes compared to a widely studied immortalized cell model. RNA sequencing was performed in primary normal human astrocytes (NHAs) to analyze the transcriptomic profile that emerges during JCPyV infection. Through a comparative analysis, it was validated that JCPyV requires the mitogen-activated protein kinase, extracellular signal-regulated kinase (MAPK/ERK) pathway, and additionally requires the expression of dual-specificity phosphatases (DUSPs). Specifically, the expression of DUSP1 is needed to establish a successful infection in NHAs, yet this was not observed in an immortalized cell model of JCPyV infection. Additional analyses demonstrated immune activation uniquely observed in NHAs. These results support the hypothesis that DUSPs within the MAPK/ERK pathway impact viral infection and influence potential downstream targets and cellular pathways. Collectively, this research implicates DUSP1 in JCPyV infection of primary human astrocytes, and most importantly, further resolves the signaling events that lead to successful JCPyV infection in the CNS.

Among the numerous biological constraints that hinder cassava (Manihot esculenta Crantz) production, foremost is cassava mosaic disease (CMD) caused by virus members of the family Geminiviridae, genus Begomovirus. The mechanisms of CMD tolerance and susceptibility are not fully understood; however, CMD susceptible T200 and tolerant TME3 cassava landraces have been shown to exhibit different large-scale transcriptional reprogramming in response to South African cassava mosaic virus (SACMV). Recent identification of 85 MeWRKY transcription factors in cassava demonstrated high orthology with those in Arabidopsis, however, little is known about their roles in virus responses in this non-model crop. Significant differences in MeWRKY expression and regulatory networks between the T200 and TME3 landraces were demonstrated. Overall, WRKY expression and associated hormone and enriched biological processes in both landraces reflect oxidative and other biotic stress responses to SACMV. Notably, MeWRKY11 and MeWRKY81 were uniquely up and downregulated at 12 and 67 days post infection (dpi) respectively in TME3, implicating a role in tolerance and symptom recovery. AtWRKY28 and AtWRKY40 homologs of MeWRKY81 and MeWRKY11, respectively, have been shown to be involved in regulation of jasmonic and salicylic acid signaling in Arabidopsis. AtWRKY28 is an interactor in the RPW8-NBS resistance (R) protein network and downregulation of its homolog MeWRKY81 at 67 dpi in TME3 suggests a negative role for this WRKY in SACMV tolerance. In contrast, in T200, nine MeWRKYs were differentially expressed from early (12 dpi), middle (32 dpi) to late (67 dpi) infection. MeWRKY27 (homolog AtWRKY33) and MeWRKY55 (homolog AtWRKY53) were uniquely up-regulated at 12, 32 and 67 dpi in T200. AtWRKY33 and AtWRKY53 are positive regulators of leaf senescence and oxidative stress in Arabidopsis, suggesting MeWRKY55 and 27 contribute to susceptibility in T200.

BACKGROUND: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. METHODS: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. RESULTS: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. CONCLUSIONS: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

The cellular metabolism of host tRNAs and life cycle of HIV-1 cross paths at several key virus-host interfaces. Emerging data suggest a multi-faceted interplay between host tRNAs and HIV-1 that plays essential roles, both structural and regulatory, in viral genome replication, genome packaging, and virion biogenesis. HIV-1 not only hijacks host tRNAs and transforms them into obligatory reverse transcription primers but further commandeers tRNAs to regulate the localization of its major structural protein, Gag, via a specific interface. This review highlights recent advances in understanding tRNA-HIV-1 interactions, primarily from a structural perspective, which start to elucidate their underlying molecular mechanisms, intrinsic specificities, and biological significances. Such understanding may provide new avenues toward developing HIV/AIDS treatments and therapeutics including small molecules and RNA biologics that target these host-virus interfaces.

Over the decades, the world has witnessed diverse virus associated pandemics. The significant inhibitory effects of marine sulfated polysaccharides against SARS-CoV-2 shows its therapeutic potential in future biomedical applications and drug development. Algal polysaccharides exhibited significant role in antimicrobial, antitumor, antioxidative, antiviral, anticoagulant, antihepatotoxic and immunomodulating activities. Owing to their health benefits, the sulfated polysaccharides from marine algae are a great deal of interest globally. Algal polysaccharides such as agar, alginate, carrageenans, porphyran, fucoidan, laminaran and ulvans are investigated for their nutraceutical potential at different stages of infection processes, structural diversity, complexity and mechanism of action. In this review, we focus on the recent antiviral studies of the marine algae-based polysaccharides and their potential towards antiviral medicines.

Central and peripheral hormone deficiencies have been documented during and after acute hantavirus infection. Thrombocytopenia and coagulation abnormalities are common findings in haemorrhagic fever with renal syndrome (HFRS). The associations between coagulation and hormonal abnormalities in HFRS have not been studied yet. Forty-two patients diagnosed with Puumala virus (PUUV) infection were examined during the acute phase and on a follow-up visit approximately one month later. Hormonal defects were common during acute PUUV infection. Overt (clinical) hypogonadism was identified in 80% of the men and approximately 20% of the patients had overt hypothyroidism. At the one-month follow-up visit, six patients had central hormone deficits. Acute peripheral hormone deficits associated with a more severe acute kidney injury (AKI), longer hospital stay and more severe thrombocytopenia. Half of the patients with bleeding symptoms had also peripheral hormonal deficiencies. Patients with free thyroxine levels below the reference range had higher D-dimer level than patients with normal thyroid function, but no thromboembolic events occurred. Acute phase hormonal abnormalities associate with severe disease and altered haemostasis in PUUV infection.

We introduce an explicit function that describes virus-load curves on a patient-specific level. This function is based on simple and intuitive model parameters. It allows virus load analysis of acute viral infections without solving a full virus load dynamic model. We validate our model on data from mice influenza A, human rhinovirus data, human influenza A data, and monkey and human SARS-CoV-2 data. We find wide distributions for the model parameters, reflecting large variability in the disease outcomes between individuals. Further, we compare the virus load function to an established target model of virus dynamics, and we provide a new way to estimate the exponential growth rates of the corresponding infection phases. The virus load function, the target model, and the exponential approximations show excellent fits for the data considered. Our virus-load function offers a new way to analyze patient-specific virus load data, and it can be used as input for higher level models for the physiological effects of a virus infection, for models of tissue damage, and to estimate patient risks.

Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pets during a COVID-19 household transmission investigation. Pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April-May 2020. We enrolled 37 dogs and 19 cats from 34 households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog's fur swabs (2%) tested positive by rRT-PCR at the first sampling. Among 47 pets with serological results, eight (17%) pets (four dogs, four cats) from 6/30 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40-100%) compared to households with no seropositive pet (median 37%; range: 13-100%) (p = 0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 h) with the index patient before the person's COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the index patient after diagnosis and none were seropositive; of the 19 (58%) pets with continued contact, four (21%) were seropositive. Seropositive pets likely acquired infection after contact with people with COVID-19. People with COVID-19 should restrict contact with pets and other animals.

Mosquito-borne West Nile virus (WNV) is the causative agent of West Nile disease in humans, horses, and some bird species. Since the initial introduction of WNV to the United States (US), approximately 30,000 horses have been impacted by West Nile neurologic disease and hundreds of additional horses are infected each year. Research describing the drivers of West Nile disease in horses is greatly needed to better anticipate the spatial and temporal extent of disease risk, improve disease surveillance, and alleviate future economic impacts to the equine industry and private horse owners. To help meet this need, we integrated techniques from spatiotemporal epidemiology, eco-phylogenetics, and distributional ecology to assess West Nile disease risk in horses throughout the contiguous US. Our integrated approach considered horse abundance and virus exposure, vector and host distributions, and a variety of extrinsic climatic, socio-economic, and environmental risk factors. Birds are WNV reservoir hosts, and therefore we quantified avian host community dynamics across the continental US to show intra-annual variability in host phylogenetic structure and demonstrate host phylodiversity as a mechanism for virus amplification in time and virus dilution in space. We identified drought as a potential amplifier of virus transmission and demonstrated the importance of accounting for spatial non-stationarity when quantifying interaction between disease risk and meteorological influences such as temperature and precipitation. Our results delineated the timing and location of several areas at high risk of West Nile disease and can be used to prioritize vaccination programs and optimize virus surveillance and monitoring.

Feline calicivirus (FCV) is an important pathogen of cats that has two genogroups (GI and GII). To investigate the prevalence and molecular characteristics of FCVs in southwestern China, 162 nasal swab samples were collected from cats in animal shelters and pet hospitals. In total, 38 of the clinical samples (23.46%) were identified as FCV positive using nested RT-PCR. Phylogenetic analyses using 10 capsid protein VP1 sequences revealed that 8 GI and 2 GII strains formed two independent clusters. Additionally, three separated FCVs that were not clustered phylogenetically (two GI and one GII strains) were successfully isolated from clinical samples and their full-length genomes were obtained. Phylogenetic and recombinant analyses of a GI FCV revealed genomic breakpoints in ORF1 and ORF2 regions with evidence for recombinant events between GI sub-genogroups, which is reported in China for the first time. Furthermore, sera obtained from mice immunized independently with the three FCV isolates and a commercial vaccine were used to evaluate the cross-reactivity of neutralizing antibodies. The three separate FCVs were neutralized by each other at a 1:19 to 1:775 titer range, whereas the triple-inactivated vaccine was at a titer of 1:16, which suggested that different genogroup/sub-genogroup FCV strains exhibit significantly different titers of neutralizing antibodies, including the commercial FCV vaccine. Thus, our study revealed the genetic diversity and complex cross-reactivity levels of FCVs in southwestern China, which provides new insights for application in vaccination strategies.

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.

Bovine viral diarrhea virus (BVDV) causes a severe threat to the cattle industry due to ineffective control measures. Gypenoside is the primary component of Gynostemma pentaphyllum, which has potential medicinal value and has been widely applied as a food additive and herbal supplement. However, little is known about the antiviral effects of gypenoside. The present study aimed to explore the antiviral activities of gypenoside against BVDV infection. The inhibitory activity of gypenoside against BVDV was assessed by using virus titration and performing Western blotting, quantitative reverse transcription PCR (RT-qPCR), and immunofluorescence assays in MDBK cells. We found that gypenoside exhibited high anti-BVDV activity by interfering with the viral attachment to and internalization in cells. The study showed that BVDV infection inhibits apoptosis of infected cells from escaping the innate defense of host cells. Our data further demonstrated that gypenoside inhibited BVDV infection by electively activating the apoptosis of BVDV-infected cells for execution, as evidenced by the regulation of the expression of the apoptosis-related protein, promotion of caspase-3 activation, and display of positive TUNEL staining; no toxicity was observed in non-infected cells. Collectively, the data identified that gypenoside exerts an anti-BVDV-infection role by inhibiting viral attachment and internalization and selectively purging virally infected cells. Therefore, our study will contribute to the development of a novel prophylactic and therapeutic strategy against BVDV infection.

Zika virus (ZIKV) infection during pregnancy causes a wide spectrum of congenital abnormalities and postnatal developmental sequelae such as fetal loss, intrauterine growth restriction (IUGR), microcephaly, or motor and neurodevelopmental disorders. Here, we investigated whether a mouse pregnancy model recapitulated a wide range of symptoms after congenital ZIKV infection, and whether the embryonic age of congenital infection changed the fetal or postnatal outcomes. Infection with ZIKV strain PRVABC59 from embryonic day 6.5 (E6.5) to E8.5, corresponding to the mid-first trimester in humans, caused fetal death, fetal resorption, or severe IUGR, whereas infection from E9.5 to E14.5, corresponding to the late-first to second trimester in humans, caused stillbirth, neonatal death, microcephaly, and postnatal growth deficiency. Furthermore, 4-week-old offspring born to dams infected at E12.5 showed abnormalities in neuropsychiatric state, motor behavior, autonomic function, or reflex and sensory function. Thus, our model recapitulated the multiple symptoms seen in human cases, and the embryonic age of congenital infection was one of the determinant factors of offspring outcomes in mice. Furthermore, maternal neutralizing antibodies protected the offspring from neonatal death after congenital infection at E9.5, suggesting that neonatal death in our model could serve as criteria for screening of vaccine candidates.

Many plant viruses depend on insect vectors for their transmission and dissemination. The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is one of the most important virus vectors, transmitting more than four hundred virus species, the majority belonging to begomoviruses (Geminiviridae), with their ssDNA genomes. Begomoviruses are transmitted by B. tabaci in a persistent, circulative manner, during which the virus breaches barriers in the digestive, hemolymph, and salivary systems, and interacts with insect proteins along the transmission pathway. These interactions and the tissue tropism in the vector body determine the efficiency and specificity of the transmission. This review describes the mechanisms involved in circulative begomovirus transmission by B. tabaci, focusing on the most studied virus in this regard, namely the tomato yellow leaf curl virus (TYLCV) and its closely related isolates. Additionally, the review aims at drawing attention to the recent knowhow of unorthodox virus-B. tabaci interactions. The recent knowledge of whitefly-mediated transmission of two recombinant poleroviruses (Luteoviridae), a virus group with an ssRNA genome and known to be strictly transmitted with aphids, is discussed with its broader context in the emergence of new whitefly-driven virus diseases.

The emergence of multi-drug resistant (MDR) bacteria is recognised today as one of the greatest challenges to public health. As traditional antimicrobials are becoming ineffective and research into new antibiotics is diminishing, a number of alternative treatments for MDR bacteria have been receiving greater attention. Bacteriophage therapies are being revisited and present a promising opportunity to reduce the burden of bacterial infection in this post-antibiotic era. This review focuses on the current evidence supporting bacteriophage therapy against prevalent or emerging multi-drug resistant bacterial pathogens in respiratory medicine and the challenges ahead in preclinical data generation. Starting with efforts to improve delivery of bacteriophages to the lung surface, the current developments in animal models for relevant efficacy data on respiratory infections are discussed before finishing with a summary of findings from the select human trials performed to date.

Monocytes are key modulators in acute viral infections, determining both inflammation and development of specific B- and T-cell responses. Recently, these cells were shown to be associated to different SARS-CoV-2 infection outcome. However, their role in acute HIV-1 infection remains unclear. We had the opportunity to evaluate the mononuclear cell compartment in an early hyper-acute HIV-1 patient in comparison with an untreated chronic HIV-1 and a cohort of SARS-CoV-2 infected patients, by high dimensional flow cytometry using an unsupervised approach. A distinct polarization of the monocyte phenotype was observed in the two viral infections, with maintenance of pro-inflammatory M1-like profile in HIV-1, in contrast to the M2-like immunosuppressive shift in SARS-CoV-2. Noticeably, both acute infections had reduced CD14low/-CD16+ non-classical monocytes, with depletion of the population expressing Slan (6-sulfo LacNac), which is thought to contribute to immune surveillance through pro-inflammatory properties. This depletion indicates a potential role of these cells in acute viral infection, which has not previously been explored. The inflammatory state accompanied by the depletion of Slan+ monocytes may provide new insights on the critical events that determine the rate of viral set-point in acute HIV-1 infection and subsequent impact on transmission and reservoir establishment.

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human-emerged virus of the 21st century from the Coronaviridae family, causing the ongoing coronavirus disease 2019 (COVID-19) pandemic. Due to the high zoonotic potential of coronaviruses, it is critical to unravel their evolutionary history of host species breadth, host-switch potential, adaptation and emergence, to identify viruses posing a pandemic risk in humans. We present here a comprehensive analysis of the composition and codon usage bias of the 82 Orthocoronavirinae members, infecting 47 different avian and mammalian hosts. Our results clearly establish that synonymous codon usage varies widely among viruses, is only weakly dependent on their primary host, and is dominated by mutational bias towards AU-enrichment and by CpG avoidance. Indeed, variation in GC3 explains around 34%, while variation in CpG frequency explains around 14% of total variation in codon usage bias. Further insight on the mutational equilibrium within Orthocoronavirinae revealed that most coronavirus genomes are close to their neutral equilibrium, the exception being the three recently infecting human coronaviruses, which lie further away from the mutational equilibrium than their endemic human coronavirus counterparts. Finally, our results suggest that, while replicating in humans, SARS-CoV-2 is slowly becoming AU-richer, likely until attaining a new mutational equilibrium.

Brazil was considered one of the emerging epicenters of the coronavirus pandemic in 2021, experiencing over 3000 daily deaths caused by the virus at the peak of the second wave. In total, the country had more than 20.8 million confirmed cases of COVID-19, including over 582,764 fatalities. A set of emerging variants arose in the country, some of them posing new challenges for COVID-19 control. The goal of this study was to describe mutational events across samples from Brazilian SARS-CoV-2 sequences publicly obtainable on Global Initiative on Sharing Avian Influenza Data-EpiCoV (GISAID-EpiCoV) platform and to generate indexes of new mutations by each genome. A total of 16,953 SARS-CoV-2 genomes were obtained, which were not proportionally representative of the five Brazilian geographical regions. A comparative sequence analysis was conducted to identify common mutations located at 42 positions of the genome (38 were in coding regions, whereas two were in 5' and two in 3' UTR). Moreover, 11 were synonymous variants, 27 were missense variants, and more than 44.4% were located in the spike gene. Across the total of single nucleotide variations (SNVs) identified, 32 were found in genomes obtained from all five Brazilian regions. While a high genomic diversity has been reported in Europe given the large number of sequenced genomes, Africa has demonstrated high potential for new variants. In South America, Brazil, and Chile, rates have been similar to those found in South Africa and India, providing enough "space" for new mutations to arise. Genomic surveillance is the central key to identifying the emerging variants of SARS-CoV-2 in Brazil and has shown that the country is one of the "hotspots" in the generation of new variants.

Zika virus (ZIKV) is the mosquito-transmitted virus that the WHO declared a Public Health Emergency of International Concern in 2016 due to the consequence of microcephaly from infected pregnancies. The incidence of Zika infection has been unclear in many countries because most infected people have nonspecific febrile illnesses. This study's aim is to investigate the incidence of symptomatic Zika virus infections from the archived samples of a dengue cohort study of children in central Thailand from 2006 to 2009. We performed Zika NS1 immunoglobulin (Ig)G enzyme-linked immunosorbent assay (ELISA) screening to identify symptomatic Zika infections in paired acute/convalescent serum samples. Symptomatic Zika infections were confirmed by reverse transcription polymerase chain reactions (RT-PCR) of acute serum samples. The comparison of the Zika NS1 IgG ELISA results between acute and convalescent samples showed 290/955 (30.4%) seropositive cases. Zika RT-PCR results were positive in 28 febrile cases (15 females, 13 males). Zika RT-PCR showed that symptomatic Zika infection occurred in children aged 4-11 years in Ratchaburi province, Thailand (2007-2009, first case in April 2007), and the symptomatic Zika:dengue infection ratio was 28 Zika:394 dengue (1:14). Phylogenetic analysis showed that all Zika viruses were of Asian lineage. Zika NS1 IgG ELISA identified Zika-infected patients and showed a low Zika:dengue ratio.

African swine fever virus (ASFV) is prevalent in many countries and is a contagious and lethal virus that infects pigs, posing a threat to the global pig industry and public health. The interaction between the virus and the host is key to unlocking the mystery behind viral pathogenesis. A comprehensive understanding of the viral and host protein interaction may provide clues for developing new antiviral strategies. Here, we show a network of ASFV MGF360-9L protein interactions in porcine kidney (PK-15) cells. Overall, 268 proteins that interact with MGF360-9L are identified using immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS). Accordingly, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted, and the protein-protein interaction (PPI) network was created. It was speculated that the cellular proteins interacting with MGF360-9L are involved in protein binding, metabolism, and the innate immune response. Proteasome subunit alpha type (PSMA3), 26S protease regulatory subunit 4 (PSMC1), autophagy and beclin 1 regulator 1 (AMBRA1), and DEAD-box helicase 20 (DDX20) could interact with MGF360-9L in vitro. PSMA3 and PSMC1 overexpression significantly promoted ASFV replication, and MGF360-9L maintained the transcriptional level of PSMA3 and PSMC1. Here, we show the interaction between ASFV MGF360-9L and cellular proteins and elucidate the virus-host interaction network, which effectively provides useful protein-related information that can enable further study of the potential mechanism and pathogenesis of ASFV infection.

We used wastewater-based epidemiology and amplicon-based long-read high-throughput sequencing for surveillance of enteroviruses (EVs) in Maricopa County, Arizona, Southwest United States. We collected 48 samples from 13 sites in three municipalities between 18 June and 1 October 2020, and filtered (175 mL each; 0.45 µm pore size) and extracted RNA from the filter-trapped solids. The RNA was converted to cDNA and processed through two workflows (Sanger sequencing (SSW) and long-read Illumina sequencing (LRISW)) each including a nested polymerase chain reaction (nPCR) assay. We subjected the ~350 bp amplicon from SSW to Sanger sequencing and the ~1900-2400 bp amplicon from LRISW to Illumina sequencing. We identified EV contigs from 11 of the 13 sites and 41.67% (20/48) of screened samples. Using the LRISW, we detected nine EV genotypes from three species (Enterovirus A (CVA4, EV-A76, EV-A90), Enterovirus B (E14) and Enterovirus C (CVA1, CVA11, CVA13, CVA19 and CVA24)) with Enterovirus C representing approximately 90% of the variants. However, the SSW only detected the five Enterovirus C types. Similarity and phylogenetic analysis showed that multiple Enterovirus C lineages were circulating, co-infecting and recombining in the population during the season despite the SARS-CoV-2 pandemic and the non-pharmaceutical public health measures taken to curb transmission.

Uruguay controlled the viral dissemination during the first nine months of the SARS-CoV-2 pandemic. Unfortunately, towards the end of 2020, the number of daily new cases exponentially increased. Herein, we analyzed the country-wide genetic diversity of SARS-CoV-2 between November 2020 and April 2021. We identified that the most prevalent viral variant during the first epidemic wave in Uruguay (December 2020-February 2021) was a B.1.1.28 sublineage carrying Spike mutations Q675H + Q677H, now designated as P.6, followed by lineages P.2 and P.7. P.6 probably arose around November 2020, in Montevideo, Uruguay's capital department, and rapidly spread to other departments, with evidence of further local transmission clusters; it also spread sporadically to the USA and Spain. The more efficient dissemination of lineage P.6 with respect to P.2 and P.7 and the presence of mutations (Q675H and Q677H) in the proximity of the key cleavage site at the S1/S2 boundary suggest that P.6 may be more transmissible than other lineages co-circulating in Uruguay. Although P.6 was replaced by the variant of concern (VOC) P.1 as the predominant lineage in Uruguay since April 2021, the monitoring of the concurrent emergence of Q675H + Q677H in VOCs should be of worldwide interest.

HDV infection causes severe liver disease, the global health burden of which may be underestimated due to limited epidemiological data. HDV depends on HBV for infection, but recent studies indicated that dissemination can also be supported by other helper viruses such as HCV. We used a rapid point-of-care test and an ELISA to retrospectively test for antibodies against the Hepatitis Delta antigen (anti-HDV-Ab) in 4103 HBsAg-positive and 1661 HBsAg-negative, anti-HCV-positive sera from China and Germany. We found that the HDV seroprevalence in HBsAg-positive patients in China is limited to geographic hotspots (Inner Mongolia: 35/251, 13.9%; Xinjiang: 7/180, 3.9%) and high-risk intravenous drug users (HBV mono-infected: 23/247, 9.3%; HBV-HCV co-infected: 34/107, 31.8%), while none of the 2634 HBsAg carriers from other metropolitan regions were anti-HDV-Ab-positive. In Germany, we recorded an HDV seroprevalence of 5.3% in a university hospital environment. In a cohort of HBsAg-negative, anti-HCV-positive patients that were not exposed to HBV before (anti-HBc-negative), HDV was not associated with HCV mono-infection (Chinese high-risk cohort: 0/365, 0.0%; German mixed cohort: 0/263, 0.0%). However, 21/1033 (2.0%) high-risk HCV patients in China with markers of a previously cleared HBV infection (anti-HBc-positive) were positive for anti-HDV-Ab, with two of them being positive for both HDV and HCV RNA but negative for HBV DNA. The absence of anti-HDV-Ab in HCV mono-infected patients shows that HCV cannot promote HDV transmission in humans.

We report on the efficacy of the non-attenuated HER2-retargeted oHSV named R-337 against the immunologically hot CT26-HER2 tumor, and an insight into the basis of the immune protection. Preliminarily, we conducted an RNA immune profiling and immune cell content characterization of CT26-HER2 tumor in comparison to the immunologically cold LLC1-HER2 tumor. CT26-HER2 tumor was implanted into HER2-transgenic BALB/c mice. Hallmarks of R-337 effects were the protection from primary tumor, long-term adaptive vaccination directed to both HER2 and CT26-wt cell neoantigens. The latter effect differentiated R-337 from OncoVEXGM-CSF. As to the basis of the immune protection, R-337 orchestrated several changes to the tumor immune profile, which cumulatively reversed the immunosuppression typical of this tumor (graphical abstract). Thus, Ido1 (inhibitor of T cell anticancer immunity) levels and T regulatory cell infiltration were decreased; Cd40 and Cd27 co-immunostimulatory markers were increased; the IFNγ cascade was activated. Of note was the dampening of IFN-I response, which we attribute to the fact that R-337 is fully equipped with genes that contrast the host innate response. The IFN-I shut-down likely favored viral replication and the expression of the mIL-12 payload, which, in turn, boosted the antitumor response. The results call for a characterization of tumor immune markers to employ oncolytic herpesviruses more precisely.

Mumps virus (MuV) is an important human pathogen that causes parotitis, orchitis, oophoritis, meningitis, encephalitis, and sensorineural hearing loss. Although mumps is a vaccine-preventable disease, sporadic outbreaks have occurred worldwide, even in highly vaccinated populations. MuV not only causes systemic infection but also has a unique tropism to glandular tissues and the central nervous system. In general, tropism can be defined by multiple factors in the viral life cycle, including its entry, interaction with host factors, and host-cell immune responses. Although the underlying mechanisms of MuV tropism remain to be fully understood, recent studies on virus-host interactions have provided insights into viral pathogenesis. This review was aimed at summarizing the entry process of MuV by focusing on the glycan receptors, particularly the recently identified receptors with a trisaccharide core motif, and their interactions with the viral attachment proteins. Here, we describe the receptor structures, their distribution in the human body, and the recently identified host factors for MuV and analyze their relationship with MuV tropism.

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

African swine fever (ASF) is one of the most important viral diseases of pigs caused by the ASF virus (ASFV). The virus is highly stable over a wide range of temperatures and pH and can survive in meat and meat products for several months, leading to long-distance transmission of ASF. Whole blood, serum, and organs from infected pigs are used routinely as approved sample types in the laboratory diagnosis of ASF. However, these sample types may not always be available. Here, we investigated meat exudate as an alternative sample type for the detection of ASFV-specific nucleic acids and antibodies. Pigs were infected with various ASFV strains: the highly virulent ASFV Malawi LIL 18/2 strain, the moderately-virulent ASFV Estonia 2014 strain, or the low-virulent ASFV OURT/88/3 strain. The animals were euthanized on different days post-infection (dpi), and meat exudates were collected and tested for the presence of ASFV-specific nucleic acids and antibodies. Animals infected with the ASFV Malawi LIL 18/2 developed severe clinical signs and succumbed to the infection within seven dpi, while pigs infected with ASFV Estonia 2014 also developed clinical signs but survived longer, with a few animals seroconverting before succumbing to the ASFV infection or being euthanized as they reached humane endpoints. Pigs infected with ASFV OURT/88/3 developed transient fever and seroconverted without mortality. ASFV genomic material was detected in meat exudate from pigs infected with ASFV Malawi LIL 18/2 and ASFV Estonia 2014 at the onset of viremia but at a lower amount when compared to the corresponding whole blood samples. Low levels of ASFV genomic material were detected in the whole blood of ASFV OURT/88/3-infected pigs, and no ASFV genomic material was detected in the meat exudate of these animals. Anti-ASFV antibodies were detected in the serum and meat exudate derived from ASFV OURT/88/3-infected pigs and in some of the samples derived from the ASFV Estonia 2014-infected pigs. These results indicate that ASFV genomic material and anti-ASFV antibodies can be detected in meat exudate, indicating that this sample can be used as an alternative sample type for ASF surveillance when routine sample types are unavailable or are not easily accessible.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are considered important for the entry of many different viruses. Previously, we demonstrated that heparanase (HPSE), the host enzyme responsible for cleaving HS chains, is upregulated by herpes simplex virus-1 (HSV-1) infection. Higher levels of HPSE accelerate HS removal from the cell surface, facilitating viral release from infected cells. Here, we study the effects of overexpressing HPSE on viral entry, cell-to-cell fusion, plaque formation, and viral egress. We provide new information that higher levels of HPSE reduce syncytial plaque formation while promoting egress and extracellular release of the virions. We also found that transiently enhanced expression of HPSE did not affect HSV-1 entry into host cells or HSV-1-induced cell-to-cell fusion, suggesting that HPSE activation is tightly regulated and facilitates extracellular release of the maturing virions. We demonstrate that an HSPG-shedding agonist, PMA; a protease, thrombin; and a growth factor, EGF as well as bacterially produced recombinant heparinases resulted in enhanced HSV-1 release from HeLa and human corneal epithelial (HCE) cells. Our findings here underscore the significance of syndecan-1 functions in the HSV-1 lifecycle, provide evidence that the shedding of syndecan-1 ectodomain is another way HPSE works to facilitate HSV-1 release, and add new evidence on the significance of various HSPG shedding agonists in HSV-1 release from infected cells.

The pandemic of COVID-19 caused by SARS-CoV-2 infection continues to spread around the world. Vaccines that elicit protective immunity have reduced infection and mortality, however new viral variants are arising that may evade vaccine-induced immunity or cause disease in individuals who are unable to develop robust vaccine-induced responses. Investigating the role of viral variants in causing severe disease, evading vaccine-elicited immunity, and infecting vulnerable individuals is important for developing strategies to control the pandemic. Here, we report fourteen breakthrough infections of SARS-CoV-2 in vaccinated individuals with symptoms ranging from asymptomatic/mild (6/14) to severe disease (8/14). High viral loads with a median Ct value of 19.6 were detected in the nasopharyngeal specimens from subjects regardless of disease severity. Sequence analysis revealed four distinct virus lineages, including alpha and gamma variants of concern. Immunosuppressed individuals were more likely to be hospitalized after infection (p = 0.047), however no specific variant was associated with severe disease. Our results highlight the high viral load that can occur in asymptomatic breakthrough infections and the vulnerability of immunosuppressed individuals to post-vaccination infections by diverse variants of SARS-CoV-2.