Enteric viruses, such as poliovirus, are a leading cause of gastroenteritis, which causes 2-3 million deaths annually. Environmental surveillance of wastewater supplements clinical surveillance for monitoring enteric virus circulation. However, while many environmental surveillance methods require liquid samples, some at-risk locations utilize pit latrines with waste characterized by high solids content. This study's objective was to develop and evaluate enteric virus concentration protocols for high solids content samples. Two existing protocols were modified and tested using poliovirus type 1 (PV1) seeded into primary sludge. Method 1 (M1) utilized acid adsorption, followed by 2 or 3 elutions (glycine/sodium chloride and/or threonine/sodium chloride), and skimmed milk flocculation. Method 2 (M2) began with centrifugation. The liquid fraction was filtered through a ViroCap filter and eluted (beef extract/glycine). The solid fraction was eluted (beef extract/disodium hydrogen phosphate/citric acid) and concentrated by skimmed milk flocculation. Recovery was enumerated by plaque assay. M1 yielded higher PV1 recovery than M2, though this result was not statistically significant (26.1% and 15.9%, respectively). M1 was further optimized, resulting in significantly greater PV1 recovery when compared to the original protocol (p < 0.05). This method can be used to improve understanding of enteric virus presence in communities without liquid waste streams.

SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus 2) has accumulated multiple mutations during its global circulation. Recently, three SARS-CoV-2 lineages, B.1.1.7 (501Y.V1), B.1.351 (501Y.V2) and B.1.1.28.1 (P.1), have emerged in the United Kingdom, South Africa and Brazil, respectively. Here, we have presented global viewpoint on implications of emerging SARS-CoV-2 variants based on structural-function impact of crucial mutations occurring in its spike (S), ORF8 and nucleocapsid (N) proteins. While the N501Y mutation was observed in all three lineages, the 501Y.V1 and P.1 accumulated a different set of mutations in the S protein. The missense mutational effects were predicted through a COVID-19 dedicated resource followed by atomistic molecular dynamics simulations. Current findings indicate that some mutations in the S protein might lead to higher affinity with host receptors and resistance against antibodies, but not all are due to different antibody binding (epitope) regions. Mutations may, however, result in diagnostic tests failures and possible interference with binding of newly identified anti-viral candidates against SARS-CoV-2, likely necessitating roll out of recurring "flu-like shots" annually for tackling COVID-19. The functional relevance of these mutations has been described in terms of modulation of host tropism, antibody resistance, diagnostic sensitivity and therapeutic candidates. Besides global economic losses, post-vaccine reinfections with emerging variants can have significant clinical, therapeutic and public health impacts.

Experiments in which complex virome sequencing data is generated remain difficult to explore and unpack for scientists without a background in data science. The processing of raw sequencing data by high throughput sequencing workflows usually results in contigs in FASTA format coupled to an annotation file linking the contigs to a reference sequence or taxonomic identifier. The next step is to compare the virome of different samples based on the metadata of the experimental setup and extract sequences of interest that can be used in subsequent analyses. The viromeBrowser is an application written in the opensource R shiny framework that was developed in collaboration with end-users and is focused on three common data analysis steps. First, the application allows interactive filtering of annotations by default or custom quality thresholds. Next, multiple samples can be visualized to facilitate comparison of contig annotations based on sample specific metadata values. Last, the application makes it easy for users to extract sequences of interest in FASTA format. With the interactive features in the viromeBrowser we aim to enable scientists without a data science background to compare and extract annotation data and sequences from virome sequencing analysis results.

Viruses have evolved numerous strategies to maximize the use of their limited genetic material, including proteolytic cleavage of polyproteins to yield products with different functions. The poliovirus polyprotein 3CD is involved in important protein-protein, protein-RNA and protein-lipid interactions in viral replication and infection. It is a precursor to the 3C protease and 3D RNA-dependent RNA polymerase, but has different protease specificity, is not an active polymerase, and participates in other interactions differently than its processed products. These functional differences are poorly explained by the known X-ray crystal structures. It has been proposed that functional differences might be due to differences in conformational dynamics between 3C, 3D and 3CD. To address this possibility, we conducted nuclear magnetic resonance spectroscopy experiments, including multiple quantum relaxation dispersion, chemical exchange saturation transfer and methyl spin-spin relaxation, to probe conformational dynamics across multiple timescales. Indeed, these studies identified differences in conformational dynamics in functionally important regions, including enzyme active sites, and RNA and lipid binding sites. Expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not observed in 3C and 3D alone despite having nearly identical lowest-energy structures.

The host range of SARS-CoV-2 and the susceptibility of animal species to the virus are topics of great interest to the international scientific community. The angiotensin I converting enzyme 2 (ACE2) protein is the major receptor for the virus, and sequence and structural analysis of the protein has been performed to determine its cross-species conservation. Based on these analyses, cattle have been implicated as a potential susceptible species to SARS-CoV-2 and have been reported to have increased ACE2 receptor distribution in the liver and kidney, and lower levels in the lungs. The goal of the current study was to determine the susceptibility of cattle to SARS-CoV-2 utilizing inoculation routes that facilitated exposure to tissues with increased ACE2 receptor distribution. For this, colostrum-deprived calves approximately 6 weeks of age were inoculated via the intratracheal or intravenous routes. Nasal and rectal swab samples, as well as blood and urine samples, were collected over the course of the study to evaluate viral shedding, viremia, and seroconversion. Pyrexia was used as the primary criteria for euthanasia and tissue samples were collected during necropsy. Importantly, SARS-CoV-2 RNA was detected in only two nasal swab samples collected on days 3 and 10 post-inoculation (pi) in two calves; one calf in the intratracheal group and the other calf in the intravenous group, respectively. Additionally, the calf in the intratracheal group that was positive on the nasal swab on day 3 pi also had a positive tracheobronchial lymph node on day 9 pi. Viral nucleic acid load on these samples, based on PCR cycle threshold values, were low and infectious virus was not recovered from the samples. These results suggest that there was no productive replication of SARS-CoV-2 in calves following intratracheal and intravenous inoculation.

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CA16) are major etiological agents of hand foot and mouth disease (HFMD) in children, which may result in fatal neurological complications. The development of safe, cost effective vaccines against HFMD, especially for use in developing countries, is still a top public health priority. We have successfully generated a stable, cold-adapted, temperature sensitive/conditional lethal EV-A71 through adaptive culturing in Vero cells at incrementally lower cultivation temperatures. An additional 40 passages at an incubation temperature of 28 °C, and a temperature reversion study at an incubation temperature of 37 °C and 39.5 °C, reveals the virus's phenotypic and genetic stability at the predefined culture conditions. Six unique mutations (two in noncoding regions and four in nonstructural protein-coding genes) in combination may have contributed to its stable phenotype and inability to fully revert to its original wild phenotype. The safety and immunogenicity of this stable, cold-adapted, temperature sensitive/conditional lethal EV-A71 was performed in six monkeys. None of the inoculated monkeys developed any obvious clinical illness except one which developed a transient spike of fever. No gross postmortem lesion or abnormal histological finding was noted for all monkeys at autopsy. No virus was reisolated although EV-A71 specific RNA was detected in serum samples collected on both day 4 and day 8 postinoculation. Only EV-A71 RNA and viral antigen were detected in the spleen homogenate and peripheral blood mononuclear cells, respectively, collected on day 4. The two remaining monkeys developed good humoral immune response on day 14 and day 30 post-inoculation.

Pyruvate kinase M2 (PKM2) mainly catalyzes glycolysis, but it also exerts non-glycolytic functions in several cancers. While it has been shown to interact with the human papillomavirus 16 (HPV16) E7 oncoprotein, the functional significance of PKM2 in HPV-associated cervical cancer has been elusive. Here, we show that HPV16 E7 increased the expression of PKM2 in cervical cancer cells. TCGA data analyses revealed a higher level of PKM2 in HPV+ than HPV- cervical cancers and a worse prognosis for patients with high PKM2 expression. Functionally, we demonstrate that shRNA-mediated PKM2 knockdown decreased the proliferation of HPV+ SiHa cervical cancer cells. PKM2 knockdown also inhibited the E7-induced proliferation of cervical cancer cells. ML265 activating the pyruvate kinase function of PKM2 inhibited cell cycle progression and colony formation. ML265 treatments decreased phosphorylation of PKM2 at the Y105 position that has been associated with non-glycolytic functions. On the contrary, HPV16 E7 increased the PKM2 phosphorylation. Our results indicate that E7 increases PKM2 expression and activates a non-glycolytic function of PKM2 to promote cervical cancer cell proliferation.

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.

Picobirnaviruses (PBVs) are small non-enveloped bisegmented double-stranded RNA viruses found in humans, mammals, and birds. Increasing molecular epidemiology studies suggest a high sequence diversity of PBVs in numerous hosts and the environment. In this study, using 229 fecal samples from dromedary camels in Dubai, 52.8% were positive for PBVs, of which 77.7% and 41.3% were positive for genogroup I and II, respectively, and 19.0% were positive for both genotypes. Phylogenetic analysis showed high diversity among the sequences of genogroup I and II dromedary PBVs. Marked nucleotide polymorphisms were observed in 75.5% and 46.0% of genogroup I and II RNA-dependent RNA polymerase (RdRp) sequences, respectively, suggesting the co-existence of multiple strains in the same specimen. Both high genetic diversity and prevalence of genogroup I and II PBV in dromedaries were observed. In fact, the prevalence of genogroup II PBV in dromedaries is the highest among all animals to date. The complete/near-complete core genomes of five genogroup I and one genogroup II dromedary PBVs and partial segment 1 and 2 of both genotypes were also sequenced. The dromedary PBV genome organizations were similar to those of other animals. Genetic reassortment and mutation are both important in the ecology and evolution of PBVs.

The use of convalescent plasma in the treatment of COVID-19 may lead to a milder course of infection and has been associated with improved outcomes. Determining optimal treatments in high risk populations is crucial, as is the case in those with hematological malignancies. We analyzed a cohort of 23 patients with hematological malignancies and COVID-19 who had received plasma 48-72 h after the diagnosis of infection and compared it with a historical group of 22 patients who received other therapy. Overall survival in those who received convalescent plasma was significantly higher than in the historical group (p = 0.03460). The plasma-treated group also showed a significantly milder course of infection (p = 0.03807), characterized by less severe symptoms and faster recovery (p = 0.00001). In conclusion, we have demonstrated that convalescent plasma is an effective treatment and its early administration leads to clinical improvement, increased viral clearance and longer overall survival in patients with hematological malignancies and COVID-19. To our knowledge, this is the first report to analyze the efficacy of convalescent plasma in a cohort of patients with hematological malignancies.

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.

Enteroviruses (EVs) are highly prevalent viruses world-wide, causing a wide range of diseases in both children and adults. Insight in the global prevalence of EVs is important to define their clinical significance and total disease burden, and assists in making therapeutic decisions. While many studies have been conducted to describe epidemiology of EVs in specific (sub)populations and patient cohorts, little effort has been made to aggregate the available evidence. In the current study, we conducted a search in the PubMed and Embase (Ovid) databases to identify articles reporting EV prevalence and type distribution. We summarized the findings of 153 included studies. We found that EVs are highly prevalent viruses in all continents. Enterovirus B was the most detected species worldwide, while the other species showed continent-specific differences, with Enterovirus C more detected in Africa and Enterovirus A more detected in Asia. Echovirus 30 was by far the most detected type, especially in studies conducted in Europe. EV types in species Enterovirus B-including echovirus 30-were often detected in patient groups with neurological infections and in cerebrospinal fluid, while Enterovirus C types were often found in stool samples.

The availability of all oral direct acting antiviral agents (DAAs) has revolutionized the management of HCV infections in recent years, allowing to achieve a sustained virological response (SVR) in more than 95% of cases, irrespective of hepatitis C Virus (HCV) genotype or staging of liver disease. Although rare, the failure to the latest-generation regimens (grazoprevir/elbasvir, sofosbuvir/velpatasvir, pibrentasvir/glecaprevir) represents a serious clinical problem, since the data available in the literature on the virological characteristics and management of these patients are few. The aim of the present narrative review was to provide an overview of the impact of baseline RASs in patients treated with the latest-generation DAAs and to analyze the efficacy of the available retreatment strategies in those who have failed these regimens.

With deep sadness, we have to accept that two cornerstones, not just of British immunology, but also world-famous scientists in the field of complement research, passed away within the margin of a few weeks: Peter Julius Lachmann on 26 December 2020 (Figure 1), and Robert Braidwood Sim (known to everyone as Bob) on 6 February 2021 (Figure 2) [...].

Tomato spotted wilt virus (TSWV) is one of the most destructive plant viruses, causing severe losses in many important crops worldwide. The non-structural protein NSm of TSWV is a viral movement protein that induces viral symptoms. However, the molecular mechanisms by which NSm contributes to symptom development are unclear. Here, we present evidence that NSm directly interacts with Nicotiana benthamiana chloroplast thylakoid membrane protein TMP14 (NbTMP14) by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. The interaction between NSm and NbTMP14 led to the translocation of the NbTMP14 protein from the chloroplast to the cytoplasm in TSWV-infected plants, and overexpressing NSm decreased NbTMP14 mRNA accumulation. In addition, abnormal chloroplasts and starch accumulation were observed in TSWV-infected plants. Silencing of NbTMP14 by TRV VIGS also showed similar results to those of TSWV-infected plants. Overexpressing NbTMP14 in transgenic N. benthamiana plants impeded TSWV infection, and silencing NbTMP14 in N. benthamiana plants increased disease symptom severity and virus accumulation. To our knowledge, this is the first report showing that the plant chloroplast TMP14 protein is involved in viral infection. Knowledge of the interaction between NSm and NbTMP14 advances our understanding of the molecular mechanisms underlying TSWV symptom development and infection.

Enterococcus faecalis is a Gram-positive pathogen which colonizes human intestinal surfaces, forming biofilms, and demonstrates a high resistance to many antibiotics. Especially, antibiotics are less effective for eradicating biofilms and better alternatives are needed. In this study, we have isolated and characterized a bacteriophage, PBEF129, infecting E. faecalis. PBEF129 infected a variety of strains of E. faecalis, including those exhibiting antibiotic resistance. Its genome is a linear double-stranded DNA, 144,230 base pairs in length. Its GC content is 35.9%. The closest genomic DNA sequence was found in Enterococcus phage vB_EfaM_Ef2.3, with a sequence identity of 99.06% over 95% query coverage. Furthermore, 75 open reading frames (ORFs) were functionally annotated and five tRNA-encoding genes were found. ORF 6 was annotated as a phage endolysin having an L-acetylmuramoyl-l-alanine amidase activity. We purified the enzyme as a recombinant protein and confirmed its enzymatic activity. The endolysin's host range was observed to be wider than its parent phage PBEF129. When applied to bacterial biofilm on the surface of in vitro cultured human intestinal cells, it demonstrated a removal efficacy of the same degree as cefotaxime, but much lower than its parent bacteriophage.

Retroviruses belong to an important and diverse family of RNA viruses capable of causing neoplastic disease in their hosts. Feline leukaemia virus (FeLV) is a gammaretrovirus that infects domestic and wild cats, causing immunodeficiency, cytopenia and neoplasia in progressively infected cats. The outcome of FeLV infection is influenced by the host immune response; progressively infected cats demonstrate weaker immune responses compared to regressively infected cats. In this study, humoral immune responses were examined in 180 samples collected from 123 domestic cats that had been naturally exposed to FeLV, using a novel ELISA to measure antibodies recognizing the FeLV surface unit (SU) glycoprotein in plasma samples. A correlation was demonstrated between the strength of the humoral immune response to the SU protein and the outcome of exposure. Cats with regressive infection demonstrated higher antibody responses to the SU protein compared to cats belonging to other outcome groups, and samples from cats with regressive infection contained virus neutralising antibodies. These results demonstrate that an ELISA that assesses the humoral response to FeLV SU complements the use of viral diagnostic tests to define the outcome of exposure to FeLV. Together these tests could allow the rapid identification of regressively infected cats that are unlikely to develop FeLV-related disease.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa. Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide bonds were reduced by Acetylcysteine. In in vitro whole virus culture of both wild-type and spike mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.

Reactivations of BK polyoma virus (BKPyV) and human cytomegalovirus (HCMV) frequently cause life- and graft-threatening complications after renal transplantation. Both viruses are dependent on the mTOR pathway for replication. In this study we investigated the association of viral replication with mTOR activity in peripheral lymphocytes of renal transplant recipients. A flow-cytometry based assay for the measurement of Thr389 p70S6k phosphorylation, a surrogate marker of the mTOR pathway was established. Forty-eight adult renal transplant recipients were recruited to measure p70S6k activity in their peripheral blood mononuclear cells. This data set in conjunction with information concerning previous replication of BKPyV and HCMV was examined for correlations. Episodes of BKPyV replication were significantly associated with increased p70S6k phosphorylation in CD4+ T lymphocytes (p = 0.0002) and CD19+ B lymphocytes (p = 0.0073). HCMV infection of patients with a high-risk HCMV constellation of donor and recipient (D+/R-) was associated with increased p70S6k phosphorylation in CD19+ B lymphocytes (p = 0.0325). These associations were found to be independent of the trough levels of the immunosuppressive drugs. Conclusion: P70S6k phosphorylation in peripheral lymphocytes is associated with BKPyV reactivations and to a lesser extent with HCMV infections in renal transplant recipients.

Nef is a multifunctional viral protein that has the ability to downregulate cell surface molecules, including CD4 and major histocompatibility complex class I (MHC-I) and, as recently shown, also members of the serine incorporator family (SERINC). Here, we analyzed the impact of naturally occurring mutations in HIV-1 Nef on its ability to counteract SERINC restriction and the clinical course of infection. HIV-1 Nef sequences were obtained from 123 participants of the Amsterdam Cohort Studies and showed multiple amino acid variations and mutations. Most of the primary Nef proteins showed increased activity to counteract SERINC3 and SERINC5 as compared to NL4-3 Nef. Several mutations in Nef were associated with either an increased or decreased infectivity of Bal26-pseudotyped HIV-1 produced in the presence of SERINC3 or SERINC5. The 8R, 157N and R178G Nef mutations were shown to have an effect on disease progression. Survival analysis showed an accelerated disease progression of individuals infected with HIV-1 carrying arginine or asparagine at position 8 or 157 in Nef, respectively, or the R178G Nef mutation. Here, we observed that naturally occurring mutations in Nef affect the ability of Nef to counteract SERINC3- and SERINC5-mediated inhibition of viral infectivity. The majority of these Nef mutations had no significant effect on HIV-1 pathogenesis and only the 8R, 157N and R178G mutations were associated with disease course.

Plant viral diseases are the foremost threat to sustainable agriculture, leading to several billion dollars in losses every year. Many viruses infecting several crops have been described in the literature; however, new infectious viruses are emerging frequently through outbreaks. For the effective treatment and prevention of viral diseases, there is great demand for new techniques that can provide accurate identification on the causative agents. With the advancements in biochemical and molecular biology techniques, several diagnostic methods with improved sensitivity and specificity for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. New portable devices and technologies that could provide real-time analyses in a relatively short period of time are prime important for in-field diagnostics. Current development and application of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail.

Although antibody levels progressively decrease following SARS-CoV-2 infection, the immune memory persists for months. Thus, individuals who naturally contracted SARS-CoV-2 are expected to develop a more rapid and sustained response to COVID-19 vaccines than naïve individuals. In this study, we analyzed the dynamics of the antibody response to the BNT162b2 mRNA COVID-19 vaccine in six healthcare workers who contracted SARS-CoV-2 in March 2020, in comparison to nine control subjects without a previous infection. The vaccine was well tolerated by both groups, with no significant difference in the frequency of vaccine-associated side effects, with the exception of local pain, which was more common in previously infected subjects. Overall, the titers of neutralizing antibodies were markedly higher in response to the vaccine than after natural infection. In all subjects with pre-existing immunity, a rapid increase in anti-spike receptor-binding domain (RBD) IgG antibodies and neutralizing antibody titers was observed one week after the first dose, which seemed to act as a booster. Notably, in previously infected individuals, neutralizing antibody titers 7 days after the first vaccine dose were not significantly different from those observed in naïve subjects 7 days after the second vaccine dose. These results suggest that, in previously infected people, a single dose of the vaccine might be sufficient to induce an effective response.

Cytokine storm syndrome in patients with COVID-19 is mediated by pro-inflammatory cytokines resulting in acute lung injury and multiorgan failure. Elevation in serum ferritin and D-dimer is observed in COVID-19 patients. To determine prognostic values of optimal serum cutoff with trajectory plots for both serum ferritin and D-dimer in COVID-19 patients with invasive ventilator dependence and in-hospital mortality. We used retrospective longitudinal data from the Cerner COVID-19 de-identified cohort. COVID-19 infected patients with valid repeated values of serum ferritin and D-dimer during hospitalization were used in mixed-effects logistic-regression models. Among 52,411 patients, 28.5% (14,958) had valid serum ferritin and 28.6% (15,005) D-dimer laboratory results. Optimal cutoffs of ferritin (714 ng/mL) and D-dimer (2.1 mg/L) revealed AUCs ≥ 0.99 for in-hospital mortality. Optimal cutoffs for ferritin (502 ng/mL) and D-dimer (2.0 mg/L) revealed AUCs ≥ 0.99 for invasive ventilator dependence. Optimal cutoffs for in-house mortality, among females, were lower in serum ferritin (433 ng/mL) and D-dimer (1.9 mg/L) compared to males (740 ng/mL and 2.5 mg/L, respectively). Optimal cutoffs for invasive ventilator dependence, among females, were lower in ferritin (270 ng/mL) and D-dimer (1.3 mg/L) compared to males (860 ng/mL and 2.3 mg/L, respectively). Optimal prognostic cutoffs for serum ferritin and D-dimer require considering the entire trajectory of laboratory values during the disease course. Females have an overall lower optimal cutoff for both serum ferritin and D-dimer. The presented research allows health professionals to predict clinical outcomes and appropriate allocation of resources during the COVID-19 pandemic, especially early recognition of COVID-19 patients needing higher levels of care.

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

The Human Immunodeficiency Virus type 1 (HIV-1) virion contains a conical shell, termed capsid, encasing the viral RNA genome. After cellular entry of the virion, the capsid is released and ensures the protection and delivery of the HIV-1 genome to the host nucleus for integration. The capsid relies on many virus-host factor interactions which are regulated spatiotemporally throughout the course of infection. In this paper, we will review the current understanding of the highly dynamic HIV-1 capsid-host interplay during the early stages of viral replication, namely intracellular capsid trafficking after viral fusion, nuclear import, uncoating, and integration of the viral genome into host chromatin. Conventional anti-retroviral therapies primarily target HIV-1 enzymes. Insights of capsid structure have resulted in a first-in-class, long-acting capsid-targeting inhibitor, GS-6207 (Lenacapavir). This inhibitor binds at the interface between capsid protein subunits, a site known to bind host factors, interferes with capsid nuclear import, HIV particle assembly, and ordered assembly. Our review will highlight capsid structure, the host factors that interact with capsid, and high-throughput screening techniques, specifically genomic and proteomic approaches, that have been and can be used to identify host factors that interact with capsid. Better structural and mechanistic insights into the capsid-host factor interactions will significantly inform the understanding of HIV-1 pathogenesis and the development of capsid-centric antiretroviral therapeutics.

The year 2020 was profoundly marked by the emergence and spread of SARS-CoV-2, causing COVID-19, which represents the greatest pandemic of the 21st century until now, and a major challenge for virologists in the scientific and medical communities. Increased numbers of SARS-CoV-2 infection all over the world imposed social and travel restrictions, including avoidance of face-to-face scientific meetings. Therefore, for the first time in history, the 2020 edition of the Brazilian Society of Virology (SBV) congress was totally online. Despite the challenge of the new format, the Brazilian society board and collaborators were successful in virtually congregating more than 921 attendees, which was the greatest SBV participant number ever reached. Seminal talks from prominent national and international researchers were presented every night, during a week, and included discussions about environmental, basic, animal, human, plant and invertebrate virology. A special roundtable debated exclusively new data and perspectives regarding COVID-19 by some of the greatest Brazilian virologists. Women scientists were very well represented in another special roundtable called "Young Women Inspiring Research", which was one of the most viewed and commented section during the meeting, given the extraordinary quality of the presented work. Finally, SBV offered the Helio Gelli Pereira award for one graduate and one undergraduate student, which has also been a fruitful collaboration between the society and Viruses journal. The annual SBV meeting has, therefore, reached its goals to inspire young scientists, stimulate high-quality scientific discussion and to encourage global collaboration between virologists.

The aim of this study was to establish the persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on inanimate surfaces such as plastic, stainless steel, and glass during UV-C irradiation which is a physical means commonly utilized in sanitization procedures. The viral inactivation rate, virus half-life, and percentage of titer reduction after UV-C irradiation were assessed. Infectivity was maintained on plastic and glass until 120 h and on stainless steel until 72 h. The virus half-life was 5.3, 4.4, and 4.2 h on plastic, stainless steel, and glass, respectively. In all cases, titer decay was >99% after drop drying. UV-C irradiation efficiently reduced virus titer (99.99%), with doses ranging from 10.25 to 23.71 mJ/cm2. Plastic and stainless steel needed higher doses to achieve target reduction. The total inactivation of SARS-CoV-2 on glass was obtained with the lower dose applied. SARS-CoV-2 survival can be long lasting on inanimate surfaces. It is worth recommending efficient disinfection protocols as a measure of prevention of viral spread. UV-C can provide rapid, efficient and sustainable sanitization procedures of different materials and surfaces. The dosages and mode of irradiation are important parameters to consider in their implementation as an important means to fight the SARS-CoV-2 pandemic.

The coronavirus SARS-CoV-2, which causes Coronavirus disease 2019 (COVID-19), has infected more than 100 million people globally and caused over 2.5 million deaths in just over one year since its discovery in Wuhan, China in December 2019. The pandemic has evoked widespread collateral damage to societies and economies, and has destabilized mental health and well-being. Early in 2020, unprecedented efforts went into the development of vaccines that generate effective antibodies to the SARS-CoV-2 virus. Teams developing twelve candidate vaccines, based on four platforms (messenger RNA, non-replicating viral vector, protein/virus-like particle, and inactivated virus) had initiated or announced the Phase III clinical trial stage by early November 2020, with several having received emergency use authorization in less than a year. Vaccine rollout has proceeded around the globe. Previously, we and others had proposed a target product profile (TPP) for ideal/optimal and acceptable/minimal COVID-19 vaccines. How well do these candidate vaccines stack up to a harmonized TPP? Here, we perform a comparative analysis in several categories of these candidate vaccines based on the latest available trial data and highlight the early successes as well as the hurdles and barriers yet to be overcome for ending the global COVID-19 pandemic.

Glycan-protein interactions are highly specific yet transient, rendering glycans ideal recognition signals in a variety of biological processes. In human norovirus (HuNoV) infection, histo-blood group antigens (HBGAs) play an essential but poorly understood role. For murine norovirus infection (MNV), sialylated glycolipids or glycoproteins appear to be important. It has also been suggested that HuNoV capsid proteins bind to sialylated ganglioside head groups. Here, we study the binding of HBGAs and sialoglycans to HuNoV and MNV capsid proteins using NMR experiments. Surprisingly, the experiments show that none of the norovirus P-domains bind to sialoglycans. Notably, MNV P-domains do not bind to any of the glycans studied, and MNV-1 infection of cells deficient in surface sialoglycans shows no significant difference compared to cells expressing respective glycans. These findings redefine glycan recognition by noroviruses, challenging present models of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of novel coronavirus disease 2019 (COVID-19), has become a severe threat to global public health. There are currently no antiviral therapies approved for the treatment or prevention of mild to moderate COVID-19 as remdesivir is only approved for severe COVID-19 cases. Here, we evaluated the antiviral potential of a Propylamylatin formula, which is a mixture of propionic acid and isoamyl hexanoates. The Propylamylatin formula was investigated in gaseous and liquid phases against 1 mL viral suspensions containing 105 PFU of SARS-CoV-2. Viral suspensions were sampled at various times post-exposure and infectious virus was quantified by plaque assay on Vero E6 cells. Propylamylatin formula vapors were effective at inactivating infectious SARS-CoV-2 to undetectable levels at room temperature and body temperature, but the decline in virus was substantially faster at the higher temperature (15 min versus 24 h). The direct injection of liquid Propylamylatin formula into viral suspensions also completely inactivated SARS-CoV-2 and the rapidity of inactivation occurred in an exposure dependent manner. The overall volume that resulted in 90% viral inactivation over the course of the direct injection experiment (EC90) was 4.28 µls. Further investigation revealed that the majority of the antiviral effect was attributed to the propionic acid which yielded an overall EC90 value of 11.50 µls whereas the isoamyl hexanoates provided at most a 10-fold reduction in infectious virus. The combination of propionic acid and isoamyl hexanoates was much more potent than the individual components alone, suggesting synergy between these components. These findings illustrate the therapeutic promise of the Propylamylatin formula as a potential treatment strategy for COVID-19 and future studies are warranted.

The treatment of coronavirus disease 2019 (COVID-19) has been a challenge. The efficacy of several drugs has been evaluated and variability in drug response has been observed. Pharmacogenetics could explain this variation and improve patients' outcomes with this complex disease; nevertheless, several disease-related issues must be carefully reviewed in the pharmacogenetic study of COVID-19 treatment. We aimed to describe the pharmacogenetic variants reported for drugs used for COVID-19 treatment (remdesivir, oseltamivir, lopinavir, ritonavir, azithromycin, chloroquine, hydroxychloroquine, ivermectin, and dexamethasone). In addition, other factors relevant to the design of pharmacogenetic studies were mentioned. Variants in CYP3A4, CYP3A5, CYP2C8, CY2D6, ABCB1, ABCC2, and SLCO1B1, among other variants, could be included in pharmacogenetic studies of COVID-19 treatment. Besides, nongenetic factors such as drug-drug interactions and inflammation should be considered in the search for personalized therapy of COVID-19.

Genetic variations in dengue virus (DENV) play a distinct role in epidemic emergence. The DENV 3' UTR has become a recent interest in research. The objective of the study was to examine the genetic variation in the domain II, 3' UTR region of human and mosquito-derived DENV. DENV-infected human sera were orally infected to laboratory reared Aedes aegypti mosquitoes. The domain II, 3' UTR of each human- and mosquito-derived sample was amplified. The nucleotide sequence variation, phylogenetic and secondary structure analysis was carried out incorporating respective regions of so far recorded Sri Lankan and the reference genotype strains of the DENV3 and DENV1 serotypes. The human- and mosquito-derived domain II, 3' UTR were identical in nucleotide sequences within the serotypes isolated, indicating the conserved nature of the region during host switch. The sequence analysis revealed distinct variations in study isolates compared to so far recorded Sri Lankan isolates. However, despite single nucleotide variations, the maintenance of structural integrity was evident in related strains within the serotypes in the secondary structure analysis. The phylogenetic analysis revealed distinct clade segregation of the study sequences from so far reported Sri Lankan isolates and illustrated the phylogenetic relations of the study sequences to the available global isolates of respective serotypes.

The risk of polio importation and re-emergence persists since epidemic polio still occurs in some countries, and the resurgence of polio occurring almost 20 years after polio eradication was declared in Asia has been reported. We analyzed the results of acute flaccid paralysis (AFP) surveillance in Korea to assess the quality of AFP surveillance and understand the etiology of non-polio enterovirus (NPEV)-associated central nervous system diseases in a polio-free area. We investigated 637 AFP patients under 15 years of age whose cases were confirmed during 2012-2019 by virus isolation, real-time reverse transcription polymerase chain reaction, and VP1 gene sequencing. Among the 637 AFP cases, NPEV was detected in 213 (33.4%) patients, with the majority observed in EV-A71, with 54.9% of NPEV positives. EV-A71 has been shown to play a role as a major causative agent in most neurological diseases except for Guillain-Barré syndrome (GBS), acute disseminated encephalomyelitis (ADEM), and meningitis. This study provides information on the AFP surveillance situation in Korea and highlights the polio eradication stage in the monitoring and characterization of NPEV against the outbreak of neurological infectious diseases such as polio.

Epizootic hemorrhagic disease virus (EHDV; family Reoviridae, genus Orbivirus) is an arthropod-borne virus of ungulates, primarily white-tailed deer in North America. Culicoides sonorensis, the only confirmed North American vector of EHDV, is rarely collected from Florida despite annual virus outbreaks. Culicoides insignis is an abundant species in Florida and is also a confirmed vector of the closely related Bluetongue virus. In this study, oral challenge of C. insignis was performed to determine vector competence for EHDV serotype-2. Field-collected female midges were provided bovine blood spiked with three different titers of EHDV-2 (5.05, 4.00, or 2.94 log10PFUe/mL). After an incubation period of 10 days or after death, bodies and legs were collected. Saliva was collected daily from all females from 3 days post feeding until their death using honey card assays. All samples were tested for EHDV RNA using RT-qPCR. Our results suggest that C. insignis is a weakly competent vector of EHDV-2 that can support a transmissible infection when it ingests a high virus titer (29% of midges had virus positive saliva when infected at 5.05 log10PFUe/mL), but not lower virus titers. Nevertheless, due to the high density of this species, particularly in peninsular Florida, it is likely that C. insignis plays a role in the transmission of EHDV-2.

The mitochondrial antiviral-signaling protein (MAVS, also known as VISA, IPS-1, or CARDIF) plays an essential role in the type I interferon (IFN) response and in retinoic acid-inducible gene I (RIG-I) mediated antiviral innate immunity in mammals. In this study, the caprine MAVS gene (caMAVS, 1566 bp) was identified and cloned. The caMAVS shares the highest amino acid similarity (98.1%) with the predicted sheep MAVS. Confocal microscopy analysis of partial deletion mutants of caMAVS revealed that the transmembrane and the so-called Non-Characterized domains are indispensable for intracellular localization to mitochondria. Overexpression of caMAVS in caprine endometrial epithelial cells up-regulated the mRNA levels of caprine interferon-stimulated genes. We concluded that caprine MAVS mediates the activation of the type I IFN pathway. We further demonstrated that both the CARD-like domain and the transmembrane domain of caMAVS were essential for the activation of the IFN-β promotor. The interaction between caMAVS and caprine RIG-I and the vital role of the CARD and NC domain in this interaction was demonstrated by co-immunoprecipitation. Upon infection with the Peste des Petits Ruminants Virus (PPRV, genus Morbillivirus), the level of MAVS was greatly reduced. This reduction was prevented by the addition of the proteasome inhibitor MG132. Moreover, we found that viral protein V could interact and colocalize with MAVS. Together, we identified caMAVS as a RIG-I interactive protein involved in the activation of type I IFN pathways in caprine cells and as a target for PPRV immune evasion.

Using reverse genetics, we analyzed a chikungunya virus (CHIKV) isolate of the Indian Ocean lineage lacking direct repeat (DR) elements in the 3' untranslated region, namely DR1a and DR2a. While this deletion mutant CHIKV-∆DR exhibited growth characteristics comparable to the wild-type virus in Baby Hamster Kidney cells, replication of the mutant was reduced in Aedes albopictus C6/36 and Ae. aegypti Aag2 cells. Using oral and intrathoracic infection of mosquitoes, viral infectivity, dissemination, and transmission of CHIKV-∆DR could be shown for the well-known CHIKV vectors Ae. aegypti and Ae. albopictus. Oral infection of Ae. vexans and Culex pipiens mosquitoes with mutant or wild-type CHIKV showed very limited infectivity. Dissemination, transmission, and transmission efficiencies as determined via viral RNA in the saliva were slightly higher in Ae. vexans for the wild-type virus than for CHIKV-∆DR. However, both Ae. vexans and Cx. pipiens allowed efficient viral replication after intrathoracic injection confirming that the midgut barrier is an important determinant for the compromised infectivity after oral infection. Transmission efficiencies were neither significantly different between Ae. vexans and Cx. pipiens nor between wild-type and CHIKV-∆DR. With a combined transmission efficiency of 6%, both Ae. vexans and Cx. pipiens might serve as potential vectors in temperate regions.

HIV-1 replication capacity is an important characteristic to understand the replication competence of single variants or virus populations. It can further aid in the understanding of HIV-1 pathogenicity, disease progression, and drug resistance mutations. To effectively study RC, many assays have been established. However, there is still demand for a high throughput replication capacity assay using primary cells which is robust and reproducible. In this study, we established such an assay and validated it using 346 primary HIV-1 isolates from patients enrolled in the Zurich Primary HIV Infection study (ZPHI) and two control viruses, HIV-1 JR-CSFWT and HIV-1 JR-CSFK65R_M184V. Replication capacity was determined by measuring the viral growth on PBMCs over 10 days by longitudinally transferring cell culture supernatant to TZM-bl reporter cells. By utilizing the TZM-bl luciferase reporter assay, we determined replication capacity by measuring viral infectivity. The simplicity of the experimental setup allowed for all 346 primary HIV-1 isolates to be replicated at one time. Although the infectious input dose for each virus was normalized, a broad range of replication capacity values over 4 logs was observed. The approach was confirmed by two repeated experiments and we demonstrated that the reproducibility of the replication capacity values is statistically comparable between the two separate experiments. In summary, these results endorse our high throughput replication capacity assay as reproducible and robust and can be utilized for large scale HIV-1 replication capacity experiments in primary cells.

Sylvatic rabies was present in Slovenia between 1973 and 2013, with the red fox as the main reservoir of the rabies virus. The first oral rabies vaccination (ORV) control program in foxes started in 1988, using the manual distribution of baits. Significant improvement of fox vaccination was achieved with the aerial distribution of baits, starting in 1995 and successfully finished with the final, fifty-ninth vaccination campaign in 2019. Between 1979 and 2019, a total of 86,471 samples were tested, and 10,975 (12.69%) rabies-positive animals were identified. Within the ORV, two different vaccines were used, containing modified live virus strain Street Alabama Dufferin (SAD) B19 and SAD Bern, while the last ORV campaigns were completed in 2019, with a vaccine containing a genetically modified strain of SPBN GASGAS. Molecular epidemiological studies of 95 rabies-positive samples, originating from red foxes, badgers, cattle, dogs, martens, cats, and horses, revealed a low genetic diversity of circulating strains and high similarity to strains from neighboring countries. During the elimination program, few vaccine-induced rabies cases were detected: three in red foxes and one case in a marten, with no epidemiological relevance. Slovenia has been officially declared a country free of rabies since 2016.

During 2019, five carcasses of juvenile Egyptian fruit bats (Rousettus aegyptiacus) were submitted to the Kimron Veterinary Institute. These bats exhibited typical poxvirus like lesion plaques of different sizes on the skin, abdomen and the ventral side of the wings. Clinical and histopathological findings suggested a poxvirus infection. Infectious virus was isolated from skin swabs, skin tissue and tongue of the dead bats and was further confirmed to be a Poxvirus by molecular diagnosis using PCR with pan-chordopoxviruses primers. All the dead bats were found positive for two Poxvirus genes encoding a metalloproteinase and DNA dependent DNA polymerase. In this study, a novel real time quantitative PCR (qPCR) assay was established to further confirmed the presence of specific poxvirus viral DNA in all pathologically tested tissues. Moreover, according to sequence analysis, the virus was found to be highly similar to the recently discovered Israeli Rousettus aegyptiacus Pox Virus (IsrRAPXV).

Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.

Flaviviruses circulate worldwide and cause a number of medically relevant human diseases, such as dengue, Zika, yellow fever, and tick-borne encephalitis (TBE). Serology plays an important role in the diagnosis of flavivirus infections, but can be impeded by antigenic cross-reactivities among flaviviruses. Therefore, serological diagnosis of a recent infection can be insufficiently specific, especially in areas where flaviviruses co-circulate and/or vaccination coverage against certain flaviviruses is high. In this study, we developed a new IgM assay format, which is well suited for the specific diagnosis of TBE, Zika and dengue virus infections. In the case of TBE and Zika, the IgM response proved to be highly specific for the infecting virus. In contrast, primary dengue virus infections induced substantial amounts of cross-reactive IgM antibodies, which is most likely explained by structural peculiarities of dengue virus particles. Despite the presence of cross-reactive IgM, the standardized nature and the quantitative read-out of the assay even allowed the serotype-specific diagnosis of recent dengue virus infections in most instances.

There are limited real-world mutational and virological outcomes data of treatment-experienced persons diagnosed with HIV-1 subtype C (HIV-1 C) who are failing Integrase Strand Transfer Inhibitor-based regimens. Requisition forms sent for HIV-1 genotypic resistance testing (GRT) between May 2015 and September 2019 were reviewed and participants experiencing virologic failure while on dolutegravir (DTG) or raltegravir (RAL) cART at sampling recruited. Sanger sequencing of the HIV-1 Pol gene was performed from residual plasma samples and drug resistance mutational (DRM) analysis performed using the Stanford University HIV drug resistance database. 40 HIV-1C integrase sequences were generated from 34 individuals, 24 of whom were on DTG cART, three on RAL cART and seven on an unknown (DTG or RAL)-anchored cART at time of GRT. 11/34 (32%) individuals had DRMs to DTG and other integrase inhibitors. 7/11 (64%) patients had exposure to a RAL-based cART at the time of sampling. Out of the 11 individuals with DRMs, one (9%) had 2-class, 6 (55%) had 3-class, and 4 (36%) had 4-class multidrug-resistant HIV-1C. 7/11 individuals (64%) are currently virologically suppressed. Of the four individuals not virologically suppressed, three had extensive DRMs involving 4-classes of ARV drugs and one individual has demised. Resistance to DTG occurs more often in patients exposed to RAL cART. Individuals with 4-class DRMs plus integrase T97 and E157Q mutations appear to have worse outcomes. There is a need for frequent VL monitoring and GRT amongst treatment-experienced HIV-1C diagnosed individuals.

Middle East respiratory syndrome-related coronavirus (MERS-CoV) is a persistent zoonotic pathogen with frequent spillover from dromedary camels to humans in the Arabian Peninsula, resulting in limited outbreaks of MERS with a high case-fatality rate. Full genome sequence data from camel-derived MERS-CoV variants show diverse lineages circulating in domestic camels with frequent recombination. More than 90% of the available full MERS-CoV genome sequences derived from camels are from just two countries, the Kingdom of Saudi Arabia (KSA) and United Arab Emirates (UAE). In this study, we employ a novel method to amplify and sequence the partial MERS-CoV genome with high sensitivity from nasal swabs of infected camels. We recovered more than 99% of the MERS-CoV genome from field-collected samples with greater than 500 TCID50 equivalent per nasal swab from camel herds sampled in Jordan in May 2016. Our subsequent analyses of 14 camel-derived MERS-CoV genomes show a striking lack of genetic diversity circulating in Jordan camels relative to MERS-CoV genome sequences derived from large camel markets in KSA and UAE. The low genetic diversity detected in Jordan camels during our study is consistent with a lack of endemic circulation in these camel herds and reflective of data from MERS outbreaks in humans dominated by nosocomial transmission following a single introduction as reported during the 2015 MERS outbreak in South Korea. Our data suggest transmission of MERS-CoV among two camel herds in Jordan in 2016 following a single introduction event.

The renal involvement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported. The etiology of kidney injury appears to be tubular, mainly due to the expression of angiotensin-converting enzyme 2, the key joint receptor for SARS-CoV-2; however, cases with glomerular implication have also been documented. The multifactorial origin of this renal involvement could include virus-mediated injury, cytokine storm, angiotensin II pathway activation, complement dysregulation, hyper-coagulation, and microangiopathy. We present the renal histological findings from a patient who developed acute kidney injury and de novo nephrotic syndrome, highly suggestive of acute IgA-dominant infection-associated glomerulonephritis (IgA-DIAGN) after SARS-CoV-2 infection, as evidenced by the presence of this virus detected in the renal tissue of the patient via immunohistochemistry assay. In summary, we document the first case of IgA-DIAGN associated to SARS-CoV-2. Thus, SARS-CoV-2 S may act as a super antigen driving the development of multisystem inflammatory syndrome as well as cytokine storm in patients affected by COVID-19, reaching the glomerulus and leading to the development of this novel IgA-DIAGN.

Rubella virus (RuV) is the causative agent of rubella ("German measles") and remains a global health concern. Until recently, RuV was the only known member of the genus Rubivirus and the only virus species classified within the Matonaviridae family of positive-sense RNA viruses. Recently, two new rubella-like matonaviruses, Rustrela virus and Ruhugu virus, have been identified in several mammalian species, along with more divergent viruses in fish and reptiles. To screen for the presence of additional novel rubella-like viruses, we mined published transcriptome data using genome sequences from Rubella, Rustrela, and Ruhugu viruses as baits. From this, we identified a novel rubella-like virus in a transcriptome of Tetronarce californica-order Torpediniformes (Pacific electric ray)-that is more closely related to mammalian Rustrela virus than to the divergent fish matonavirus and indicative of a complex pattern of cross-species virus transmission. Analysis of host reads confirmed that the sample analysed was indeed from a Pacific electric ray, and two other viruses identified in this animal, from the Arenaviridae and Reoviridae, grouped with other fish viruses. These findings indicate that the evolutionary history of the Matonaviridae is more complex than previously thought and highlights the vast number of viruses that remain undiscovered.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted from person to person by close contact, small aerosol respiratory droplets, and potentially via contact with contaminated surfaces. Herein, we investigated the effectiveness of commercial UVC-LED disinfection boxes in inactivating SARS-CoV-2-contaminated surfaces of personal items. We contaminated glass, metal, and plastic samples representing the surfaces of personal items such as smartphones, coins, or credit cards with SARS-CoV-2 formulated in an organic matrix mimicking human respiratory secretions. For disinfection, the samples were placed at different distances from UVC emitting LEDs inside commercial UVC-LED disinfection boxes and irradiated for different time periods (up to 10 min). High viral loads of SARS-CoV-2 were effectively inactivated on all surfaces after 3 min of irradiation. Even 10 s of UVC-exposure strongly reduced viral loads. Thus, UVC-LED boxes proved to be an effective method for disinfecting SARS-CoV-2-contaminated surfaces that are typically found on personal items.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection 'dead end'.

Jumbo phages have DNA genomes larger than 200 kbp in large virions composed of an icosahedral head, tail, and other adsorption structures, and they are known to be abundant biological substances in nature. In this study, phages in leaf litter compost were screened for their potential to suppress rice seedling rot disease caused by the bacterium Burkholderia glumae, and a novel phage was identified in a filtrate-enriched suspension of leaf litter compost. The phage particles consisted of a rigid tailed icosahedral head and contained a DNA genome of 227,105 bp. The phage could lyse five strains of B. glumae and six strains of Burkholderia plantarii. The phage was named jumbo Burkholderia phage FLC6. Proteomic tree analysis revealed that phage FLC6 belongs to the same clade as two jumbo Ralstonia phages, namely RSF1 and RSL2, which are members of the genus Chiangmaivirus (family: Myoviridae; order: Caudovirales). Interestingly, FLC6 could also lyse two strains of Ralstonia pseudosolanacearum, the causal agent of bacterial wilt, suggesting that FLC6 has a broad host range that may make it especially advantageous as a bio-control agent for several bacterial diseases in economically important crops. The novel jumbo phage FLC6 may enable leaf litter compost to suppress several bacterial diseases and may itself be useful for controlling plant diseases in crop cultivation.

Mastomys natalensis are a ubiquitous and often dominant rodent across sub-Saharan Africa. Importantly, they are a natural reservoir for microbial pathogens including Lassa virus (LASV), the etiological agent of Lassa fever in humans. Lassa-infected rodents have been documented across West Africa and coincide with regions where annual outbreaks occur. Zoonotic transmission to humans most often occurs directly from infected rodents. Little is known about LASV infection kinetics and transmissibility in M.natalensis, primarily due to available animals. Here, we describe the establishment of a laboratory breeding colony of genetically confirmed M.natalensis from wild-captured rodents. This colony will provide a convenient source of animals to study LASV and other emerging pathogens that utilize M. natalensis in their enzootic lifecycles.

Type I interferons (IFNs) are produced by most cells in response to virus infection and stimulate a program of anti-viral gene expression in neighboring cells to suppress virus replication. Type III IFNs have similar properties, however their effects are limited to epithelial cells at mucosal surfaces due to restricted expression of the type III IFN receptor. Rotavirus (RV) replicates in intestinal epithelial cells that respond predominantly to type III IFNs, and it has been shown that type III rather than type I IFNs are important for controlling RV infections in vivo. The RV NSP1 protein antagonizes the host type I IFN response by targeting IRF-3, IRF-5, IRF-7, or β-TrCP for proteasome-mediated degradation in a strain-specific manner. Here we provide the first demonstration that NSP1 proteins from several human and animal RV strains antagonize type III as well as type I IFN induction. We also show that NSP1 is a potent inhibitor of IRF-1, a previously undescribed property of NSP1 which is conserved among human and animal RVs. Interestingly, all NSP1 proteins were substantially more effective inhibitors of IRF-1 than either IRF-3 or IRF-7 which has significance for evasion of basal anti-viral immunity and type III IFN induction in the intestinal epithelium.

The discovery of sodium taurocholate co-transporting polypeptide (NTCP) as a hepatitis B (HBV) and delta virus (HDV) entry receptor has encouraged the development of new animal models of infection. This review provides an overview of the different in vivo models that are currently available to study HDV either in the absence or presence of HBV. By presenting new advances and remaining drawbacks, we will discuss human host factors which, in addition to NTCP, need to be investigated or identified to enable a persistent HDV infection in murine hepatocytes. Detailed knowledge on species-specific factors involved in HDV persistence also shall contribute to the development of therapeutic strategies.

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids.

Echoviruses (E) are a diverse group of viruses responsible for various pathological conditions in humans including aseptic meningitis, myocarditis, and acute flaccid paralysis. The detection and identification of echovirus genotypes in clinical samples is challenging due to its high genetic diversity. Here, we report the complete genome sequences of nine echoviruses, obtained by next-generation sequencing of 238 fecal samples from individuals with gastroenteritis in regions of Brazil. Detected viruses were classified into six genotypes: Three E1 sequences (BRA/TO-028, BRA/TO-069 and BRA/TO-236), one E3 (BRA/TO-018), one E11 (BRA/TO-086), one E20 (BRA/TO-016), two E29 (BRA/TO-030 and BRA/TO-193), and one E30 sequence (BRA/TO-032). Phylogenetic analysis indicated that the echoviruses E1 and E29 circulating in Brazil are divergent from strains circulating worldwide. The genotype diversity identified in our study may under-represent the total echovirus diversity in Brazil because of the small sample size and the restricted geographical distribution covered by the survey.

Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops.

Chronic hepatitis B infection remains a globally important cause of morbidity and mortality and has recently undergone a renaissance in therapeutic interest with increased pre-clinical and clinical testing of new drug classes. One of the first new classes in the clinic was RNA interference agents, which have the potential to impact the entire viral life cycle by reducing all virus-produced mRNA. Early clinical testing with the first of these agents in the clinic, ARC-520, demonstrated that rapid and deep reductions in viral proteins, RNA and DNA could be produced with this approach, but also the surprising insight that HBsAg production from incomplete HBV DNA integrated into the host genome appears to play a heretofore unappreciated and important role in maintaining circulating HBsAg, thought to play a fundamental role in preventing host clearance of the virus. Thus, accounting for viral DNA integration in novel HBV treatment approaches may prove to be essential to achieving successful finite therapies of this difficult to treat chronic infection.

The rapid and dynamic activation of the innate immune system is achieved through complex signaling networks regulated by post-translational modifications modulating the subcellular localization, activity, and abundance of signaling molecules. Many constitutively expressed signaling molecules are present in the cell in inactive forms, and become functionally activated once they are modified with ubiquitin, and, in turn, inactivated by removal of the same post-translational mark. Moreover, upon infection resolution a rapid remodeling of the proteome needs to occur, ensuring the removal of induced response proteins to prevent hyperactivation. This review discusses the current knowledge on the negative regulation of innate immune signaling pathways by deubiquitinating enzymes, and through degradative ubiquitination. It focusses on spatiotemporal regulation of deubiquitinase and E3 ligase activities, mechanisms for re-establishing proteostasis, and degradation through immune-specific feedback mechanisms vs. general protein quality control pathways.

Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of; Pea early browning virus (PEBV), Cucumber mosaic virus (CMV), Bean yellow mosaic virus (BYMV) and Pea seedborne mosaic virus (PSbMV). TG-Seq detected all the expected viral amplicons within multiplex PCR (mPCR) reactions. In contrast, the expected PCR amplicons were not detected by gel electrophoresis (GE). For example, for CMV, GE only detected RNA1 and RNA2 while TG-Seq detected all the three RNA components of CMV. In an mPCR to amplify all four viruses, TG-Seq readily detected each virus with more than 732,277 sequence reads mapping to each amplicon. In addition, TG-Seq also detected all four amplicons within a 10-8 serial dilution that were not detectable by GE. Our current findings reveal that the TG-Seq approach offers significant potential and is a highly sensitive targeted approach for detecting multiple plant viruses within a given biological sample. This is the first study describing direct HTS of plant virus mPCR products. These findings have major implications for grain germplasm healthy certification programs and biosecurity management in relation to pathogen entry into Australia and elsewhere.

Feline morbilliviruses (FeMV) are fairly newly discovered paramyxoviruses found in cats. The first description indicated an association with widely distributed chronic kidney disease (CKD) in the host species. In various studies, a global prevalence and a further genotype, designated FeMV-2, and the involvement of other organ systems in infected individuals were shown. Using an immunofluorescence assay, we detected an overall seroprevalence of FeMV in almost half of the cats investigated (n = 380), with a significantly increased proportion in younger animals. In comparison to European Shorthair cats, the rate of seropositivity is higher in pedigree cats. Regardless of the breed, FeMV infection was associated with increased blood creatinine concentrations, suggesting an association with CKD. Further analysis indicated that this association was the strongest in animals having high IFA titers against FeMV-2. In addition, a significant association between FeMV-positive status and the prevalence of feline lower urinary tract disease (FLUTD, or idiopathic cystitis) was detected. This association was dominated by cats having antibodies against FeMV-1 only. To further evaluate the positive correlation between FeMV seroprevalence and CKD as well as FLUTD, consideration of additional clinical characteristics and laboratory parameters is warranted, and controlled infection studies with both FeMV genotypes are necessary. Clinicians should, however, be aware of a possible link between renal and lower urinary tract disease and FeMV infections.

African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Short-read sequencing (SRS) can produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for comprehensively annotating transcriptomes. Long-read sequencing (LRS) can overcome some of SRS's limitations, but it also has drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the ASFV transcriptomic atlas. This work identified many novel transcripts and transcript isoforms and annotated the precise termini of previously described RNAs. This study identified a novel species of ASFV transcripts, the replication origin-associated RNAs. Additionally, we discovered several nested genes embedded into larger canonical genes. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism. A multifaceted meshwork of transcriptional overlaps was also discovered.

The poly-adenosine diphosphate (ADP)-ribose polymerases (PARPs) are responsible for ADP-ribosylation, a reversible post-translational modification involved in many cellular processes including DNA damage repair, chromatin remodeling, regulation of translation and cell death. In addition to these physiological functions, recent studies have highlighted the role of PARPs in host defenses against viruses, either by direct antiviral activity, targeting certain steps of virus replication cycle, or indirect antiviral activity, via modulation of the innate immune response. This review focuses on the antiviral activity of PARPs, as well as strategies developed by viruses to escape their action.

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. Owing to the lack of effective vaccines and specific therapeutic options for PEDV, it is pertinent to develop new and available antivirals. This study identified, for the first time, a salinomycin that actively inhibited PEDV replication in Vero cells in a dose-dependent manner. Furthermore, salinomycin significantly inhibited PEDV infection by suppressing the entry and post-entry of PEDV in Vero cells. It did not directly interact with or inactivate PEDV particles, but it significantly ameliorated the activation of Erk1/2, JNK and p38MAPK signaling pathways that are associated with PEDV infection. This implied that salinomycin inhibits PEDV replication by altering MAPK pathway activation. Notably, the PEDV induced increase in reactive oxidative species (ROS) was not decreased, indicating that salinomycin suppresses PEDV replication through a pathway that is an independent pathway of viral-induced ROS. Therefore, salinomycin is a potential drug that can be used for treating PEDV infection.

Molecular cluster detection analyzes HIV sequences to identify rapid HIV transmission and inform public health responses. We describe changes in the capability to detect molecular clusters and in geographic variation in transmission dynamics. We examined the reporting completeness of HIV-1 polymerase sequences in quarterly National HIV Surveillance System datasets from December 2015 to December 2019. Priority clusters were identified quarterly. To understand populations recently affected by rapid transmission, we described the transmission risk and race/ethnicity of people in clusters first detected in 2018-2019. During December 2015 to December 2019, national sequence completeness increased from 26% to 45%. Of the 1212 people in the 136 clusters first detected in 2018-2019, 69% were men who have sex with men (MSM) and 11% were people who inject drugs (PWID). State-by-state analysis showed substantial variation in transmission risk and racial/ethnic groups in clusters of rapid transmission. HIV sequence reporting has increased nationwide. Molecular cluster analysis identifies rapid transmission in varied populations and identifies emerging patterns of rapid transmission in specific population groups, such as PWID, who, in 2015-2016, comprised only 1% of people in such molecular clusters. These data can guide efforts to focus, tailor, and scale up prevention and care services for these populations.

The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues (i.e., 125, 176, 192) in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability.

BACKGROUND: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. METHODS: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). RESULTS: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. CONCLUSIONS: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB.

The recently emerged SARS-CoV-2 virus is responsible for the ongoing COVID-19 pandemic that has rapidly developed into a global public health threat. Patients severely affected with COVID-19 present distinct clinical features, including acute respiratory disorder, neutrophilia, cytokine storm, and sepsis. In addition, multiple pro-inflammatory cytokines are found in the plasma of such patients. Transcriptome sequencing of different specimens obtained from patients suffering from severe episodes of COVID-19 shows dynamics in terms of their immune responses. However, those host factors required for SARS-CoV-2 propagation and the underlying molecular mechanisms responsible for dysfunctional immune responses during COVID-19 infection remain elusive. In the present study, we analyzed the mRNA-long non-coding RNA (lncRNA) co-expression network derived from publicly available SARS-CoV-2-infected transcriptome data of human lung epithelial cell lines and bronchoalveolar lavage fluid (BALF) from COVID-19 patients. Through co-expression network analysis, we identified four differentially expressed lncRNAs strongly correlated with genes involved in various immune-related pathways crucial for cytokine signaling. Our findings suggest that the aberrant expression of these four lncRNAs can be associated with cytokine storms and anti-viral responses during severe SARS-CoV-2 infection of the lungs. Thus, the present study uncovers molecular interactions behind the cytokine storm activation potentially responsible for hyper-inflammatory responses in critical COVID-19 patients.

Mass mortality events of common carp (Cyprinus carpio, carp) associated with carp edema virus (CEV) alone or in coinfections with koi herpesvirus (KHV), is an emerging issue. Despite recent outbreaks of CEV in wild carp populations, the host range of North American species has not been well studied. To that end, we intensively sampled carp (n = 106) and co-habiting native fish species (n = 5 species; n = 156 total fish) from a CEV-suspect mass-mortality event of carp in a small Minnesota lake (Lake Swartout). Additionally, fecal and regurgitant samples (n = 73 each) from double-crested cormorants (Phalacrocorax auritus, DCCO) were sampled to test the potential of DCCO to act as a vector for virus transmission. CEV was confirmed to be widespread in the Lake Swartout carp population during the outbreak with high viral loads and histological confirmation, suggesting that CEV was the cause of the mortality event. There were no detections of CEV in any native fish species; however, DCCO regurgitant and fecal samples were positive for CEV DNA. In addition, three CEV-positive and one CEV + KHV-positive mortality events were confirmed with no observed mortality or morbidity of non-carp species in other lakes. This study provides evidence that CEV infection and disease may be specific to carp during mortality events with mixed-species populations, identifies DCCO as a potential vector for CEV, and further expands the known range of CEV, as well as coinfections with KHV, in North America.

(1) Background: Thailand has made significant progress in reducing the number of human and animal rabies cases. However, control and elimination of the last remaining pockets of dog-mediated rabies have shown to be burdensome, predominantly as a result of the large numbers of free-roaming dogs without an owner that cannot be restrained without special efforts and therefore remain unvaccinated. To reach these dogs, the feasibility, and benefits of oral rabies vaccination (ORV) as a complementary tool has been examined under field conditions. (2) Methods: ORV of dogs was tested in five study areas of four provinces in Thailand. In these areas, sites with free-roaming dogs were identified with the support of local municipal workers and dog caretakers. ORV teams visited each of five study areas and distributed rabies vaccine (SPBN GASGAS) in three bait formats that were offered to the dogs using a hand-out and retrieval model. The three bait types tested included: egg-flavored baits, egg-flavored baits pasted with commercially available cat liquid snack, and boiled-intestine baits. A dog offered a vaccine bait was considered vaccinated when the discarded sachet was perforated or if a dog chewed vaccine bait at least 5 times before it swallowed the bait, including the sachet. (3) Results: A total of 2444 free-roaming dogs considered inaccessible for parenteral vaccination were identified at 338 sites. As not all dogs were approachable, 79.0% were offered a bait; of these dogs, 91.6% accepted the bait and subsequently 83.0% were considered successfully vaccinated. (4) Conclusion: Overall, 65.6% of the free-roaming dogs at these sites were successfully vaccinated by the oral route. Such a significant increase of the vaccination coverage of the free-roaming dog population could interrupt the rabies transmission cycle and offers a unique opportunity to reach the goal to eliminate dog-mediated human rabies in Thailand by 2030.

Lagos bat virus (LBV), one of the 17 accepted viral species of the Lyssavirus genus, was the first rabies-related virus described in 1956. This virus is endemic to the African continent and is rarely encountered. There are currently four lineages, although the observed genetic diversity exceeds existing lyssavirus species demarcation criteria. Several exposures to rabid bats infected with LBV have been reported; however, no known human cases have been reported to date. This review provides the history of LBV and summarizes previous knowledge as well as new detections. Genetic diversity, pathogenesis and prevention are re-evaluated and discussed.

The immune system defends against invading pathogens through the rapid activation of innate immune signaling pathways. Interferon regulatory factor 3 (IRF3) is a key transcription factor activated in response to virus infection and is largely responsible for establishing an antiviral state in the infected host. Studies in Irf3-/- mice have demonstrated the absence of IRF3 imparts a high degree of susceptibility to a wide range of viral infections. Virus infection causes the activation of IRF3 to transcribe type-I interferon (e.g., IFNβ), which is responsible for inducing the interferon-stimulated genes (ISGs), which act at specific stages to limit virus replication. In addition to its transcriptional function, IRF3 is also activated to trigger apoptosis of virus-infected cells, as a mechanism to restrict virus spread within the host, in a pathway called RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA). These dual functions of IRF3 work in concert to mediate protective immunity against virus infection. These two pathways are activated differentially by the posttranslational modifications (PTMs) of IRF3. Moreover, PTMs regulate not only IRF3 activation and function, but also protein stability. Consequently, many viruses utilize viral proteins or hijack cellular enzymes to inhibit IRF3 functions. This review will describe the PTMs that regulate IRF3's RIPA and transcriptional activities and use coronavirus as a model virus capable of antagonizing IRF3-mediated innate immune responses. A thorough understanding of the cellular control of IRF3 and the mechanisms that viruses use to subvert this system is critical for developing novel therapies for virus-induced pathologies.

The genus Flavivirus contains pathogenic vertebrate-infecting flaviviruses (VIFs) and insect-specific flaviviruses (ISF). ISF transmission to vertebrates is inhibited at multiple stages of the cellular infection cycle, via yet to be elucidated specific antiviral responses. The zinc-finger antiviral protein (ZAP) in vertebrate cells can bind CpG dinucleotides in viral RNA, limiting virus replication. Interestingly, the genomes of ISFs contain more CpG dinucleotides compared to VIFs. In this study, we investigated whether ZAP prevents two recently discovered lineage II ISFs, Binjari (BinJV) and Hidden Valley viruses (HVV) from replicating in vertebrate cells. BinJV protein and dsRNA replication intermediates were readily observed in human ZAP knockout cells when cultured at 34 °C. In ZAP-expressing cells, inhibition of the interferon response via interferon response factors 3/7 did not improve BinJV protein expression, whereas treatment with kinase inhibitor C16, known to reduce ZAP's antiviral function, did. Importantly, at 34 °C, both BinJV and HVV successfully completed the infection cycle in human ZAP knockout cells evident from infectious progeny virus in the cell culture supernatant. Therefore, we identify vertebrate ZAP as an important barrier that protects vertebrate cells from ISF infection. This provides new insights into flavivirus evolution and the mechanisms associated with host switching.

The natural hosts of Orthohantaviruses are rodents, soricomorphs and bats, and it is well known that they may cause serious or even fatal diseases among humans worldwide. The virus is persistent among animals and it is shed via urine, saliva and feces throughout the entirety of their lives. We aim to identify the effectiveness of hantavirus detection in rodent tissue samples and urine originating from naturally infected rodents. Initially, animals were trapped at five distinct locations throughout the Transdanubian region in Hungary. Lung, liver, kidney and urine samples were obtained from 163 deceased animals. All organs and urine were tested using nested reverse transcription polymerase chain reaction (nRT-PCR). Furthermore, sera were examined for IgG antibodies against Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) by Western blot assay. IgG antibodies against hantaviruses and/or nucleic acid were detected in 25 (15.3%) cases. Among Apodemus, Myodes, and Microtus rodent species, DOBV, PUUV and Tula virus (TULV) were clearly identified. Amid the PCR-positive samples, the nucleic acid of the viruses was detected most effectively in the kidney (100%), while only 55% of screened lung tissues were positive. Interestingly, only three out of 20 rodent urine samples were positive when tested using nRT-PCR. Moreover, five rodents were seropositive without detectable virus nucleic acid in any of the tested organs.

The Covid-19 pandemic has required nonpharmaceutical interventions, primarily physical distancing, personal hygiene and face mask use, to limit community transmission, irrespective of seasons. In fact, the seasonality attributes of this pandemic remain one of its biggest unknowns. Early studies based on past experience from respiratory diseases focused on temperature or humidity, with disappointing results. Our hypothesis that ultraviolet (UV) radiation levels might be a factor and a more appropriate parameter has emerged as an alternative to assess seasonality and exploit it for public health policies. Using geographical, socioeconomic and epidemiological criteria, we selected twelve North-equatorial-South countries with similar characteristics. We then obtained UV levels, mobility and Covid-19 daily incidence rates for nearly the entire 2020. Using machine learning, we demonstrated that UV radiation strongly associated with incidence rates, more so than mobility did, indicating that UV is a key seasonality indicator for Covid-19, irrespective of the initial conditions of the epidemic. Our findings can inform the implementation of public health emergency measures, partly based on seasons in the Northern and Southern Hemispheres, as the pandemic unfolds into 2021.

Plant viruses can be genetically modified to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo mosaic virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.

The eradication of infectious diseases has been achieved only once in history, in 1980, with smallpox. Since 1988, significant effort has been made to eliminate poliomyelitis viruses, but eradication is still just out of reach. As the goal of viral disease eradication approaches, the ability to recreate historically eradicated viruses using synthetic biology has the potential to jeopardize the long-term sustainability of eradication. However, the emergence of the severe acute respiratory syndrome-coronavirus (SARS-CoV)-2 pandemic has highlighted our ability to swiftly and resolutely respond to a potential outbreak. This virus has been synthetized faster than any other in the past and is resulting in vaccines before most attenuated candidates reach clinical trials. Here, synthetic biology has the opportunity to demonstrate its truest potential to the public and solidify a footing in the world of vaccines.

Phage display technology involves the surface genetic engineering of phages to expose desirable proteins or peptides whose gene sequences are packaged within phage genomes, thereby rendering direct linkage between genotype with phenotype feasible. This has resulted in phage display systems becoming invaluable components of directed evolutionary biotechnology. The M13 is a DNA phage display system which dominates this technology and usually involves selected proteins or peptides being displayed through surface engineering of its minor coat proteins. The displayed protein or peptide's functionality is often highly reduced due to harsh treatment of M13 variants. Recently, we developed a novel phage display system using the coliphage Qβ as a nano-biotechnology platform. The coliphage Qβ is an RNA phage belonging to the family of Leviviridae, a long investigated virus. Qβ phages exist as a quasispecies and possess features making them comparatively more suitable and unique for directed evolutionary biotechnology. As a quasispecies, Qβ benefits from the promiscuity of its RNA dependent RNA polymerase replicase, which lacks proofreading activity, and thereby permits rapid variant generation, mutation, and adaptation. The minor coat protein of Qβ is the readthrough protein, A1. It shares the same initiation codon with the major coat protein and is produced each time the ribosome translates the UGA stop codon of the major coat protein with the of misincorporation of tryptophan. This misincorporation occurs at a low level (1/15). Per convention and definition, A1 is the target for display technology, as this minor coat protein does not play a role in initiating the life cycle of Qβ phage like the pIII of M13. The maturation protein A2 of Qβ initiates the life cycle by binding to the pilus of the F+ host bacteria. The extension of the A1 protein with a foreign peptide probe recognizes and binds to the target freely, while the A2 initiates the infection. This avoids any disturbance of the complex and the necessity for acidic elution and neutralization prior to infection. The combined use of both the A1 and A2 proteins of Qβ in this display system allows for novel bio-panning, in vitro maturation, and evolution. Additionally, methods for large library size construction have been improved with our directed evolutionary phage display system. This novel phage display technology allows 12 copies of a specific desired peptide to be displayed on the exterior surface of Qβ in uniform distribution at the corners of the phage icosahedron. Through the recently optimized subtractive bio-panning strategy, fusion probes containing up to 80 amino acids altogether with linkers, can be displayed for target selection. Thus, combined uniqueness of its genome, structure, and proteins make the Qβ phage a desirable suitable innovation applicable in affinity maturation and directed evolutionary biotechnology. The evolutionary adaptability of the Qβ phage display strategy is still in its infancy. However, it has the potential to evolve functional domains of the desirable proteins, glycoproteins, and lipoproteins, rendering them superior to their natural counterparts.

The hepatitis B virus (HBV) capsid is an attractive drug target, relevant to combating viral hepatitis as a major public health concern. Among small molecules known to interfere with capsid assembly, the phenylpropenamides, including AT130, represent an important antiviral paradigm based on disrupting the timing of genome packaging. Here, all-atom molecular dynamics simulations of an intact AT130-bound HBV capsid reveal that the compound increases spike flexibility and improves recovery of helical secondary structure in the spike tips. Regions of the capsid-incorporated dimer that undergo correlated motion correspond to established sub-domains that pivot around the central chassis. AT130 alters patterns of correlated motion and other essential dynamics. A new conformational state of the dimer is identified, which can lead to dramatic opening of the intradimer interface and disruption of communication within the spike tip. A novel salt bridge is also discovered, which can mediate contact between the spike tip and fulcrum even in closed conformations, revealing a mechanism of direct communication across these sub-domains. Altogether, results describe a dynamical connection between the intra- and interdimer interfaces and enable mapping of allostery traversing the entire core protein dimer.

Monoclonal antibodies represent an important avenue for COVID-19 therapy and are routinely used for rapid and accessible diagnosis of SARS-CoV-2 infection. The recent emergence of SARS-CoV-2 genetic variants emphasized the need to enlarge the repertoire of antibodies that target diverse epitopes, the combination of which may improve immune-diagnostics, augment the efficiency of the immunotherapy and prevent selection of escape-mutants. Antigen-specific controlled immunization of experimental animals may elicit antibody repertoires that significantly differ from those generated in the context of the immune response mounted in the course of disease. Accordingly, rabbits were immunized by several recombinant antigens representing distinct domains of the viral spike protein and monoclonal antibodies were isolated from single cells obtained by cell sorting. Characterization of a panel of successfully isolated anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies demonstrated that they exhibit high specificity and affinity profiles. Anti-RBD antibodies revealing significant neutralizing potency against SARS-CoV-2 in vitro were found to target at least three distinct epitopes. Epitope mapping established that two of these antibodies recognized a novel epitope located on the surface of the RBD. We suggest that the antibodies isolated in this study are useful for designing SARS-CoV-2 diagnosis and therapy approaches.

This study is focused on the epidemiological characteristics and biomolecular mechanisms that lead to the development of precancerous and cancerous conditions of oral lesions related to Human Papilloma Virus (HPV) infections. Current evidence from the literature demonstrates the role of HPV in potentially malignant oral disorders. Therefore, the underlying biomolecular processes can give arise, or contribute to, benign lesions as well as to oral carcinogenesis.

A central feature of vertebrate immune systems is the ability to form antigen-specific immune memory in response to microbial challenge and so provide protection against future infection. In conflict with this process is the ability that many viruses have to mutate their antigens to escape infection- or vaccine-induced antibody memory responses. Mutable viruses such as dengue virus, influenza virus and of course coronavirus have a major global health impact, exacerbated by this ability to evade immune responses through mutation. There have been several outstanding recent studies on B-cell memory that also shed light on the potential and limitations of antibody memory to protect against viral antigen variation, and so promise to inform new strategies for vaccine design. For the purposes of this review, the current understanding of the different memory B-cell (MBC) populations, and their potential to recognize mutant antigens, will be described prior to some examples from antibody responses against the highly mutable RNA based flaviviruses, influenza virus and SARS-CoV-2.

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.

The cold season is usually accompanied by an increased incidence of respiratory infections and increased air pollution from combustion sources. As we are facing growing numbers of COVID-19 cases caused by the novel SARS-CoV-2 coronavirus, an understanding of the impact of air pollutants and meteorological variables on the incidence of respiratory infections is crucial. The incidence of influenza-like illness (ILI) can be used as a close proxy for the circulation of influenza viruses. Recently, SARS-CoV-2 has also been detected in patients with ILI. Using distributed lag nonlinear models, we analyzed the association between ILI, meteorological variables and particulate matter concentration in Bialystok, Poland, from 2013-2019. We found an exponential relationship between cumulative PM2.5 pollution and the incidence of ILI, which remained significant after adjusting for air temperatures and a long-term trend. Pollution had the greatest effect during the same week, but the risk of ILI was increased for the four following weeks. The risk of ILI was also increased by low air temperatures, low absolute humidity, and high wind speed. Altogether, our results show that all measures implemented to decrease PM2.5 concentrations would be beneficial to reduce the transmission of SARS-CoV-2 and other respiratory infections.

In this Special Issue, a wide variety of original and review articles provide a timely overview of how viruses are recognized by and evade from cellular innate immunity, which represents the first line of defense against viruses [...].

Diverse paramyxoviruses have coevolved with their bat hosts, including fruit bats such as flying foxes (Chiroptera: Pteropodidae). Several of these viruses are zoonotic, but the diversity and distribution of Paramyxoviridae are poorly understood. We screened pooled feces samples from three Pteropus vampyrus colonies and assayed tissues, rectal swabs, and oral swabs from 95 individuals of 23 pteropodid species sampled at 17 sites across the Indonesian archipelago with a conventional paramyxovirus PCR; all tested negative. Samples from 43 individuals were screened with next generation sequencing (NGS), and a single Pteropus vampyrus collected near Flores had Tioman virus sequencing reads. Tioman virus is a bat-borne virus in the genus Pararubulavirus with prior evidence of spillover to humans. This work expands the known range of Tioman virus, and it is likely that this isolated colony likely has sustained intergenerational transmission over a long period.

Coronaviruses are a large family of zoonotic RNA viruses, whose infection can lead to mild or lethal respiratory tract disease. Severe Acute Respiratory Syndrome-Coronavirus-1 (SARS-CoV-1) first emerged in Guangdong, China in 2002 and spread to 29 countries, infecting 8089 individuals and causing 774 deaths. In 2012, Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) emerged in Saudi Arabia and has spread to 27 countries, with a mortality rate of ~34%. In 2019, SARS-CoV-2 emerged and has spread to 220 countries, infecting over 100,000,000 people and causing more than 2,000,000 deaths to date. These three human coronaviruses cause diseases of varying severity. Most people develop mild, common cold-like symptoms, while some develop acute respiratory distress syndrome (ARDS). The success of all viruses, including coronaviruses, relies on their evolved abilities to evade and modulate the host anti-viral and pro-inflammatory immune responses. However, we still do not fully understand the transmission, phylogeny, epidemiology, and pathogenesis of MERS-CoV and SARS-CoV-1 and -2. Despite the rapid application of a range of therapies for SARS-CoV-2, such as convalescent plasma, remdesivir, hydroxychloroquine and type I interferon, no fully effective treatment has been determined. Remarkably, COVID-19 vaccine research and development have produced several offerings that are now been administered worldwide. Here, we summarise an up-to-date understanding of epidemiology, immunomodulation and ongoing anti-viral and immunosuppressive treatment strategies. Indeed, understanding the interplay between coronaviruses and the anti-viral immune response is crucial to identifying novel targets for therapeutic intervention, which may even prove invaluable for the control of future emerging coronavirus.

The 2019 coronavirus infectious disease (COVID-19) is caused by infection with the new severe acute respiratory syndrome coronavirus (SARS-CoV-2). Currently, the treatment options for COVID-19 are limited. The purpose of the experiments presented here was to investigate the effectiveness of ketotifen, naproxen and indomethacin, alone or in combination, in reducing SARS-CoV-2 replication. In addition, the cytotoxicity of the drugs was evaluated. The findings showed that the combination of ketotifen with indomethacin (SJP-002C) or naproxen both reduce viral yield. Compared to ketotifen alone (60% inhibition at EC50), an increase in percentage inhibition of SARS-CoV-2 to 79%, 83% and 93% was found when co-administered with 25, 50 and 100 μM indomethacin, respectively. Compared to ketotifen alone, an increase in percentage inhibition of SARS-CoV-2 to 68%, 68% and 92% was found when co-administered with 25, 50 and 100 μM naproxen, respectively. For both drug combinations the observations suggest an additive or synergistic effect, compared to administering the drugs alone. No cytotoxic effects were observed for the administered dosages of ketotifen, naproxen, and indomethacin. Further research is warranted to investigate the efficacy of the combination of ketotifen with indomethacin (SJP-002C) or naproxen in the treatment of SARS-CoV-2 infection in humans.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-CoV-2 RNA is an essential test to monitor the occurrence of COVID-19. A methodology is proposed for the determination of maximum pool size and adjustments of cut-off values of cycle threshold (Ct in RT-qPCR pool testing, to compensate for the dilution caused by pooling. The trade-off between pool size and test sensitivity is stated explicitly. The procedure was designed to ensure that samples that would be detectable in individual testing remain detectable in pool testing. The proposed relaxation in cut-off is dependent on the pool size, allowing a relatively tight correction to avoid loss of detection of positive samples. The methodology was evaluated in a study of pool testing of adults attending a public emergency care unit, reference for COVID-19 in Belo Horizonte, Brazil, and presenting flu-like symptoms. Even samples on the edge of detectability in individual testing were detected correctly. The proposed procedure enhances the consistency of RT-qPCR pool testing by enforcing that the scales of detectability in pool processing and in individual sample processing are compatible. This may enhance the contribution of pool testing to large-scale testing for COVID-19.

The discovery of the Australia Antigen in the mid-1960s led, in a few years, to the identification of the virus of Hepatitis B [...].

Intrauterine transmission of the Chikungunya virus (CHIKV) during early pregnancy has rarely been reported, although vertical transmission has been observed in newborns. Here, we report four cases of spontaneous abortion in women who became infected with CHIKV between the 11th and 17th weeks of pregnancy. Laboratorial confirmation of the infection was conducted by RT-PCR on a urine sample for one case, and the other three were by detection of IgM anti-CHIKV antibodies. Hematoxylin and eosin (H&E) staining and an electron microscopy assay allowed us to find histopathological, such as inflammatory infiltrate in the decidua and chorionic villi, as well as areas of calcification, edema and the deposition of fibrinoid material, and ultrastructural changes, such as mitochondria with fewer cristae and ruptured membranes, endoplasmic reticulum with dilated cisterns, dispersed chromatin in the nuclei and the presence of an apoptotic body in case 1. In addition, by immunohistochemistry (IHC), we found a positivity for the anti-CHIKV antibody in cells of the endometrial glands, decidual cells, syncytiotrophoblasts, cytotrophoblasts, Hofbauer cells and decidual macrophages. Electron microscopy also helped in identifying virus-like particles in the aborted material with a diameter of 40-50 nm, which was consistent with the size of CHIKV particles in the literature. Our findings in this study suggest early maternal fetal transmission, adding more evidence on the role of CHIKV in fetal death.

The Asian tiger mosquito Aedes albopictus is contributing to the (re)-emergence of Chikungunya virus (CHIKV). To gain insights into the molecular underpinning of viral persistence, which renders a mosquito a life-long vector, we coupled small RNA and whole genome sequencing approaches on carcasses and ovaries of mosquitoes sampled 14 days post CHIKV infection and investigated the profile of small RNAs and the presence of vDNA fragments. Since Aedes genomes harbor nonretroviral Endogenous Viral Elements (nrEVEs) which confers tolerance to cognate viral infections in ovaries, we also tested whether nrEVEs are formed after CHIKV infection. We show that while small interfering (si)RNAs are evenly distributed along the full viral genome, PIWI-interacting (pi)RNAs mostly arise from a ~1000 bp window, from which a unique vDNA fragment is identified. CHIKV infection does not result in the formation of new nrEVEs, but piRNAs derived from existing nrEVEs correlate with differential expression of an endogenous transcript. These results demonstrate that all three RNAi pathways contribute to the homeostasis during the late stage of CHIKV infection, but in different ways, ranging from directly targeting the viral sequence to regulating the expression of mosquito transcripts and expand the role of nrEVEs beyond immunity against cognate viruses.

Bats are natural reservoirs for many viruses, including several that are zoonotic. Two unusual H17N10 and H18N11 influenza viruses have been found in New World bats. Although neither of these viruses have been isolated, infectious clone technology has permitted significant progress to understand their biology, which include unique features compared to all other known influenza A viruses. In addition, an H9N2-like influenza A virus was isolated from Old World bats and it shows similar characteristics of normal influenza A viruses. In this review, current status and perspective on influenza A viruses identified in bats is reviewed and discussed.

The results of experimental and clinical trials of the agents based on oncolytic Newcastle disease virus (NDV) strains provided hope for the development of virotherapy as a promising method for treating human tumors. However, the mechanism of the antitumor effect of NDV and realization of its cytotoxic potential in a cancer cell remains to be elucidated. In the current work, we have studied the antitumor effect of NDV in a syngeneic model of mouse Krebs-2 carcinoma treated with intratumoral injections of a wild-type strain NDV/Altai/pigeon/770/2011. Virological methods were used for preparation of a virus-containing sample. Colorimetric MTS assay was used to assess the viability of Krebs-2 tumor cells infected with a viral strain in vitro. In vivo virotherapy was performed in eight-week-old male BALB/c mice treated with serial intratumoral injections of NDV in an experimental model of Krebs-2 solid carcinoma. Changes in the tumor nodes of Krebs-2 carcinoma after virotherapy were visualized by MRI and immunohistological staining. Light microscopy examination, immunohistochemical and morphometric analyses have shown that intratumoral viral injections contribute to the inhibition of tumor growth, appearance of necrosis-like changes in the tumor tissue and the antiangiogenic effect of the virus. It has been established that a course of intratumoral virotherapy with NDV/Altai/pigeon/770/2011 strain in a mouse Krebs-2 carcinoma resulted in increased destructive changes in the tumor tissue, in the volume density of necrotic foci and numerical density of endothelial cells expressing CD34 and VEGFR. These results indicate that intratumoral NDV injection reduces tumor progression of an aggressive tumor.

Update of bioRxiv. 2021 Feb 10;:

Hemorrhagic disease (HD) is considered one of the most significant infectious diseases of white-tailed deer in North America. Investigations into environmental conditions associated with outbreaks suggest drought conditions are strongly correlated with outbreaks in some regions of the United States. However, during 2017, an HD outbreak occurred in the Eastern United States which appeared to be associated with a specific physiographic region, the Appalachian Plateau, and not drought conditions. The objective of this study was to determine if reported HD in white-tailed deer in 2017 was correlated with physiographic region. There were 456 reports of HD from 1605 counties across 26 states and 12 physiographic regions. Of the 93 HD reports confirmed by virus isolation, 76.3% (71/93) were identified as EHDV-2 and 66.2% (47/71) were from the Appalachian Plateau. A report of HD was 4.4 times more likely to occur in the Appalachian Plateau than not in 2017. Autologistic regression models suggested a statistically significant spatial dependence. The underlying factors explaining this correlation are unknown, but may be related to a variety of host, vector, or environmental factors. This unique outbreak and its implications for HD epidemiology highlight the importance for increased surveillance and reporting efforts in the future.

Novel SARS-CoV-2 variants with potential impacts on diagnostics, antivirals, and vaccines are spreading in Italy. In this editorial, we highlight the role that veterinary public health institutes may have in this global crisis, as their expertise in genomic/antigenic surveillance and animal studies are crucial to tackle SARS-CoV-2 pandemic.

Sargassum fusiforme, a plant used as a medicine and food, is regarded as a marine vegetable and health supplement to improve life expectancy. Here, we demonstrate that S. fusiforme extract (SFE) has antiviral effects against respiratory syncytial virus (RSV) in vitro and in vivo mouse model. Treatment of HEp2 cells with a non-cytotoxic concentration of SFE significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and syncytium formation. Moreover, oral inoculation of SFE significantly improved RSV clearance from the lungs of BALB/c mice. Interestingly, the phenolic compounds eicosane, docosane, and tetracosane were identified as active components of SFE. Treatment with a non-cytotoxic concentration of these three components elicited similar antiviral effects against RSV infection as SFE in vitro. Together, these results suggest that SFE and its potential components are a promising natural antiviral agent candidate against RSV infection.

Influenza virus, a highly mutable respiratory pathogen, causes significant disease nearly every year. Current vaccines are designed to protect against circulating influenza strains of a given season. However, mismatches between vaccine strains and circulating strains, as well as inferior vaccine effectiveness in immunodeficient populations, represent major obstacles. In an effort to expand the breadth of protection elicited by influenza vaccination, one of the major surface glycoproteins, hemagglutinin (HA), has been modified to develop immunogens that display conserved regions from multiple viruses or elicit a highly polyclonal antibody response to broaden protection. These approaches, which target either the head or the stalk domain of HA, or both domains, have shown promise in recent preclinical and clinical studies. Furthermore, the role of adjuvants in bolstering the robustness of the humoral response has been studied, and their effects on the vaccine-elicited antibody repertoire are currently being investigated. This review will discuss the progress made in the universal influenza vaccine field with respect to influenza A viruses from the perspectives of both antigen and adjuvant, with a focus on the elicitation of broadly neutralizing antibodies.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes an important disease in ruminants, with great economic losses. The infection can be also transmitted to humans; therefore, it is considered a major threat to both human and animal health. In a previous work, we described a novel RVFV variant selected in cell culture in the presence of the antiviral agent favipiravir that was highly attenuated in vivo. This variant displayed 24 amino acid substitutions in different viral proteins when compared to its parental viral strain, two of them located in the NSs protein that is known to be the major virulence factor of RVFV. By means of a reverse genetics system, in this work we have analyzed the effect that one of these substitutions, P82L, has in viral attenuation in vivo. Rescued viruses carrying this single amino acid change were clearly attenuated in BALB/c mice while their growth in an interferon (IFN)-competent cell line as well as the production of interferon beta (IFN-β) did not seem to be affected. However, the pattern of nuclear NSs accumulation was modified in cells infected with the mutant viruses. These results highlight the key role of the NSs protein in the modulation of viral infectivity.

Human parvovirus (B19V) is the causative agent of erythema infectiosum in children and is linked to a wide range of clinical manifestations. Studies related to B19V prevalence in the Middle East and North Africa (MENA) region and other parts of Asia are very scarce. The objectives of this study were to estimate the seroprevalence (anti-B19V IgM and IgG), the viremia rate (B19V DNA), and the circulating genotypes of B19V among blood donors in Qatar. METHODS: Donors' blood samples (n = 5026) from different nationalities, mainly from the MENA region and South East Asia, were collected from 2014-2016. Samples were tested for the B19V DNA using RT-PCR. Furthermore, 1000 selected samples were tested to determine the seroprevalence of B19V antibodies using enzyme-linked immunosorbent assay (ELISA). Genotyping was performed on 65 DNA positive samples by sequencing of nested PCR fragments (NS1-VP1u region, 927 nt). RESULTS: Only 1.4% (70/5026) of the samples had detectible B19V DNA in their blood. B19V DNA prevalence statistically decreased with age (p = 0.03). Anti-B19V IgG was detected in 60.3% (561/930) of the tested samples, while only 2.1% (20/930) were IgM-positive and 1.2% (11/930) were both IgM- and IgG-positive. B19V genotyping showed a predominance of Genotype 1 (100%). Sequence analysis of the NS1-VP1u region revealed 139 mutation sites, some of which were amino acid substitutions. CONCLUSION: Our results indicated a relatively high seroprevalence of B19V in Qatar. Most importantly, B19 DNA was detected among Qatari and non-Qatari blood donors. Therefore, blood banks in Qatar might need to consider screening for B19V, especially when transfusion is intended for high-risk populations, including immunocompromised patients.

Several life-threatening viruses have recently appeared, including the coronavirus, infecting a variety of human and animal hosts and causing a range of diseases like human upper respiratory tract infections. They not only cause serious human and animal deaths, but also cause serious public health problems worldwide. Currently, seven species are known to infect humans, namely SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1. The coronavirus nonstructural protein 16 (NSP16) structure is similar to the 5'-end capping system of mRNA used by eukaryotic hosts and plays a vital role in evading host immunity response and protects the nascent viral mRNA from degradation. NSP16 is also well-conserved among related coronaviruses and requires its binding partner NSP10 to activate its enzymatic activity. With the continued threat of viral emergence highlighted by human coronaviruses and SARS-CoV-2, mutant strains continue to appear, affecting the highly conserved NSP16: this provides a possible therapeutic approach applicable to any novel coronavirus. To this end, current information on the 2'-O-MTase activity mechanism, the differences between NSP16 and NSP10 in human coronaviruses, and the current potential prevention and treatment strategies related to NSP16 are summarized in this review.

Viruses play a primary role as etiological agents of pandemics worldwide. Although there has been progress in identifying the molecular features of both viruses and hosts, the extent of the impact these and other factors have that contribute to interspecies transmission and their relationship with the emergence of diseases are poorly understood. The objective of this review was to analyze the factors related to the characteristics inherent to RNA viruses accountable for pandemics in the last 20 years which facilitate infection, promote interspecies jump, and assist in the generation of zoonotic infections with pandemic potential. The search resulted in 48 research articles that met the inclusion criteria. Changes adopted by RNA viruses are influenced by environmental and host-related factors, which define their ability to adapt. Population density, host distribution, migration patterns, and the loss of natural habitats, among others, have been associated as factors in the virus-host interaction. This review also included a critical analysis of the Latin American context, considering its diverse and unique social, cultural, and biodiversity characteristics. The scarcity of scientific information is striking, thus, a call to local institutions and governments to invest more resources and efforts to the study of these factors in the region is key.

The disinfection of surfaces in medical facilities is an important element of infection control, including the control of viral infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Preparations used for surface disinfection are typically characterized via their activity against test organisms (i.e., viruses, bacteria and fungi) in the laboratory. Typically, these methods use a suspension of the test organism to assess the bactericidal, fungicidal or virucidal activity of a given preparation. However, such suspension methods do not fully imitate real-life conditions. To address this issue, carrier methods have been developed, in which microorganisms are applied to the surface of a carrier (e.g., stainless steel, glass and polyvinyl chloride (PVC)) and then dried. Such methods more accurately reflect the applications in real-life clinical practice. This article summarizes the available methods for assessing the virucidal activity of chemical disinfectants for use in medical facilities based on the current European standards, including the activity against coronaviruses.

Comment in Viruses. 2022 Feb 28;14(3):

Enteric viruses are the leading cause of diarrhea in children globally. Identifying viral agents and understanding their genetic diversity could help to develop effective preventive measures. This study aimed to determine the detection rate and genetic diversity of four enteric viruses in Gabonese children aged below five years. Stool samples from children <5 years with (n = 177) and without (n = 67) diarrhea were collected from April 2018 to November 2019. Norovirus, astrovirus, sapovirus, and aichivirus A were identified using PCR techniques followed by sequencing and phylogenetic analyses. At least one viral agent was identified in 23.2% and 14.9% of the symptomatic and asymptomatic participants, respectively. Norovirus (14.7%) and astrovirus (7.3%) were the most prevalent in children with diarrhea, whereas in the healthy group norovirus (9%) followed by the first reported aichivirus A in Gabon (6%) were predominant. The predominant norovirus genogroup was GII, consisting mostly of genotype GII.P31-GII.4 Sydney. Phylogenetic analysis of the 3CD region of the aichivirus A genome revealed the presence of two genotypes (A and C) in the study cohort. Astrovirus and sapovirus showed a high diversity, with five different astrovirus genotypes and four sapovirus genotypes, respectively. Our findings give new insights into the circulation and genetic diversity of enteric viruses in Gabonese children.

We describe for the first time the genetic and antigenic characterization of 18 avian avulavirus type-6 viruses (AAvV-6) that were isolated from wild waterfowl in the Americas over the span of 12 years. Only one of the AAvV-6 viruses isolated failed to hemagglutinate chicken red blood cells. We were able to obtain full genome sequences of 16 and 2 fusion gene sequences from the remaining 2 isolates. This is more than double the number of full genome sequences available at the NCBI database. These AAvV-6 viruses phylogenetically grouped into the 2 existing AAvV-6 genotype subgroups indicating the existence of an intercontinental epidemiological link with other AAvV-6 viruses isolated from migratory waterfowl from different Eurasian countries. Antigenic maps made using HI assay data for these isolates showed that the two genetic groups were also antigenically distinct. An isolate representing each genotype was inoculated in specific pathogen free (SPF) chickens, however, no clinical symptoms were observed. A duplex fusion gene based real-time assay for the detection and genotyping of AAvV-6 to genotype 1 and 2 was developed. Using the developed assay, the viral shedding pattern in the infected chickens was examined. The chickens infected with both genotypes were able to shed the virus orally for about a week, however, no significant cloacal shedding was detected in chickens of both groups. Chickens in both groups developed detectable levels of anti-hemagglutinin antibodies 7 days after infection.

Redondoviridae is a recently discovered DNA virus family consisting of two species, vientovirus and brisavirus. Here we used PCR amplification and sequencing to characterize redondoviruses in nasal/throat swabs collected longitudinally from a cohort of 58 individuals working with animals in Vietnam. We additionally analyzed samples from animals to which redondovirus DNA-positive participants were exposed. Redondoviruses were detected in approximately 60% of study participants, including 33% (30/91) of samples collected during episodes of acute respiratory disease and in 50% (29/58) of baseline samples (with no respiratory symptoms). Vientovirus (73%; 24/33) was detected more frequently in samples than brisaviruses (27%; 9/33). In the 23 participants with at least 2 redondovirus-positive samples among their longitudinal samples, 10 (43.5%) had identical redondovirus replication-gene sequences detected (sampling duration: 35-132 days). We found no identical redondovirus replication genes in samples from different participants, and no redondoviruses were detected in 53 pooled nasal/throat swabs collected from domestic animals. Phylogenetic analysis described no large-scale geographical clustering between viruses from Vietnam, the US, Spain, and China, indicating that redondoviruses are highly genetically diverse and have a wide geographical distribution. Collectively, our study provides novel insights into the Redondoviridae family in humans, describing a high prevalence, potentially associated with chronic shedding in the respiratory tract with lack of evidence of zoonotic transmission from close animal contacts. The tropism and potential pathogenicity of this viral family remain to be determined.

Human postmortem specimens are extremely valuable resources for investigating translational hypotheses. Tissue repositories collect clinically assessed specimens from people with and without HIV, including age, viral load, treatments, substance use patterns and cognitive functions. One challenge is the limited number of specimens suitable for transcriptional studies, mainly due to poor RNA quality resulting from long postmortem intervals. We hypothesized that epigenomic signatures would be more stable than RNA for assessing global changes associated with outcomes of interest. We found that H3K27Ac or RNA Polymerase (Pol) were not consistently detected by Chromatin Immunoprecipitation (ChIP), while the enhancer H3K4me3 histone modification was abundant and stable up to the 72 h postmortem. We tested our ability to use HeK4me3 in human prefrontal cortex from HIV+ individuals meeting criteria for methamphetamine use disorder or not (Meth +/-) which exhibited poor RNA quality and were not suitable for transcriptional profiling. Systems strategies that are typically used in transcriptional metadata were applied to H3K4me3 peaks revealing consistent genomic activity differences in regions where addiction and neuronal synapses pathway genes are represented, including genes of the dopaminergic system, as well as inflammatory pathways. The resulting comparisons mirrored previously observed effects of Meth on suppressing gene expression and provided insights on neurological processes affected by Meth. The results suggested that H3K4me3 detection in chromatin may reflect transcriptional patterns, thus providing opportunities for analysis of larger numbers of specimens from cases with substance use and neurological deficits. In conclusion, the detection of H3K4me3 in isolated chromatin can be an alternative to transcriptome strategies to increase the power of association using specimens with long postmortem intervals and low RNA quality.

Infectious laryngotracheitis (ILT) is an infectious upper respiratory tract disease that impacts the poultry industry worldwide. ILT is caused by an alphaherpesvirus commonly referred to as infectious laryngotracheitis virus (ILTV). Vaccination with live attenuated vaccines is practiced regularly for the control of ILT. However, extensive and improper use of live attenuated vaccines is related to vaccine viruses reverting to virulence. An increase in mortality and pathogenicity has been attributed to these vaccine revertant viruses. Recent studies characterized Canadian ILTV strains originating from ILT outbreaks as related to live attenuated vaccine virus revertants. However, information is scarce on the pathogenicity and transmission potential of these Canadian isolates. Hence, in this study, the pathogenicity and transmission potential of two wildtype ILTVs and a chicken embryo origin (CEO) vaccine revertant ILTV of Canadian origin were evaluated. To this end, 3-week-old specific pathogen-free chickens were experimentally infected with each of the ILTV isolates and compared to uninfected controls. Additionally, naïve chickens were exposed to the experimentally infected chickens to mimic naturally occurring infection. Pathogenicity of each of these ILTV isolates was evaluated by the severity of clinical signs, weight loss, mortality, and lesions observed at the necropsy. The transmission potential was evaluated by quantification of ILTV genome loads in oropharyngeal and cloacal swabs and tissue samples of the experimentally infected and contact-exposed chickens, as well as in the capacity to produce ILT in contact-exposed chickens. We observed that the CEO vaccine revertant ILTV isolate induced severe disease in comparison to the two wildtype ILTV isolates used in this study. According to ILTV genome load data, CEO vaccine revertant ILTV isolate was successfully transmitted to naïve contact-exposed chickens in comparison to the tested wildtype ILTV isolates. Overall, the Canadian origin CEO vaccine revertant ILTV isolate possesses higher virulence, and dissemination potential, when compared to the wildtype ILTV isolates used in this study. These findings have serious implications in ILT control in chickens.

Dengue contributes a significant burden on global public health and economies. In Africa, the burden of dengue virus (DENV) infection is not well described. This review was undertaken to determine the prevalence of dengue and associated risk factors. A literature search was done on PubMed/MEDLINE, Scopus, Embase, and Google Scholar databases to identify articles published between 1960 and 2020. Meta-analysis was performed using a random-effect model at a 95% confidence interval, followed by subgroup meta-analysis to determine the overall prevalence. Between 1960 and 2020, 45 outbreaks were identified, of which 17 and 16 occurred in East and West Africa, respectively. Dengue virus serotype 1 (DENV-1) and DENV-2 were the dominant serotypes contributing to 60% of the epidemics. Of 2211 cases reported between 2009 and 2020; 1954 (88.4%) were reported during outbreaks. Overall, the prevalence of dengue was 29% (95% CI: 20-39%) and 3% (95% CI: 1-5%) during the outbreak and non-outbreak periods, respectively. Old age (6/21 studies), lack of mosquito control (6/21), urban residence (4/21), climate change (3/21), and recent history of travel (3/21) were the leading risk factors. This review reports a high burden of dengue and increased risk of severe disease in Africa. Our findings provide useful information for clinical practice and health policy decisions to implement effective interventions.

Infectious bronchitis of chicken is a high morbidity and mortality viral disease affecting the poultry industry worldwide; therefore, a better understanding of this pathogen is of utmost importance. The primary aim of this study was to obtain a deeper insight into the genomic diversity of field infectious bronchitis virus (IBV) strains using phylogenetic and recombination analysis. We sequenced the genome of 20 randomly selected strains from seven European countries. After sequencing, we created a genome sequence data set that contained 36 European origin field isolates and 33 vaccine strains. When analyzing these 69 IBV genome sequences, we identified 215 recombination events highlighting that some strains had multiple recombination breaking points. Recombination hot spots were identified mostly in the regions coding for non-structural proteins, and multiple recombination hot spots were identified in the nsp2, nsp3, nsp8, and nsp12 coding regions. Recombination occurred among different IBV genotypes and involved both field and vaccine IBV strains. Ninety percent of field strains and nearly half of vaccine strains showed evidence of recombination. Despite the low number and the scattered geographical and temporal origin of whole-genome sequence data collected from European Gammacoronaviruses, this study underlines the importance of recombination as a major evolutionary mechanism of IBVs.

The etiological agent of the COVID-19 pandemic is SARS-CoV-2. As a member of the Coronaviridae, the enveloped pathogen has several membrane proteins, of which two, E and 3a, were suggested to function as ion channels. In an effort to increase our treatment options, alongside providing new research tools, we have sought to inhibit the 3a channel by targeted drug repurposing. To that end, using three bacteria-based assays, we screened a library of 2839 approved-for-human-use drugs and identified the following potential channel-blockers: Capreomycin, Pentamidine, Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine and Fludarabine. The stage is now set for examining the activity of these compounds in detailed electrophysiological studies and their impact on the whole virus with appropriate biosafety measures.

The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host-pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (>150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and suggest clustering of infection foci in close proximity. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

The emergent human coronavirus SARS-CoV-2 and its high infectivity rate has highlighted the strong need for new disinfection systems. Evidence has proven that airborne transmission is an important route of spreading for this virus. Therefore, this short communication introduces CLODOS Technology®, a novel strategy to disinfect contaminated surfaces. It is a product based on stable and 99% pure chlorine dioxide, already certified as a bactericide, fungicide and virucide against different pathogens. In this study, CLODOS Technology®, by direct contact or thermonebulization, showed virucidal activity against the human coronavirus HCoV-229E at non-cytotoxic doses. Different conditions such as nebulization, exposure time and product concentration have been tested to standardize and optimize this new feasible method for disinfection.

SUMOylation is a highly dynamic ubiquitin-like post-translational modification that is essential for cells to respond to and resolve various genotoxic and proteotoxic stresses. Virus infections also constitute a considerable stress scenario for cells, and recent research has started to uncover the diverse roles of SUMOylation in regulating virus replication, not least by impacting antiviral defenses. Here, we review some of the key findings of this virus-host interplay, and discuss the increasingly important contribution that large-scale, unbiased, proteomic methodologies are making to discoveries in this field. We highlight the latest proteomic technologies that have been specifically developed to understand SUMOylation dynamics in response to cellular stresses, and comment on how these techniques might be best applied to dissect the biology of SUMOylation during innate immunity. Furthermore, we showcase a selection of studies that have already used SUMO proteomics to reveal novel aspects of host innate defense against viruses, such as functional cross-talk between SUMO proteins and other ubiquitin-like modifiers, viral antagonism of SUMO-modified antiviral restriction factors, and an infection-triggered SUMO-switch that releases endogenous retroelement RNAs to stimulate antiviral interferon responses. Future research in this area has the potential to provide new and diverse mechanistic insights into host immune defenses.

Effective broad-spectrum antiviral treatments are in dire need as disinfectants and therapeutic alternatives. One such method of disinfection is photodynamic inactivation, which involves the production of reactive oxygen species from dissolved oxygen in response to light-stimulated photosensitizers. This study evaluated the efficacy of functionalized porphyrin compounds for photodynamic inactivation of bacteriophages as human virus surrogates. A blue-light light emitting diode (LED) lamp was used to activate porphyrin compounds in aqueous solution (phosphate buffer). The DNA bacteriophages ΦX174 and P22 were more resistant to porphyrin TMPyP photodynamic inactivation than RNA bacteriophage fr, with increasing rates of inactivation in the order: ΦX174 << P22 << fr. Bacteriophage ΦX174 was therefore considered a resistant virus suitable for the evaluation of three additional porphyrins. These porphyrins were synthesized from TMPyP by inclusion of a central palladium ion (PdT4) and/or the addition of a hydrophobic C14 chain (PdC14 or C14). While the inactivation rate of bacteriophage ΦX174 via TMPyP was similar to previous reports of resistant viruses, ΦX174 inactivation increased by a factor of approximately 2.5 using the metalloporphyrins PdT4 and PdC14. The order of porphyrin effectiveness was TMPyP < C14 < PdT4 < PdC14, indicating that both Pd2+ ligation and C14 functionalization aided virus inactivation.

Since the coronavirus disease (COVID-19) pandemic was first identified in early 2020, rare cases of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in pet cats have been reported worldwide. Some reports of cats with SARS-CoV-2 showed self-limiting respiratory or gastrointestinal disease after suspected human-to-feline transmission via close contact with humans with SARS-CoV-2. In the present study, we investigated a cat with SARS-CoV-2 that was presented to a private animal clinic in Northern Italy in May 2020 in a weak clinical condition due to an underlying intestinal B-cell lymphoma. The cat developed signs of respiratory tract disease, including a sneeze, a cough and ocular discharge, three days after an oropharyngeal swab tested positive for SARS-CoV-2 viral RNA using two real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays for the envelope (E) and RNA-dependent RNA polymerase (RdRp) gene. Thus, SARS-CoV-2 viral RNA was detectable prior to the onset of clinical signs. Five and six months after positive molecular results, the serological testing substantiated the presence of a SARS-CoV-2 infection in the cat with the detection of anti-SARS-CoV-2 receptor binding domain (RBD) immunoglobulin (IgG) antibodies and neutralizing activity in a surrogate virus neutralization assay (sVNT). To the best of our knowledge, this extends the known duration of seropositivity of SARS-CoV-2 in a cat. Our study provides further evidence that cats are susceptible to SARS-CoV-2 under natural conditions and strengthens the assumption that comorbidities may play a role in the development of clinical disease.

Congenital Zika virus (ZIKV) infection may present with a broad spectrum of clinical manifestations. Some sequelae, particularly neurodevelopmental problems, may have a later onset. We conducted a prospective cohort study of 799 high-risk pregnant women who were followed up until delivery. Eighty-three women and/or newborns were considered ZIKV exposed and/or infected. Laboratory diagnosis was made by polymerase chain reaction in the pregnant mothers and their respective newborns, as well as Dengue virus, Chikungunya virus, and ZIKV serology. Serology for toxoplasmosis, rubella, cytomegalovirus, herpes simplex virus, and syphilis infections were also performed in microcephalic newborns. The newborns included in the study were followed up until their third birthday. Developmental delay was observed in nine patients (13.2%): mild cognitive delay in three patients, speech delay in three patients, autism spectrum disorder in two patients, and severe neurological abnormalities in one microcephalic patient; sensorineural hearing loss, three patients and dysphagia, six patients. Microcephaly due to ZIKV occurred in three patients (3.6%). Clinical manifestations can appear after the first year of life in children infected/exposed to ZIKV, emphasizing the need for long-term follow-up.

The threat of a new influenza pandemic is real. With past pandemics claiming millions of lives, finding new ways to combat this virus is essential. Host cells have developed a multi-modular system to detect incoming pathogens, a phenomenon called sensing. The signaling cascade triggered by sensing subsequently induces protection for themselves and their surrounding neighbors, termed interferon (IFN) response. This response induces the upregulation of hundreds of interferon-stimulated genes (ISGs), including antiviral effectors, establishing an antiviral state. As well as the antiviral proteins induced through the IFN system, cells also possess a so-called intrinsic immunity, constituted of antiviral proteins that are constitutively expressed, creating a first barrier preceding the induction of the interferon system. All these combined antiviral effectors inhibit the virus at various stages of the viral lifecycle, using a wide array of mechanisms. Here, we provide a review of mammalian and avian influenza A restriction factors, detailing their mechanism of action and in vivo relevance, when known. Understanding their mode of action might help pave the way for the development of new influenza treatments, which are absolutely required if we want to be prepared to face a new pandemic.

Eukaryotic translation initiation factor 4E (eIF4E) plays a key role in the infection of potyviruses in susceptible plants by interacting with viral genome-linked protein (VPg). Sugarcane (Saccharum spp.) production is threatened by mosaic disease caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and Sugarcane streak mosaic virus (SCSMV). In this study, two eIF4Es and their isoform eIF(iso)4E and 4E-binding protein coding genes were cloned from sugarcane cultivar ROC22 and designated SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP, respectively. Real-time quantitative PCR analysis showed different expression profiles of these four genes upon SCMV challenge. A subcellular localization assay showed that SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP were distributed in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SceIF4Ea/b and SceIF(iso)4E were selectively employed by different sugarcane mosaic pathogens, i.e., SCMV-VPg interacted with SceIF4Ea/b and SceIF(iso)4E, SrMV-VPg interacted with both SceIF4Eb and SceIF(iso)4E, and SCSMV-VPg interacted only with SceIF(iso)4E. Intriguingly, the BiFC assays, but not the Y2H assays, showed that ScnCBP interacted with the VPgs of SCMV, SrMV, and SCSMV. Competitive interaction assays showed that SCMV-VPg, SrMV-VPg, and SCMV-VPg did not compete with each other to interact with SceIF(iso)4E, and SceIF(iso)4E competed with SceIF4Eb to interact with SrMV-VPg but not SCMV-VPg. This study sheds light on the molecular mechanism of sugarcane mosaic pathogen infection of sugarcane plants and benefits sugarcane breeding against the sugarcane mosaic disease.

The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.

Human metapneumovirus (hMPV) is one of the main pathogens responsible for acute respiratory infections in children up to 5 years of age, contributing substantially to health burden. The worldwide economic and social impact of this virus is significant and must be addressed. The structural components of hMPV (either proteins or genetic material) can be detected by several receptors expressed by host cells through the engagement of pattern recognition receptors. The recognition of the structural components of hMPV can promote the signaling of the immune response to clear the infection, leading to the activation of several pathways, such as those related to the interferon response. Even so, several intrinsic factors are capable of modulating the immune response or directly inhibiting the replication of hMPV. This article will discuss the current knowledge regarding the innate and adaptive immune response during hMPV infections. Accordingly, the host intrinsic components capable of modulating the immune response and the elements capable of restricting viral replication during hMPV infections will be examined.

BACKGROUND: Interaction between host transcription factors (TFs) and the viral genome is fundamental for hepatitis B virus (HBV) gene expression regulation. Additionally, the distinct interaction of the TFs' network with the HBV genome determines the regulatory effect outcome. Hence, different HBV genotypes and their variants may display different viral replication/transcription regulation. Due to the lack of an efficient infection model suitable for all HBV genotypes, the hepatoma cell transfection model is primarily used in studies involving non-D HBV genotypes and variants. METHODS: We explored the transcriptome profile of host TFs with a regulatory effect on HBV in eight liver-derived cell lines in comparison with primary human hepatocytes (PHH). We further analyzed the suitability of these models in supporting HBV genotype B replication/transcription. RESULTS: Among studied models, HC-04, as a result of the close similarity of TFs transcriptome profile to PHH and the interaction of specific TFs including HNF4α and PPARα, showed the highest efficiency in regard to viral replication and antigen production. The absence of TFs expression in L02 transfection model resulted in its inefficiency in HBV replication/transcription. CONCLUSION: These observations help to better design studies on regulatory mechanisms involving non-D HBV genotypes and variants' gene expression and the development of more efficient therapeutical approaches.

Mojiang virus (MojV) is the first henipavirus identified in a rodent and known only by sequence data, whereas all other henipaviruses have been isolated from bats (Hendra virus, Nipah virus, Cedar virus) or discovered by sequence data from material of bat origin (Ghana virus). Ephrin-B2 and -B3 are entry receptors for Hendra and Nipah viruses, but Cedar virus can utilize human ephrin-B1, -B2, -A2 and -A5 and mouse ephrin-A1. However, the entry receptor for MojV remains unknown, and its species tropism is not well characterized. Here, we utilized recombinant full-length and soluble forms of the MojV fusion (F) and attachment (G) glycoproteins in membrane fusion and receptor tropism studies. MojV F and G were functionally competent and mediated cell-cell fusion in primate and rattine cells, albeit with low levels and slow fusion kinetics. Although a relative instability of the pre-fusion conformation of a soluble form of MojV F was observed, MojV F displayed significantly greater fusion activity when heterotypically paired with Ghana virus G. An exhaustive investigation of A- and B-class ephrins indicated that none serve as a primary receptor for MojV. The MojV cell fusion phenotype is therefore likely the result of receptor restriction rather than functional defects in recombinant MojV F and G glycoproteins.

Cape Town was the first city in South Africa to experience the full impact of the coronavirus disease 2019 (COVID-19) pandemic. We acquired samples from all suspected cases and their contacts during the first month of the pandemic from Tygerberg Hospital. Nanopore sequencing generated SARS-CoV-2 whole genomes. Phylogenetic inference with maximum likelihood and Bayesian methods were used to determine lineages that seeded the local epidemic. Three patients were known to have travelled internationally and an outbreak was detected in a nearby supermarket. Sequencing of 50 samples produced 46 high-quality genomes. The sequences were classified as lineages: B, B.1, B.1.1.1, B.1.1.161, B.1.1.29, B.1.8, B.39, and B.40. All the sequences from persons under investigation (PUIs) in the supermarket outbreak (lineage B.1.8) fall within a clade from the Netherlands with good support (p > 0.9). In addition, a new mutation, 5209A>G, emerged within the Cape Town cluster. The molecular clock analysis suggests that this occurred around 13 March 2020 (95% confidence interval: 9-17 March). The phylogenetic reconstruction suggests at least nine early introductions of SARS-CoV-2 into Cape Town and an early localized transmission in a shopping environment. Genomic surveillance was successfully used to investigate and track the spread of early introductions of SARS-CoV-2 in Cape Town.

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

This study aimed to indicate the influence of infection caused by genotype II African swine fever virus (ASFV)-isolate Pol18_28298_O111, currently circulating in Poland, on blood counts, biochemical parameters, as well as inflammatory and immune responses. Blood and sera collected from 21 domestic pigs infected intranasally with different doses of virulent ASFV were analysed. The infection led to variable changes in blood counts depending on the stage of the disease with a tendency towards leukopenia and thrombocytopenia. The elevated C-reactive protein (CRP) concentrations and microscopic lesions in organs confirmed the development of the inflammation process, which also resulted in an increased level of biochemical markers such as: Aspartate transaminase (AST), creatine kinase (CK), creatinine, and urea. Antibodies could be detected from 9 to 18 days post infection (dpi). Two survivors presented the highest titer of antibodies (>5 log10/mL) with a simultaneous increase in the lymphocyte T (CD3+) percentage-revealed by flow cytometry. Results confirmed a progressive inflammatory process occurring during the ASFV infection, which may lead to multiple organs failure and death of the majority of affected animals.

While SARS-CoV-2 specific neutralizing antibodies have been developed for therapeutic purposes, the specific viral triggers that drive the generation of SARS-CoV-2 specific IgG and IgM antibodies remain only partially characterized. Moreover, it is unknown whether endogenously derived antibodies drive viral clearance that might result in mitigation of clinical severity during natural infection. We developed a series of non-linear mathematical models to investigate whether SARS-CoV-2 viral and antibody kinetics are coupled or governed by separate processes. Patients with severe disease had a higher production rate of IgG but not IgM antibodies. Maximal levels of both isotypes were governed by their production rate rather than different saturation levels between people. Our results suggest that an exponential surge in IgG levels occurs approximately 5-10 days after symptom onset with no requirement for continual antigenic stimulation. SARS-CoV-2 specific IgG antibodies appear to have limited to no effect on viral dynamics but may enhance viral clearance late during primary infection resulting from the binding effect of antibody to virus, rather than neutralization. In conclusion, SARS-CoV-2 specific IgG antibodies may play only a limited role in clearing infection from the nasal passages despite providing long-term immunity against infection following vaccination or prior infection.

The common vampire bat (Desmodus rotundus) is a hematophagous species responsible for paralytic rabies and bite damage that affects livestock, humans and wildlife from Mexico to Argentina. Current measures to control vampires, based upon coumarin-derived poisons, are not used extensively due in part to the high cost of application, risks for bats that share roosts with vampires and residual environmental contamination. Observations that vampire bat bites may induce resistance in livestock against vampire bat salivary anticoagulants encourage research into novel vaccine-based alternatives particularly focused upon increasing livestock resistance to vampire salivary components. We evaluated the action of vampire bat saliva-Freund's incomplete adjuvant administered to sheep with anticoagulant responses induced by repeated vampire bites in a control group and examined characteristics of vampire bat salivary secretion. We observed that injections induced a response against vampire bat salivary anticoagulants stronger than by repeated vampire bat bites. Based upon these preliminary findings, we hypothesize the utility of developing a control technique based on induction of an immunologically mediated resistance against vampire bat anticoagulants and rabies virus via dual delivery of appropriate host and pathogen antigens. Fundamental characteristics of host biology favor alternative strategies than simple culling by poisons for practical, economical, and ecologically relevant management of vampire populations within a One Health context.

The circulation in Europe of novel reassortant strains of infectious bursal disease virus (IBDV), containing a unique genetic background composition, represents a serious problem for animal health. Since the emergence of this novel IBDV mosaic was first described in Poland, this scenario has become particularly attractive to uncover the evolutionary forces driving the genetic diversity of IBDV populations. This study additionally addressed the phenotypic characterization of these emergent strains, as well as the main features affecting the viral fitness during the competition process of IBDV lineages in the field. Our results showed how different evolutionary mechanisms modulate the genetic diversity of co-existent IBDV lineages, leading to the error catastrophe effect, Muller ratchet effect, or prevalence, depending on their genetic compositions. We also determined that the action of the positive selection pressure, depending on the genomic segment on which it is acting, can drive two main phenotypes for IBDV: immune-escaping strains from the selection on segment A or strains with functional advantages from the selection on segment B. This last group seems to possess an increased fitness landscape in the viral quasispecies composition, presenting better adaptability to dissimilar environmental conditions and likely becoming the dominant population. The reassortant strains also exhibited a lower mortality rate compared with the well-known vvIBDV strains, which can facilitate their spreading.

Understanding SARS-CoV-2 evolution is a fundamental effort in coping with the COVID-19 pandemic. The virus genomes have been broadly evolving due to the high number of infected hosts world-wide. Mutagenesis and selection are two inter-dependent mechanisms of virus diversification. However, which mechanisms contribute to the mutation profiles of SARS-CoV-2 remain under-explored. Here, we delineate the contribution of mutagenesis and selection to the genome diversity of SARS-CoV-2 isolates. We generated a comprehensive phylogenetic tree with representative genomes. Instead of counting mutations relative to the reference genome, we identified each mutation event at the nodes of the phylogenetic tree. With this approach, we obtained the mutation events that are independent of each other and generated the mutation profile of SARS-CoV-2 genomes. The results suggest that the heterogeneous mutation patterns are mainly reflections of host (i) antiviral mechanisms that are achieved through APOBEC, ADAR, and ZAP proteins, and (ii) probable adaptation against reactive oxygen species.

Rhesus macaques can be readily infected with chimeric simian-human immunodeficiency viruses (SHIV) as a suitable virus challenge system for testing the efficacy of HIV vaccines. Three Chinese-origin rhesus macaques (ChRM) were inoculated intravenously (IV) with SHIVC109P4 in a rapid serial in vivo passage. SHIV recovered from the peripheral blood of the final ChRM was used to generate a ChRM-adapted virus challenge stock. This stock was titrated for the intrarectal route (IR) in 8 ChRMs using undiluted, 1:10 or 1:100 dilutions, to determine a suitable dose for use in future vaccine efficacy testing via repeated low-dose IR challenges. All 11 ChRMs were successfully infected, reaching similar median peak viraemias at 1-2 weeks post inoculation but undetectable levels by 8 weeks post inoculation. T-cell responses were detected in all animals and Tier 1 neutralizing antibodies (Nab) developed in 10 of 11 infected ChRMs. All ChRMs remained healthy and maintained normal CD4+ T cell counts. Sequence analyses showed >98% amino acid identity between the original inoculum and virus recovered at peak viraemia indicating only minimal changes in the env gene. Thus, while replication is limited over time, our adapted SHIV can be used to test for protection of virus acquisition in ChRMs.

The SAM and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase that plays a crucial role for a variety of different cellular functions. Besides balancing intracellular dNTP concentrations, facilitating DNA damage repair, and dampening excessive immune responses, SAMHD1 has been shown to act as a major restriction factor against various virus species. In addition to its well-described activity against retroviruses such as HIV-1, SAMHD1 has been identified to reduce the infectivity of different DNA viruses such as the herpesviruses CMV and EBV, the poxvirus VACV, or the hepadnavirus HBV. While some viruses are efficiently restricted by SAMHD1, others have developed evasion mechanisms that antagonize the antiviral activity of SAMHD1. Within this review, we summarize the different cellular functions of SAMHD1 and highlight the countermeasures viruses have evolved to neutralize the restriction factor SAMHD1.

Equine encephalosis virus (EEV) is a neglected virus endemic to South Africa and is considered to generally result in mild disease in equines. Specimens were analyzed from live horses that presented with undefined neurological, febrile, or respiratory signs, or sudden and unexpected death. Between 2010 and 2017, 111 of 1523 (7.3%) horse samples tested positive for EEV using a nested real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Clinical signs were reported in 106 (7.2%) EEV positive and 1360 negative horses and included pyrexia (77/106, 72.6%), icterus (20/106, 18.9%) and dyspnea (12/106, 11.3%). Neurological signs were inversely associated with EEV infection (OR < 1, p < 0.05) relative to EEV negative cases despite a high percentage of animals presenting with neurological abnormalities (51/106, 48.1%). Seventeen of the EEV positive horses also had coinfections with either West Nile (5/106, 4.7%), Middelburg (4/106, 3.8%) or African Horse sickness virus (8/106, 7.6%). To investigate a possible genetic link between EEV strains causing the observed clinical signs in horses, the full genomes of six isolates were compared to the reference strains. Based on the outer capsid protein (VP2), serotype 1 and 4 were identified as the predominant serotypes with widespread reassortment between the seven different serotypes.

Understanding within-host evolution is critical for predicting viral evolutionary outcomes, yet such studies are currently lacking due to difficulty involving human subjects. Hepatitis C virus (HCV) is an RNA virus with high mutation rates. Its complex evolutionary dynamics and extensive genetic diversity are demonstrated in over 67 known subtypes. In this study, we analyzed within-host mutation frequency patterns of three HCV subtypes, using a large number of samples obtained from treatment-naïve participants by next-generation sequencing. We report that overall mutation frequency patterns are similar among subtypes, yet subtype 3a consistently had lower mutation frequencies and nucleotide diversity, while subtype 1a had the highest. We found that about 50% of genomic sites are highly conserved across subtypes, which are likely under strong purifying selection. We also compared within-host and between-host selective pressures, which revealed that Hyper Variable Region 1 within hosts was under positive selection, but was under slightly negative selection between hosts, which indicates that many mutations created within hosts are removed during the transmission bottleneck. Examining the natural prevalence of known resistance-associated variants showed their consistent existence in the treatment-naïve participants. These results provide insights into the differences and similarities among HCV subtypes that may be used to develop and improve HCV therapies.

The rapid spread of the virus in Latin America and the association of the infection with microcephaly in newborns or Guillain-Barré Syndrome in adults prompted the WHO to declare the Zika virus (ZIKV) epidemic to be an international public health emergency in 2016. As the virus was first discovered in monkeys and is spread not only by mosquitos but also from human to human, we investigated the stability to the human complement of ZIKV derived from mosquito (ZIKVInsect), monkey (ZIKVVero), or human cells (ZIKVA549 and ZIKVFibro), respectively. At a low serum concentration (10%), which refers to complement concentrations found on mucosal surfaces, the virus was relatively stable at 37 °C. At higher complement levels (up to 50% serum concentration), ZIKV titers differed significantly depending on the cell line used for the propagation of the virus. While the viral titer of ZIKVInsect decreased about two orders in magnitude, when incubated with human serum, the virus derived from human cells was more resistant to complement-mediated lysis (CML). By virus-capture assay and Western blots, the complement regulator protein CD55 was identified to be incorporated into the viral envelope. Blocking of CD55 by neutralizing Abs significantly increased the sensitivity to human complement. Taken together, these data indicate that the incorporation of CD55 from human cells contributes to the stability of ZIKV against complement-mediated virolysis.

The heterogeneity of immune response to COVID-19 has been reported to correlate with disease severity and prognosis. While so, how the immune response progress along the period of viral RNA-shedding (VRS), which determines the infectiousness of disease, is yet to be elucidated. We aim to exhaustively evaluate the peripheral immune cells to expose the interplay of the immune system in uncomplicated COVID-19 cases with different VRS periods and dynamic changes of the immune cell profile in the prolonged cases. We prospectively recruited four uncomplicated COVID-19 patients and four healthy controls (HCs) and evaluated the immune cell profile throughout the disease course. Peripheral blood mononuclear cells (PBMCs) were collected and submitted to a multi-panel flowcytometric assay. CD19+-B cells were upregulated, while CD4, CD8, and NK cells were downregulated in prolonged VRS patients. Additionally, the pro-inflammatory-Th1 population showed downregulation, followed by improvement along the disease course, while the immunoregulatory cells showed upregulation with subsequent decline. COVID-19 patients with longer VRS expressed an immune profile comparable to those with severe disease, although they remained clinically stable. Further studies of immune signature in a larger cohort are warranted.

We had access to both components of a couple who became infected with human immunodeficiency virus (HIV)-1 through sexual behavior during the early initial phase of infection and before initiation of therapy. We analyzed blood samples obtained at the time of diagnosis and after six months of combined antiretroviral therapy. Next-generation sequencing (NGS) and phylogenetic analyses were used to investigate the transmission and evolution of HIV-1 quasispecies. Phylogenetic analyses were conducted using Bayesian inference methods. Both partners were infected with an HIV-1 B subtype. No evidence of viral recombination was observed. The lowest intrapersonal genetic distances were observed at baseline, before initiation of therapy, and in particular in the V1V2 fragment (distances ranging from 0.102 to 0.148). One HIV-1 single variant was concluded to be dominant in all of the HIV-1 regions analyzed, although some minor variants could be observed. The same tree structure was observed both at baseline and after six months of therapy. These are the first extended phylogenetic analyses performed on both members of a therapy-naïve couple within a few weeks of infection, and in which the effect of antiretroviral therapy on viral evolution was analyzed. Understanding which HIV-1 variants are most likely to be transmitted would allow a better understanding of viral evolution, possibly playing a role in vaccine design and prevention strategies.

Repeated positivity and reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is a significant concern. Our study aimed to evaluate the clinical significance of repeatedly positive testing after coronavirus disease 2019 (COVID-19) recovery. We performed a systematic literature search following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. With available individual patient data reporting on repeatedly SARS-CoV-2 positive (RSP) patients, case reports, and case series were included in this analysis. We performed a descriptive analysis of baseline characteristics of repeatedly positive cases. We assessed the cases according to the length of their polymerase chain reaction (PCR) negative interval between the two episodes. Risk factors for the severity of second episodes were evaluated. Overall, we included 123 patients with repeated positivity from 56 publications, with a mean repeated positivity length of 47.8 ± 29.9 days. Younger patients were predominant in the delayed (>90 days) recurrent positive group. Furthermore, comparing patients with RSP intervals of below 60 and above 60 days, we found that a more severe disease course can be expected if the repeated positivity interval is shorter. Severe and critical disease courses might predict future repeatedly positive severe and critical COVID-19 episodes. In conclusion, our results show that the second episode of SARS-CoV-2 positivity is more severe if it happens within 60 days after the first positive PCR. On the other hand, the second episode's severity correlates with the first.

Over the last months, as oncology specialists, we have frequently been contacted for estimating prognosis for cancer patients affected by COVID-19 infection. Until now, there have been no clear markers to guide decision making regarding the appropriateness of invasive ventilation in cancer patients affected by COVID-19 infection. We developed a practical tool encompassing a prognostic score, "The Milano Policlinico ONCOVID-ICU score." The score is composed of three groups of variables: patient's characteristics such as sex, age, BMI, and comorbidities; oncological variables (treatment intent, life expectancy, on or off-treatment status); and clinical parameters in association with laboratory values (the Sequential Organ Failure Assessment (SOFA) score and D-dimer). The SOFA score includes six different clinical parameters and during the first few days of ICU admissions has an important prognostic role. The oncological history should never represent, per se, a contraindication to intensive care and must be considered together with other variables, such as laboratory values, clinical parameters, and patient characteristics, in order to make the hardest but best possible choice. To our knowledge, "The Milano Policlinico ONCOVID-ICU score" is the first prognostic score proposed in this setting of patients and requires further validation. This tool may be useful to assess the prognosis of cancer patients in critical conditions.

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4-74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.

Reassortment among co-infecting influenza A viruses (IAVs) is an important source of viral diversity and can facilitate expansion into novel host species. Indeed, reassortment played a key role in the evolution of the last three pandemic IAVs. Observed patterns of reassortment within a coinfected host are likely to be shaped by several factors, including viral load, the extent of viral mixing within the host and the stringency of selection. These factors in turn are expected to vary among the diverse host species that IAV infects. To investigate host differences in IAV reassortment, here we examined reassortment of two distinct avian IAVs within their natural host (mallards) and a mammalian model system (guinea pigs). Animals were co-inoculated with A/wildbird/California/187718-36/2008 (H3N8) and A/mallard/Colorado/P66F1-5/2008 (H4N6) viruses. Longitudinal samples were collected from the cloaca of mallards or the nasal tract of guinea pigs and viral genetic exchange was monitored by genotyping clonal isolates from these samples. Relative to those in guinea pigs, viral populations in mallards showed higher frequencies of reassortant genotypes and were characterized by higher genotype richness and diversity. In line with these observations, analysis of pairwise segment combinations revealed lower linkage disequilibrium in mallards as compared to guinea pigs. No clear longitudinal patterns in richness, diversity or linkage disequilibrium were present in either host. Our results reveal mallards to be a highly permissive host for IAV reassortment and suggest that reduced viral mixing limits avian IAV reassortment in a mammalian host.

Non-human primates (NHP) are an important source of viruses that can spillover to humans and, after adaptation, spread through the host population. Whereas HIV-1 and HTLV-1 emerged as retroviral pathogens in humans, a unique class of retroviruses called foamy viruses (FV) with zoonotic potential are occasionally detected in bushmeat hunters or zookeepers. Various FVs are endemic in numerous mammalian natural hosts, such as primates, felines, bovines, and equines, and other animals, but not in humans. They are apathogenic, and significant differences exist between the viral life cycles of FV and other retroviruses. Importantly, FVs replicate in the presence of many well-defined retroviral restriction factors such as TRIM5α, BST2 (Tetherin), MX2, and APOBEC3 (A3). While the interaction of A3s with HIV-1 is well studied, the escape mechanisms of FVs from restriction by A3 is much less explored. Here we review the current knowledge of FV biology, host restriction factors, and FV-host interactions with an emphasis on the consequences of FV regulatory protein Bet binding to A3s and outline crucial open questions for future studies.

Over 36 million people worldwide are infected with HIV. Antiretroviral therapy (ART) has proven to be highly effective to prevent HIV-1 transmission, clinical progression and death. Despite this success, the number of HIV-1 infected individuals continues increasing and ART should be taken for life. Therefore, there are two main priorities: the development of preventive vaccines to protect from HIV acquisition and achieve an efficient control of HIV infection in the absence of ART (functional cure). In this sense, in the last few years, there has been a broad interest in new and innovative approaches such as mRNA-based vaccines. RNA-based immunogens represent a promising alternative to conventional vaccines because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. Some mRNA-based vaccines platforms against infectious diseases have demonstrated encouraging results in animal models and humans. However, their application is still limited because the instability and inefficient in vivo delivery of mRNA. Immunogens, design, immunogenicity, chemical modifications on the molecule or the vaccine delivery methods are all crucial interventions for improvement. In this review we, will present the current knowledge and challenges in this research field. mRNA vaccines hold great promises as part of a combined strategy, for achieving HIV functional cure.

BACKGROUND: Data suggest that pediatric patients might react differently to influenza vaccination, both in terms of immunity and side effects. We have recently shown that using a whole virion vaccine with aluminum phosphate adjuvants, reduced dose vaccines containing 6 µg of viral hemagglutinin (HA) per strain are immunogenic, and well tolerated in adult and elderly patients. Here we show the results of a multicenter clinical trial of pediatric patients, using reduced doses of a new, whole virion, aluminum phosphate adjuvanted vaccine (FluArt, Budapest, Hungary). METHODS: A total of 120 healthy volunteers were included in two age groups (3-11 years, receiving 3 µg of HA per strain, and 12-18 years, receiving 6 µg of HA per strain). We used hemagglutination inhibition testing to assess immunogenicity, based on EMA and FDA licensing criteria, including post/pre-vaccination geometric mean titer ratios, seroconversion and seropositivity rates. Safety and tolerability were assessed using CHMP guidelines. RESULTS: All subjects entered the study and were vaccinated (ITT population). All 120 subjects attended the control visit on Day 21 (PP population). All immunogenicity licensing criteria were met in both age groups for all three vaccine virus strains. No serious adverse events were detected and the vaccine was well tolerated by both age groups. DISCUSSION: Using a whole virion vaccine and aluminum phosphate adjuvants, a reduction in the amount of the viral hemmaglutinin is possible while maintaining immunogenicity, safety and tolerability in pediatric and adolescent patients.

Haïti is at risk for wild poliovirus (WPV) importation and circulation, as well as vaccine-derived poliovirus (VDPV) emergence. Environmental surveillance (ES) for polioviruses was established in Port au Prince and Gonaïves in 2016. During 2017-2019, initial ES sites were re-evaluated, and ES was expanded into Cap Haïtien and Saint Marc. Wastewater samples and data on weather, hour of collection, and sample temperature and pH were collected every 4 weeks during March 2017-December 2019 (272 sampling events) from 21 sites in Cap Haïtien, Gonaïves, Port au Prince, and Saint Marc. Samples were processed for the detection of polio and non-polio enteroviruses using the two-phase and "Concentration and Filter Elution" methodologies. Polioviruses were serotyped and underwent intra-typic characterization. No WPV or VDPVs were isolated. Sabin-like polioviruses (oral vaccine strain) of serotypes 1 and 3 were sporadically detected. Five of six (83%), one of six (17%), five of six (83%), and two of three (67%) sites evaluated in Cap Haïtien, Gonaïves, Port au Prince, and Saint Marc, respectively, had enterovirus isolation from >50% of sampling events; these results and considerations, such as watershed population size and overlap, influence of sea water, and excessive particulates in samples, were factors in site retention or termination. The evaluation of 21 ES sampling sites in four Haïtian cities led to the termination of 11 sites. Every-four-weekly sampling continues at the remaining 10 sites across the four cities as a core Global Polio Eradication Initiative activity.

Bacteriophage (phage) taxonomy has been in flux since its inception over four decades ago. Genome sequencing has put pressure on the classification system and recent years have seen significant changes to phage taxonomy. Here, we reflect on the state of phage taxonomy and provide a roadmap for the future, including the abolition of the order Caudovirales and the families Myoviridae, Podoviridae, and Siphoviridae. Furthermore, we specify guidelines for the demarcation of species, genus, subfamily and family-level ranks of tailed phage taxonomy.

COVID-19 has become a global pandemic of the highest priority. Multiple treatment protocols have been proposed worldwide with no definitive answer for acure. A prior retrospective study showed association between bitter taste receptor 38 (T2R38) phenotypes and the severity of COVID-19. Based on this, we proposed assessing the different T2R38 phenotypes response towards a targeted treatment protocol. Starting July 2020 till December 2020, we tested subjects for T2R38 phenotypic expression (supertasters, tasters, and nontasters). Subjects who were subsequently infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (diagnosed via PCR) were included. Based on their taster status, supertasters were given dexamethasone for 4 days; tasters were given azithromycin and dexamethasone +/- hydroxychloroquine for 7 days; and nontasters were given azithromycin and dexamethasone for 12 days. Subjects were followed prospectively and their outcomes were documented. Seven hundred forty-seven COVID-19 patients were included, with 184 (24.7%) supertasters, 371 (49.6%) tasters, and192 (25.7%) nontasters. The average duration of symptoms with the treatment protocol was 5 days for supertasters, 8.1 days for tasters, and 16.2 days for nontasters. Only three subjects (0.4%) required hospitalization (3/3 nontasters). Targeted treatment protocol showed significant correlation (p < 0.05) based on patients' T2R38 phenotypic expression. Assessing treatment protocols for COVID-19 patients according to their T2R38 phenotype could provide insight into the inconsistent results obtained from the different studies worldwide. Further study is warranted on the categorization of patients based on their T2R38 phenotype.

The ruminant-associated influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5' and 3' rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5' and 3' NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D/CN286) into the bidirectional pHW2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5' and 3' non-coding regions (NCR) highlighted that the terminal 3' end of all segments harbours an uracil instead of a cytosine nucleotide, similar to other influenza viruses. Subsequent analysis on the functionality of the 5' and 3' NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5' and 3' NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin, as well as by using an in vitro model recapitulating the natural replication site of IDV in bovine and swine. This revealed that the reverse genetic clone D/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.

Human metapneumovirus (HMPV) is an important cause of upper and lower respiratory tract disease in individuals of all ages. It is estimated that most individuals will be infected by HMPV by the age of five years old. Despite this burden of disease, there remain caveats in our knowledge of global genetic diversity due to a lack of HMPV sequencing, particularly at the whole-genome scale. The purpose of this study was to create a simple and robust approach for HMPV whole-genome sequencing to be used for genomic epidemiological studies. To design our assay, all available HMPV full-length genome sequences were downloaded from the National Center for Biotechnology Information (NCBI) GenBank database and used to design four primer sets to amplify long, overlapping amplicons spanning the viral genome and, importantly, specific to all known HMPV subtypes. These amplicons were then pooled and sequenced on an Illumina iSeq 100 (Illumina, San Diego, CA, USA); however, the approach is suitable to other common sequencing platforms. We demonstrate the utility of this method using a representative subset of clinical samples and examine these sequences using a phylogenetic approach. Here we present an amplicon-based method for the whole-genome sequencing of HMPV from clinical extracts that can be used to better inform genomic studies of HMPV epidemiology and evolution.

Apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) proteins belong to a family of deaminase proteins that can catalyze the deamination of cytosine to uracil on single-stranded DNA or/and RNA. APOBEC proteins are involved in diverse biological functions, including adaptive and innate immunity, which are critical for restricting viral infection and endogenous retroelements. Dysregulation of their functions can cause undesired genomic mutations and RNA modification, leading to various associated diseases, such as hyper-IgM syndrome and cancer. This review focuses on the structural and biochemical data on the multimerization status of individual APOBECs and the associated functional implications. Many APOBECs form various multimeric complexes, and multimerization is an important way to regulate functions for some of these proteins at several levels, such as deaminase activity, protein stability, subcellular localization, protein storage and activation, virion packaging, and antiviral activity. The multimerization of some APOBECs is more complicated than others, due to the associated complex RNA binding modes.

In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4%). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage Φ16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the ∆resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

In December 2019, a novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of Hubei, China. The virus infection, coronavirus disease 2019 (COVID-19), represents a global concern, as almost all countries around the world are affected. Clinical reports have confirmed several neurological manifestations in COVID-19 patients such as headaches, vomiting, and nausea, indicating the involvement of the central nervous system (CNS) and peripheral nervous system (PNS). Neuroinvasion of coronaviruses is not a new phenomenon, as it has been demonstrated by previous autopsies of severe acute respiratory syndrome coronavirus (SARS-CoV) patients who experienced similar neurologic symptoms. The hypothalamus is a complex structure that is composed of many nuclei and diverse neuronal cell groups. It is characterized by intricate intrahypothalamic circuits that orchestrate a finely tuned communication within the CNS and with the PNS. Hypothalamic circuits are critical for maintaining homeostatic challenges including immune responses to viral infections. The present article reviews the possible routes and mechanisms of neuroinvasion of SARS-CoV-2, with a specific focus on the role of the hypothalamic circuits in mediating the neurological symptoms noted during COVID-19 infection.

Since the emergence of coronavirus disease (COVID-19) in late 2019, domestic cats have been demonstrated to be susceptible to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) under natural and experimental conditions. As pet cats often live in very close contact with their owners, it is essential to investigate SARS-CoV-2 infections in cats in a One-Health context. This study reports the first SARS-CoV-2 infection in a cat in a COVID-19-affected household in Switzerland. The cat (Cat 1) demonstrated signs of an upper respiratory tract infection, including sneezing, inappetence, and apathy, while the cohabiting cat (Cat 2) remained asymptomatic. Nasal, oral, fecal, fur, and environmental swab samples were collected twice from both cats and analyzed by RT-qPCR for the presence of SARS-CoV-2 viral RNA. Both nasal swabs from Cat 1 tested positive. In addition, the first oral swab from Cat 2 and fur and bedding swabs from both cats were RT-qPCR positive. The fecal swabs tested negative. The infection of Cat 1 was confirmed by positive SARS-CoV-2 S1 receptor binding domain (RBD) antibody testing and neutralizing activity in a surrogate assay. The viral genome sequence from Cat 1, obtained by next generation sequencing, showed the closest relation to a human sequence from the B.1.1.39 lineage, with one single nucleotide polymorphism (SNP) difference. This study demonstrates not only SARS-CoV-2 infection of a cat from a COVID-19-affected household but also contamination of the cats' fur and bed with viral RNA. Our results are important to create awareness that SARS-CoV-2 infected people should observe hygienic measures to avoid infection and contamination of animal cohabitants.

The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented, streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome, named ancient Northwest territories cripavirus (aNCV), was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. The aNCV IGR sequence adopts a secondary structure similar to contemporary IGR IRES structures, however, there are subtle differences including 105 nucleotides upstream of the IRES of unknown function. Using filter binding assays, we showed that the aNCV IRES could bind to purified ribosomes, and toeprinting analysis pinpointed the start site at a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR can direct translation in vitro in a PKI-dependent manner. Lastly, a chimeric infectious clone swapping in the aNCV IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework for the importance of this viral translational mechanism.

The recent SARS-CoV-2 pandemic has brought many questions over the origin of the virus, the threat it poses to animals both in the wild and captivity, and the risks of a permanent viral reservoir developing in animals. Animal experiments have shown that a variety of animals can become infected with the virus. While coronaviruses have been known to infect animals for decades, the true intermediate host of the virus has not been identified, with no cases of SARS-CoV-2 in wild animals. The screening of wild, farmed, and domesticated animals is necessary to help us understand the virus and its origins and prevent future outbreaks of both COVID-19 and other diseases. There is intriguing evidence that farmed mink infections (acquired from humans) have led to infection of other farm workers in turn, with a recent outbreak of a mink variant in humans in Denmark. A thorough examination of the current knowledge and evidence of the ability of SARS-CoV-2 to infect different animal species is therefore vital to evaluate the threat of animal to human transmission and reverse zoonosis.

Hepatitis E virus (HEV) infection is widespread among domestic pigs, industrial swine, and wild boars in Bulgaria. The aim of the current research was to present the HEV seroprevalence among blood donors in Bulgaria. In the present study, 555 blood donors (479 males and 76 females) were enrolled from five districts in the country (Shumen, Pleven, Stara Zagora, Plovdiv, and Sofia districts). All blood samples were tested for anti-HEV IgG using the recomWell HEV IgG ELISA test (Mikrogen GmbH, Neuried, Germany). Each participating donor completed a short, structured, and specific questionnaire to document data on the current study. Anti-HEV IgG positive results were detected in 144 (25.9%) blood donors, including 129 (26.9%) males and 15 (19.7%) females. The established HEV seropositivity was 28.8% (23/80) in Shumen district, 23.2% (22/95) in Pleven district, 27.1% (38/140) in Stara Zagora district, 27.5% (44/160) in Plovdiv district, and 21.3% (17/80) in Sofia district. A high HEV seroprevalence was found for persons who declared that they were general hunters (48.7%; 19/39; p = 0.001) and hunters of wild boars (51.6%; 16/31; p = 0.001). We present the first seroprevalence rates of HEV infection in blood donors from Bulgaria. The results of our research showed high HEV seropositivity among blood donors.

BK polyomavirus nephropathy (BKVN) and allograft rejection are two closely-associated diseases on opposite ends of the immune scale in kidney transplant recipients. The principle of balancing the immune system remains the mainstay of therapeutic strategy. While patient outcomes can be improved through screening, risk factors identification, and rapid reduction of immunosuppressants, a lack of standard curative therapy is the primary concern during clinical practice. Additionally, difficulty in pathological differential diagnosis and clinicopathology's dissociation pose problems for a definite diagnosis. This article discusses the delicate evaluation needed to optimize immunosuppression and reviews recent advances in molecular diagnosis and immunological therapy for BKVN patients. New biomarkers for BKVN diagnosis are under development. For example, measurement of virus-specific T cell level may play a role in steering immunosuppressants. The development of cellular therapy may provide prevention, even a cure, for BKVN, a complex post-transplant complication.

BACKGROUND: Efficacy for cervical cancer prevention of opportunistic HPV vaccination in post-pubertal girls is lower than in 11-year-olds. METHODS: Women born between 1986 and 1992 vaccinated at 15-25 years of age (at least one dose of 4-valent HPV vaccine) and screened at 24-27 years of age were included. Frequency of opportunistic vaccination, overall and by birth cohort, was calculated; screening outcomes were compared between vaccinated and unvaccinated women. RESULTS: Overall, 4718 (4.9%) HPV-vaccinated, and 91,512 unvaccinated, women were studied. The frequency of vaccination increased by birth cohort, ranging between 1.8% and 9.8%; age at vaccination decreased progressively by birth cohort (p < 0.0001). Participation in screening was 60.8% among vaccinated, and 56.6% among unvaccinated, women (p < 0.0001). Detection rates (DR) for high-grade lesions were lower in vaccinated women (2.11‰ vs. 3.85‰ in unvaccinated, for CIN3+, p = 0.24; 0.0‰ vs. 0.22‰ for cancer). The DR of CIN3+ increased with age at vaccination, scoring respectively 0.0‰, 0.83‰, and 4.68‰ for women vaccinated when they were 15-16, 17-20, and 21-25 years old (p = 0.17). CONCLUSIONS: In comparison to unvaccinated women, higher compliance with cervical cancer screening invitation and lower CIN3+ DR among vaccinated women was observed. Age at vaccination was inversely correlated to vaccination efficacy.

On 5 November 2020, a confirmed outbreak due to an H5N8 highly pathogenic avian influenza virus (HPAIV) occurred at an egg-hen farm in Kagawa prefecture (western Japan). This virus, A/chicken/Kagawa/11C/2020 (Kagawa11C2020), was the first HPAI poultry isolate in Japan in 2020 and had multiple basic amino acids-a motif conferring high pathogenicity to chickens-at the hemagglutinin cleavage site. Mortality of chickens was 100% through intravenous inoculation tests performed according to World Organization for Animal Health criteria. Phylogenetic analysis showed that the hemagglutinin of Kagawa11C2020 belongs to clade 2.3.4.4B of the H5 Goose/Guangdong lineage and clusters with H5N8 HPAIVs isolated from wild bird feces collected in Hokkaido (Japan) and Korea in October 2020. These H5N8 HPAIVs are closely related to H5N8 HPAIVs isolated in European countries during the winter of 2019-2020. Intranasal inoculation of chickens with 106 fifty-percent egg infectious doses of Kagawa11C2020 revealed that the 50% chicken lethal dose was 104.63 and the mean time to death was 134.4 h. All infected chickens demonstrated viral shedding beginning on 2 dpi-before clinical signs were observed. These results suggest that affected chickens could transmit Kagawa11C2020 to surrounding chickens in the absence of clinical signs for several days before they died.

Passive virus surveillance was performed in twenty-nine salmon and trout farms from seven provinces and districts in China during the period 2017-2020. A total of 25 infectious pancreatic necrosis virus (IPNV) isolates were obtained, mainly from rainbow trout (Oncorhynchus mykiss). The molecular evolution of these Chinese IPNV isolates and the previously reported Chinese IPNV strains ChRtm213 and WZ2016 was analyzed, based on their VP2 gene coding region sequences (CDS). All 27 Chinese IPNV isolates clustered within genogroups I and V, with 24 of the IPNV isolates belonging to genogroup I (including ChRtm213 and WZ2016), and only three isolates clustering in genogroup V. The Chinese genogroup I IPNV isolates lacked diversity, composing six haplotypes with 41 polymorphic sites, and the identity of nucleotide and amino acid sequences among the entire VP2 gene CDS from these isolates was 97.44%-100% and 98.19%-100%, respectively. Divergence time analyses revealed that the Chinese genogroup I IPNV isolates likely diverged from Japanese IPNV isolates in 1985 (95% highest posterior density (HPD), 1965-1997), and diverged again in 2006 (95% HPD, 1996-2013) in China. Each of the three Chinese genogroup V IPNV isolates has a unique VP2 gene CDS, with a total of 21 polymorphic sites; the identity of nucleotide and amino acid sequences among all VP2 gene CDS from these isolates was 98.5%-99.5% and 98.6%-99.0%, respectively. The data demonstrate that genogroups I and V are more likely the currently prevalent Chinese IPNV genotypes.

I was fortunate to be associated with the lab of Stephen Oroszlan at the US National Cancer Institute from ~1982 until his conversion to Emeritus status in 1995. His lab made groundbreaking discoveries on retroviral proteins during that time, including many features that could not have been inferred or anticipated from straightforward sequence information. Building on the Oroszlan lab results, my colleagues and I demonstrated that the zinc fingers in nucleocapsid proteins play a crucial role in genomic RNA encapsidation; that the N-terminal myristylation of the Gag proteins of many retroviruses is important for their association with the plasma membrane before particle assembly is completed; and that gammaretroviruses initially synthesize their Env protein as an inactive precursor and then truncate the cytoplasmic tail of the transmembrane protein, activating Env fusogenicity, during virus maturation. We also elucidated several aspects of the mechanism of translational suppression in pol gene expression in gammaretroviruses; amazingly, this is a fundamentally different mechanism of suppression from that in most other retroviral genera.

During October 2020-January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0-6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.

In recent years, novel lineages of invasive non-typhoidal Salmonella (iNTS) serovars Typhimurium and Enteritidis have been identified in patients with bloodstream infection in Sub-Saharan Africa. Here, we isolated and characterised 32 phages capable of infecting S. Typhimurium and S. Enteritidis, from water sources in Malawi and the UK. The phages were classified in three major phylogenetic clusters that were geographically distributed. In terms of host range, Cluster 1 phages were able to infect all bacterial hosts tested, whereas Clusters 2 and 3 had a more restricted profile. Cluster 3 contained two sub-clusters, and 3.b contained the most novel isolates. This study represents the first exploration of the potential for phages to target the lineages of Salmonella that are responsible for bloodstream infections in Sub-Saharan Africa.

Lassa virus (LASV) is a rodent-borne arenavirus circulating in West African regions that causes Lassa fever (LF). LF is normally asymptomatic at the initial infection stage, but can progress to severe disease with multiorgan collapse and hemorrhagic fever. To date, the therapeutic choices are limited, and there is no approved vaccine for avoiding LASV infection. Adenoviral vector-based vaccines represent an effective countermeasure against LASV because of their safety and adequate immunogenicity, as demonstrated in use against other emerging viral infections. Here, we constructed and characterized a novel Ad5 (E1-, E3-) vectored vaccine containing the glycoprotein precursor (GPC) of LASV. Ad5-GPCLASV elicited both humoral and cellular immune responses in BALB/c mice. Moreover, a bioluminescent imaging-based BALB/c mouse model infected with GPC-bearing and luciferase-expressing replication-incompetent LASV pseudovirus was utilized to evaluate the vaccine efficacy. The bioluminescence intensity of immunized mice was significantly lower than that of control mice after being inoculated with LASV pseudovirus. This study suggests that Ad5-GPCLASV represents a potential vaccine candidate against LF.

Varicella-Zoster virus (VZV) is a human herpesvirus that causes varicella (chickenpox) as a primary infection, and, following a variable period of ganglionic latency in neurons, it reactivates to cause herpes zoster (shingles). An analysis of VZV infection in cultures of neural cells, in particular when these have been obtained from induced pluripotent stem cells (iPSCs) or neural stem cells consisting of highly purified neuronal cultures, has revealed much data that may be of neurobiological significance. Early studies of VZV infection of mature cultured neural cells were mainly descriptive, but more recent studies in homogeneous neural stem cell cultures have used both neuronal cell markers and advanced molecular technology. Two general findings from such studies have been that (a) VZV infection of neurons is less severe, based on several criteria, than that observed in human fibroblasts, and (b) VZV infection of neurons does not lead to apoptosis in these cells in contrast to apoptosis observed in fibroblastic cells. Insights gained from such studies in human neural stem cells suggest that a less severe initial lytic infection in neurons, which are resistant to apoptosis, is likely to facilitate a pathological pathway to a latent state of the virus in human ganglia.

This study was aimed to understand the temporal and spatial epidemiology of peste des petits ruminants (PPR) in India using national surveillance data available in the National Animal Diseases Referral Expert System (NADRES) along with its control plan undertaken. On analysis of the outbreaks/cases reports in sheep and goats in NADRES database from 1995 to 2019, it was observed that PPR features among the top ten diseases and stands first among viral diseases, and among reported deaths, PPR accounts for 36% of mortality in sheep and goats. PPR outbreaks occur round the year in all the seasons but are encountered most frequently during the lean period especially, in the winter season (January to February) in different regions/zones. The reported outbreaks have been progressively declined in most of the states in India due to the implementation of a mass vaccination strategic program since 2011. On state-wise analysis, the PPR risk-areas showed wide variations with different levels of endemicity. Andhra Pradesh, West Bengal, and Karnataka were the top three outbreaks reported states during 1995-2010, whereas Jharkhand and West Bengal states reported more outbreaks during 2011-2015 and 2016-2019 periods. The temporal and spatial distribution of PPR in India provides valuable information on the hotspot areas/zones to take appropriate policy decisions towards its prevention and control in different regions/zones of India. The study also identifies when and where intensive surveillance and vaccination along with biosecurity measures need to be implemented for the control and eradication of the disease from India in consonance with the PPR Global Control and Eradication Strategy.

Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC50 = 0.31 μM), strongly stabilized CA hexamer (ΔTm = 8.7 °C), and exhibited substantially enhanced metabolic stability (t1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.

Ross River virus (RRV) is the most medically significant mosquito-borne virus of Australia, in terms of human morbidity. RRV cases, characterised by febrile illness and potentially persistent arthralgia, have been reported from all Australian states and territories. RRV was the cause of a large-scale epidemic of multiple Pacific Island countries and territories (PICTs) from 1979 to 1980, involving at least 50,000 cases. Historical evidence of RRV seropositivity beyond Australia, in populations of Papua New Guinea (PNG), Indonesia and the Solomon Islands, has been documented. We describe the genomic characterisation and timescale analysis of the first isolate of RRV to be sampled from PNG to date. Our analysis indicates that RRV has evolved locally within PNG, independent of Australian lineages, over an approximate 40 year period. The mean time to most recent common ancestor (tMRCA) of the unique PNG clade coincides with the initiation of the PICTs epidemic in mid-1979. This may indicate that an ancestral variant of the PNG clade was seeded into the region during the epidemic, a period of high RRV transmission. Further epidemiological and molecular-based surveillance is required in PNG to better understand the molecular epidemiology of RRV in the general Australasian region.

In 2014, high-throughput sequencing of libraries of total DNA from olive trees allowed the identification of two geminivirus-like contigs. After conventional resequencing of the two genomic DNAs, their analysis revealed they belonged to the same viral entity, for which the provisional name of Olea europaea geminivirus (OEGV) was proposed. Although DNA-A showed a genome organization similar to that of New World begomoviruses, DNA-B had a peculiar ORF arrangement, consisting of a movement protein (MP) in the virion sense and a protein with unknown function on the complementary sense. Phylogenetic analysis performed either on full-length genome or on coat protein, replication associated protein (Rep), and MP sequences did not endorse the inclusion of this virus in any of the established genera in the family Geminiviridae. A survey of 55 plants revealed that the virus is widespread in Apulia (Italy) with 91% of the samples testing positive, although no correlation of OEGV with a disease or specific symptoms was encountered. Southern blot assay suggested that the virus is not integrated in the olive genome. The study of OEGV-derived siRNA obtained from small RNA libraries of leaves and fruits of three different cultivars, showed that the accumulation of the two genomic components is influenced by the plant genotype while virus-derived-siRNA profile is in line with other geminivirids reported in literature. Single-nucleotide polymorphism (SNP) analysis unveiled a low intra-specific variability.

This retrospective cohort study aims to investigate interferon (IFN)-associated retinopathy incidence in patients with chronic hepatitis C virus (HCV) infection treated with pegylated interferon (PegIFN) plus ribavirin (RBV). We selected 1688 patients undergoing PegIFN/RBV therapy for HCV (HCV-treated cohort), 3376 patients not receiving HCV treatment (HCV-untreated cohort) and 16,880 controls without HCV (non-HCV cohort) from the Taiwan Longitudinal Health Insurance Database. The patients were frequency-matched by age, sex, and index date at a 1:2:10 ratio, and followed up until the end of 2013. Cox proportional hazard regression models were used to compare the incidences of any retinal vascular events, including subtypes, among the three cohorts. Compared with the non-HCV cohort, the HCV-treated cohort had a significantly increased risk of retinopathy (hazard ratio (HR) = 4.98, 95% confidence interval (CI): 2.02-12.3). The risk was particularly prominent for retinal hemorrhage (HR = 12.7, 95% CI: 3.78-42.9). When the HCV-untreated cohort was used as the reference, the aforementioned HRs increased to 9.02 (95% CI: 3.04-26.8) and 32.3 (95% CI: 3.94-265), respectively. This study suggested that PegIFN/RBV therapy significantly increased the risk of retinal hemorrhage but not retinal vascular occlusions in the HCV-treated cohort.

Over-immunosuppressed kidney transplant recipients are susceptible to malignancies and BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN). This study aimed to verify the association between BKPyV infection and urinary tract cancers (UTC). A total of 244 kidney transplant recipients were enrolled at Chang Gung Memorial Hospital from June 2000 to February 2020. Biopsy-proven BKPyVAN patients (n = 17) had worse kidney function (eGFR: 26 ± 13.7 vs. 47.8 ± 31.0 mL/min/1.73 m2). The 5-year allograft survival rates for patients with and without BKPyVAN were 67% and 93%, respectively (p = 0.0002), while the 10-year patient survival was not different between the two groups. BKPyVAN patients had a significantly higher incidence of UTC compared to the non-BKPyVAN group (29.4% vs. 6.6%). Kaplan-Meier analysis showed that the UTC-free survival rate was significantly lower in BKPyVAN patients, and the onset of UTC was significantly shorter in BKPyVAN patients (53.4 vs. 108.9 months). The multivariate logistic regression analysis demonstrated that age (RR = 1.062) and BKVAN (RR = 6.459) were the most significant risk factors for the development of UTC. Our study demonstrates that BKPyVAN patients have greater allograft losses, higher incidence, a lower cancer-free survival rate, and an earlier onset with a higher relative risk of developing UTC compared to non-BKPyVAN patients.

Aeromonas hydrophila is an opportunistic pathogen that infects fish, amphibians, mammals, and humans. This study isolated a myophage, vB_AhyM_Ahp2 (Ahp2), that lytically infects A. hydrophila. We observed that 96% of the Ahp2 particles adsorbed to A. hydrophila within 18 min. Ahp2 also showed a latent period of 15 min with a burst size of 142 PFU/cell. This phage has a linear double-stranded DNA genome of 47,331 bp with a GC content of 57%. At least 20 Ahp2 proteins were detected by SDS-polyacrylamide gel electrophoresis; among them, a 40-kDa protein was predicted as the major capsid protein. Sequence analysis showed that Ahp2 has a genome organization closely related to a group of Aeromonas phages (13AhydR10RR, 14AhydR10RR, 85AhydR10RR, phage 3, 32 Asp37, 59.1), which infect Aeromonas hydrophila and Aeromonas salmonicida. The tail module encompassing ORF27-29 in the Ahp2 genome was present in all Aeromonas phages analyzed in this study and likely determines the host range of the virus. This study found that Ahp2 completely lyses A. hydrophila AH300206 in 3.5 h at a MOI of 0.0001 and does not lysogenize its host. Altogether, these findings show that Ahp2 is a lytic Aeromonas phage and could be a candidate for therapeutic phage cocktails.

One important factor for successful disease management is the ability to rapidly and accurately identify the causal agent. Plant viruses cause severe economic losses and pose a serious threat to sustainable agriculture. Therefore, optimization of the speed, sensitivity, feasibility, portability, and accuracy of virus detection is urgently needed. Here, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid diagnostic method utilizing the CRISPR-Cas12a system for detecting two geminiviruses, tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV), which have single-stranded DNA genomes. Our assay detected TYLCV and ToLCNDV in infected plants with high sensitivity and specificity. Our newly developed assay can be performed in ~1 h and provides easy-to-interpret visual readouts using a simple, low-cost fluorescence visualizer, making it suitable for point-of-use applications.

Human cytomegalovirus (HCMV), a member of the betaherpesvirinae, can cause life-threatening diseases. HCMV is globally widespread, with a seroprevalence in adults varying from 50 to 100%. HCMV infection is rarely of significant consequence in immunocompetent individuals. However, although immune control is efficient, it cannot achieve the clearance of the virus. HCMV persists lifelong in the infected host and reactivates in certain circumstances. In neonates and in immunocompromised adults, HCMV is a serious pathogen that can cause fatal organ damage. Different antiviral compounds alone or in combination have been used for the treatment of HCMV diseases. In clinical use, mutations in the viral DNA polymerase or the terminase confer resistance to ganciclovir, foscarnet, cidofovir, and letermovir. There is an urgent need to find new well-tolerated compounds supporting different modes of action. The list of novel small molecules that might have anti-HCMV activity has grown in recent years. In this short review, a selection of compounds in clinical trials and novel inhibitors targeting host-cell factors or viral proteins is presented, and their modes of action, described.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious and pathogenic virus causing high morbidity and mortality, especially in newborn piglets. There remain problems with contemporary PEDV vaccines, in part because of the rapid variation of PEDV, poor conferred immunity, and numerous side effects. The ability to produce PEDV-neutralizing antibodies suggests that we may be able to increase the success rate of PEDV prevention in piglets using these antibodies. In this study, we produced an anti-PEDV S protein monoclonal antibody (anti-PEDV mAb-2) that neutralized PEDV-CV777 (a G1 strain), PEDV-SDSX16 and PEDV-Aj1102 (two G2 strains). In vivo challenge experiments demonstrated that anti-PEDV mAb-2 inhibited the PEDV infection in piglets. We also produced three HEK293 cell lines that expressed anti-PEDV mAb-2. Overall, our study showed that anti-PEDV mAb-2 produced from hybridoma supernatants effectively inhibited PEDV infection in piglets, and the recombinant HEK293 cell lines expressed anti-PEDV mAb-2 genes.

Nuclear egress is an essential process in the replication of human cytomegalovirus (HCMV), as it enables the migration of newly formed viral capsids from the nucleus into the cytoplasm. Inhibition of the HCMV core nuclear egress complex (core NEC), composed of viral proteins pUL50 and pUL53, has been proposed as a potential new target for the treatment of HCMV infection and disease. Here, we present a new type of small molecule inhibitors of HCMV core NEC formation, which inhibit the pUL50-pUL53 interaction at nanomolar concentrations. These inhibitors, i.e., verteporfin and merbromin, were identified through the screening of the Prestwick Chemical Library® of approved drug compounds. The inhibitory effect of merbromin is both compound- and target-specific, as no inhibition was seen for other mercury-organic compounds. Furthermore, merbromin does not inhibit an unrelated protein-protein interaction either. More importantly, merbromin was found to inhibit HCMV infection of cells in three different assays, as well as to disrupt HCMV NEC nuclear rim formation. Thus, while not being an ideal drug candidate by itself, merbromin may serve as a blueprint for small molecules with high HCMV core NEC inhibitory potential, as candidates for novel anti-herpesviral drugs.

Porcine epidemic diarrhea virus (PEDV) can cause intestinal infection in neonatal piglets through the nasal cavity. A process in which CD3+ T cells carry PEDV plays a key role. However, the modes through which PEDV bridles CD3+ T cells as a vehicle for migration to the intestinal epithelium have not been clarified. In this study, we first demonstrated that PEDV could survive in blood-derived CD3+ T cells for several hours, depending on the multiplicity of infection. In addition, PEDV preferentially survived in CD4+ T cells over CD8+ T cells. Moreover, viral transmission was mediated by cell-to-cell contact between mesenteric lymph-node-derived CD3+ T cells, but did not occur in blood-derived CD3+ T cells. Following an increase in gut-homing integrin α4β7, blood-derived CD3+ T cells carrying PEDV migrated to the intestines via blood circulation and transferred the virus to intestinal epithelial cells through cell-to-cell contact in neonatal piglets. Our findings have significant implications for understanding PEDV pathogenesis in neonatal piglets, which is essential for developing innovative therapies to prevent PEDV infection.

Chronic hepatitis B virus (HBV) causes serious clinical problems, such as liver cirrhosis and hepatocellular carcinoma. Current antiviral treatments suppress HBV; however, the clinical cure rate remains low. Basic research on HBV is indispensable to eradicate and cure HBV. Genetic alterations are defined by nucleotide substitutions and canonical forms of structural variations (SVs), such as insertion, deletion and duplication. Additionally, genetic changes inconsistent with the canonical forms have been reported, and these have been termed complex SVs. Detailed analyses of HBV using bioinformatical applications have detected complex SVs in HBV genomes. Sequence gaps and low sequence similarity have been observed in the region containing complex SVs. Additionally, insertional motif sequences have been observed in HBV strains with complex SVs. Following the analyses of complex SVs in the HBV genome, the role of SVs in the genetic diversity of orthohepadnavirus has been investigated. SV polymorphisms have been detected in comparisons of several species of orthohepadnaviruses. As mentioned, complex SVs are composed of multiple SVs. On the contrary, SV polymorphisms are observed as insertions of different SVs. Up to a certain point, nucleotide substitutions cause genetic differences. However, at some point, the nucleotide sequences are split into several particular patterns. These SVs have been observed as polymorphic changes. Different species of orthohepadnaviruses possess SVs which are unique and specific to a certain host of the virus. Studies have shown that SVs play an important role in the HBV genome. Further studies are required to elucidate their virologic and clinical roles.

Although the antibody response induced by primary vaccination with Fel-O-Vax® FIV (three doses, 2-4 weeks apart) is well described, the antibody response induced by annual vaccination with Fel-O-Vax® FIV (single dose every 12 months after primary vaccination) and how it compares to the primary antibody response has not been studied. Residual blood samples from a primary FIV vaccination study (n = 11), and blood samples from cats given an annual FIV vaccination (n = 10), were utilized. Samples from all 21 cats were tested with a commercially available PCR assay (FIV RealPCRTM), an anti-p24 microsphere immunoassay (MIA), an anti-FIV transmembrane (TM; gp40) peptide ELISA, and a range of commercially available point-of-care (PoC) FIV antibody kits. PCR testing confirmed all 21 cats to be FIV-uninfected for the duration of this study. Results from MIA and ELISA testing showed that both vaccination regimes induced significant antibody responses against p24 and gp40, and both anti-p24 and anti-gp40 antibodies were variably present 12 months after FIV vaccination. The magnitude of the antibody response against both p24 and gp40 was significantly higher in the primary FIV vaccination group than in the annual FIV vaccination group. The differences in prime versus recall post-vaccinal antibody levels correlated with FIV PoC kit performance. Two FIV PoC kits that detect antibodies against gp40, namely Witness® and Anigen Rapid®, showed 100% specificity in cats recently administered an annual FIV vaccination, demonstrating that they can be used to accurately distinguish vaccination and infection in annually vaccinated cats. A third FIV PoC kit, SNAP® Combo, had 0% specificity in annually FIV-vaccinated cats, and should not be used in any cat with a possible history of FIV vaccination. This study outlines the antibody response to inactivated Fel-O-Vax® FIV whole-virus vaccine, and demonstrates how best to diagnose FIV infection in jurisdictions where FIV vaccination is practiced.

The use of Natalizumab in Multiple Sclerosis (MS) can cause the reactivation of the polyomavirus JC (JCPyV); this may result in the development of progressive multifocal leukoencephalopathy (PML), a rare and usually lethal disease. JCPyV infection is highly prevalent in worldwide population, but the detection of anti-JCPyV antibodies is not sufficient to identify JCPyV infection, as PML can develop even in patients with negative JCPyV serology. Better comprehension of the JCPyV biology could allow a better understanding of JCPyV infection and reactivation, possibly reducing the risk of developing PML. Here, we investigated whether JCPyV miR-J1-5p-a miRNA that down-regulates the early phase viral protein T-antigen and promotes viral latency-could be detected and quantified by digital droplet PCR (ddPCR) in urine of 25 Natalizumab-treated MS patients. A 24-month study was designed: baseline, before the first dose of Natalizumab, and after 1 (T1), 12 (T12) and 24 months (T24) of therapy. miR-J1-5p was detected in urine of 7/25 MS patients (28%); detection was possible in three cases at T24, in two cases at T12, in one case at T1 and T12, and in the last case at baseline and T1. Two of these patients were seronegative for JCPyV Ab, and viral DNA was never found in either urine or blood. To note, only in one case miR-J1-5p was detected before initiation of Natalizumab. These results suggest that the measurement of miR-J1-5p in urine, could be a biomarker to monitor JCPyV infection and to better identify the possible risk of developing PML in Natalizumab-treated MS patients.

The emergence and re-emergence of arboviruses have occurred for centuries [...].

Genetic profiles of hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC) in Asians remain elusive. A 10-year prospective cohort study was conducted with 1043 consecutive HCV Ab-positive Taiwanese surveyed with 13 single nucleotide polymorphisms (SNPs). Of 1043, 589 (56.5%) had baseline MC, 934 (89.5%) had positive HCV RNA, 796 completed anti-HCV therapy, and 715 had sustained virological responses (SVRs). SNP associations were surveyed withgenotypic, allelic, trend, permutation and multivariate analyses. At baseline, higher male sex and MC rates were noted in HCV RNA-positive than RNA-negative patients; higher female sex and positive HCV RNA rates but lower HCV RNA levels were noted in patients with than those without MC. Baseline associations were: HLA II-rs9461776 A allele, IFNL3-rs12979860 T allele, SERPINE1-rs6976053 C allele and MC with HCV RNA positivity; IFNL3-rs12979860 C allele, ARNTL-rs6486122 T allele and HCV RNA positivity with baseline MC. In SVR patients, RETN-rs1423096 C allele and SERPINE1-rs6976053 T allele were associated with 24-week and 10-year post-therapy MC, respectively. Conclusions: HCV RNA, IFNL3-rs12979860 and ARNTL-rs6486122 were associated with baseline MC; RETN-rs1423096 and SERPINE1-rs6976053 were associated with short- and long-term post-therapy MC in SVR patients, respectively. Links with HCV RNA and immune-associated SNPs suggest MC an immune reaction to expel HCV.

The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage-host systems.

Control strategies that employ real time polymerase chain reaction (RT-PCR) tests for the diagnosis and surveillance of COVID-19 epidemic are inefficient in fighting the epidemic due to high cost, delays in obtaining results, and the need of specialized personnel and equipment for laboratory processing. Cheaper and faster alternatives, such as antigen and paper-strip tests, have been proposed. They return results rapidly, but have lower sensitivity thresholds for detecting virus. To quantify the effects of the tradeoffs between sensitivity, cost, testing frequency, and delay in test return on the overall course of an outbreak, we built a multi-scale immuno-epidemiological model that connects the virus profile of infected individuals with transmission and testing at the population level. We investigated various randomized testing strategies and found that, for fixed testing capacity, lower sensitivity tests with shorter return delays slightly flatten the daily incidence curve and delay the time to the peak daily incidence. However, compared with RT-PCR testing, they do not always reduce the cumulative case count at half a year into the outbreak. When testing frequency is increased to account for the lower cost of less sensitive tests, we observe a large reduction in cumulative case counts, from 55.4% to as low as 1.22% half a year into the outbreak. The improvement is preserved even when the testing budget is reduced by one half or one third. Our results predict that surveillance testing that employs low-sensitivity tests at high frequency is an effective tool for epidemic control.

Erratum for Viruses. 2020 Sep 30;12(10):

Mayaro virus (MAYV) and chikungunya virus (CHIKV) are known for their arthrotropism, but accumulating evidence shows that CHIKV infections are occasionally associated with serious neurological complications. However, little is known about the capacity of MAYV to invade the central nervous system (CNS). We show that human neural progenitors (hNPCs), pericytes and astrocytes are susceptible to MAYV infection, resulting in the production of infectious viral particles. In primary astrocytes, MAYV, and to a lesser extent CHIKV, elicited a strong antiviral response, as demonstrated by an increased expression of several interferon-stimulated genes, including ISG15, MX1 and OAS2. Infection with either virus led to an enhanced expression of inflammatory chemokines, such as CCL5, CXCL10 and CXCL11, whereas MAYV induced higher levels of IL-6, IL-12 and IL-15 in these cells. Moreover, MAYV was more susceptible than CHIKV to the antiviral effects of both type I and type II interferons. Taken together, this study shows that although MAYV and CHIKV are phylogenetically related, they induce different types of antiviral responses in astrocytes. This work is the first to evaluate the potential neurotropism of MAYV and shows that brain cells and particularly astrocytes and hNPCs are permissive to MAYV, which, consequently, could lead to MAYV-induced neuropathology.

(1) Background: A better understanding of COVID-19 dynamics in terms of interactions among individuals would be of paramount importance to increase the effectiveness of containment measures. Despite this, the research lacks spatiotemporal statistical and mathematical analysis based on large datasets. We describe a novel methodology to extract useful spatiotemporal information from COVID-19 pandemic data. (2) Methods: We perform specific analyses based on mathematical and statistical tools, like mathematical morphology, hierarchical clustering, parametric data modeling and non-parametric statistics. These analyses are here applied to the large dataset consisting of about 19,000 COVID-19 patients in the Veneto region (Italy) during the entire Italian national lockdown. (3) Results: We estimate the COVID-19 cumulative incidence spatial distribution, significantly reducing image noise. We identify four clusters of connected provinces based on the temporal evolution of the incidence. Surprisingly, while one cluster consists of three neighboring provinces, another one contains two provinces more than 210 km apart by highway. The survival function of the local spatial incidence values is modeled here by a tapered Pareto model, also used in other applied fields like seismology and economy in connection to networks. Model's parameters could be relevant to describe quantitatively the epidemic. (4) Conclusion: The proposed methodology can be applied to a general situation, potentially helping to adopt strategic decisions such as the restriction of mobility and gatherings.

Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural-functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50-pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.

The genus Flavivirus includes related, unclassified segmented flavi-like viruses, two segments of which have homology with flavivirus RNA-dependent RNA polymerase NS5 and RNA helicase-protease NS3. This group includes such viruses as Jingmen tick virus, Alongshan virus, Yanggou tick virus and others. We detected the Yanggou tick virus in Dermacentor nuttalli and Dermacentor marginatus ticks in two neighbouring regions of Russia. The virus prevalence ranged from 0.5% to 8.0%. We detected RNA of the Alongshan virus in 44 individuals or pools of various tick species in eight regions of Russia. The virus prevalence ranged from 0.6% to 7.8%. We demonstrated the successful replication of the Yanggou tick virus and Alongshan virus in IRE/CTVM19 and HAE/CTVM8 tick cell lines without a cytopathic effect. According to the phylogenetic analysis, we divided the Alongshan virus into two groups: an Ixodes persulcatus group and an Ixodes ricinus group. In addition, the I. persulcatus group can be divided into European and Asian subgroups. We found amino acid signatures specific to the I. ricinus and I. persulcatus groups and also distinguished between the European and Asian subgroups of the I. persulcatus group.

Erratum in Viruses. 2022 Dec 21;15(1):

Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3Dpol (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the Picornaviridae and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.

Respiratory Syncytial Virus (RSV) causes severe inflammation and airway pathology in children and the elderly by infecting the epithelial cells of the upper and lower respiratory tract. RSV replication is sensed by intracellular pattern recognition receptors upstream of the IRF and NF-κB transcription factors. These proteins coordinate an innate inflammatory response via Bromodomain-containing protein 4 (BRD4), a protein that functions as a scaffold for unknown transcriptional regulators. To better understand the pleiotropic regulatory function of BRD4, we examine the BRD4 interactome and identify how RSV infection dynamically alters it. To accomplish these goals, we leverage native immunoprecipitation and Parallel Accumulation-Serial Fragmentation (PASEF) mass spectrometry to examine BRD4 complexes isolated from human alveolar epithelial cells in the absence or presence of RSV infection. In addition, we explore the role of BRD4's acetyl-lysine binding bromodomains in mediating these interactions by using a highly selective competitive bromodomain inhibitor. We identify 101 proteins that are significantly enriched in the BRD4 complex and are responsive to both RSV-infection and BRD4 inhibition. These proteins are highly enriched in transcription factors and transcriptional coactivators. Among them, we identify members of the AP1 transcription factor complex, a complex important in innate signaling and cell stress responses. We independently confirm the BRD4/AP1 interaction in primary human small airway epithelial cells. We conclude that BRD4 recruits multiple transcription factors during RSV infection in a manner dependent on acetyl-lysine binding domain interactions. This data suggests that BRD4 recruits transcription factors to target its RNA processing complex to regulate gene expression in innate immunity and inflammation.

The ongoing SARS-CoV-2 pandemic has resulted in an increased need for technologies capable of efficiently disinfecting public spaces as well as personal protective equipment. UV light disinfection is a well-established method for inactivating respiratory viruses. Here, we have determined that broad-spectrum, pulsed UV light is effective at inactivating SARS-CoV-2 on multiple surfaces in vitro. For hard, non-porous surfaces, we observed that SARS-CoV-2 was inactivated to undetectable levels on plastic and glass with a UV dose of 34.9 mJ/cm2 and stainless steel with a dose of 52.5 mJ/cm2. We also observed that broad-spectrum, pulsed UV light is effective at reducing SARS-CoV-2 on N95 respirator material to undetectable levels with a dose of 103 mJ/cm2. We included UV dosimeter cards that provide a colorimetric readout of UV dose and demonstrated their utility as a means to confirm desired levels of exposure were reached. Together, the results presented here demonstrate that broad-spectrum, pulsed UV light is an effective technology for the in vitro inactivation of SARS-CoV-2 on multiple surfaces.

Human noroviruses (hNoVs) cause heavy disease burden worldwide and there is no clinically approved vaccination or antiviral hitherto. In this study, with the use of a zebrafish larva in vivo platform, we investigated the anti-hNoV potentials of fucoidan (from brown algae Fucus vesiculosus) and 2'-Fucosyllactose (2'-FL). As a result, although both fucoidan and 2'-FL were able to block hNoV GII.4 virus-like particle (VLPs) from binding to type A saliva as expected, only fucoidan, but not 2'-FL, was able to inhibit the replication of hNoV GII.P16-GII.4 in zebrafish larvae, indicating the possible needs of higher molecular weights for fucosylated carbohydrates to exert anti-hNoV effect.

Novirhabdoviruses cause large epizootics and economic losses of farmed trout. In this study, we surveyed Viral hemorrhagic septicemia virus and Infectious hematopoietic and necrosis virus (VHSV and IHNV) through both monitoring and investigation of clinical outbreaks reported by farmers in the regions with major rainbow trout production in Iran from 2015 to 2019. RT-PCR assays of the kidney samples and cell culture (EPC/FHM cells) samples confirmed the presence of the viruses, with 9 VHSV and 4 IHNV isolates, in both endemic and new areas of Iran. Sequence analysis of the G gene revealed that VHSV isolates belonged to genogroup Ia, and IHNV isolates were clustered into genogroup E, both typical for isolates from European countries. A haplotype analysis based on non-homologous amino acids of the G gene supports the emergence of two lineages of IHNV from clade 1 (E-1), as well as VHSV clade 2 (Ia-2) of the European genogroups, confirming that VHSV and IHNV isolates in Iran, have originated from Europe possibly via imported eggs.

The effects of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in women on the gestation course and the health of the fetus, particularly in the first and second trimesters, remain very poorly explored. This report describes a case in which the normal development of pregnancy was complicated immediately after the patient had experienced Coronavirus disease 2019 (COVID-19) at the 21st week of gestation. Specific conditions included critical blood flow in the fetal umbilical artery, fetal growth restriction (1st percentile), right ventricular hypertrophy, hydropericardium, echo-characteristics of hypoxic-ischemic brain injury (leukomalacia in periventricular area) and intraventricular hemorrhage at the 25th week of gestation. Premature male neonate delivered at the 26th week of gestation died after 1 day 18 h due to asystole. The results of independent polymerase chain reaction (PCR), mass spectrometry and immunohistochemistry analyses of placenta tissue, umbilical cord blood and child blood jointly indicated vertical transmission of SARS-CoV-2 from mother to the fetus, which we conclude to be the major cause for the development of maternal vascular malperfusion in the studied case.

Classical swine fever (CSF), caused by CSF virus (CSFV), is one of the most devastating viral epizootic diseases of swine in many countries. To control the disease, highly efficacious and safe live attenuated vaccines have been used for decades. However, the main drawback of these conventional vaccines is the lack of differentiability of infected from vaccinated animals (DIVA concept). Advances in biotechnology and our detailed knowledge of multiple basic science disciplines have facilitated the development of effective and safer DIVA vaccines to control CSF. To date, two types of DIVA vaccines have been developed commercially, including the subunit vaccines based on CSFV envelope glycoprotein E2 and chimeric pestivirus vaccines based on infectious cDNA clones of CSFV or bovine viral diarrhea virus (BVDV). Although inoculation of these vaccines successfully induces solid immunity against CSFV, none of them could ideally meet all demands regarding to safety, efficacy, DIVA potential, and marketability. Due to the limitations of the available choices, researchers are still striving towards the development of more advanced DIVA vaccines against CSF. This review summarizes the present status of candidate CSFV vaccines that have been developed. The strategies and approaches revealed here may also be helpful for the development of new-generation vaccines against other diseases.

Seoul virus (SEOV) is a zoonotic orthohantavirus carried by rats. In humans, SEOV can cause hemorrhagic fever with renal syndrome. Recent human SEOV cases described in the USA, United Kingdom, France and the Netherlands were associated with contact with pet or feeder rats. The prevalence of SEOV in these types of rats is unknown. We collected 175 pet and feeder rats (Rattus norvegicus) from private owners, ratteries and commercial breeders/traders in the Netherlands. Lung tissue of the rats was tested using a SEOV real-time RT-qPCR and heart fluid was tested for the presence of antibodies against SEOV. In all three investigated groups, RT-qPCR-positive rats were found: in 1/29 rats from private owners (3.6%), 2/56 rats from ratteries (3.4%) and 11/90 rats from commercial breeders (12.2%). The seroprevalence was largely similar to the prevalence calculated from RT-qPCR-positive rats. The SEOV sequences found were highly similar to sequences previously found in domesticated rats in Europe. In conclusion, SEOV is spread throughout different populations of domesticated rats.

Heightened expression of human endogenous retrovirus (HERV) sequences has been associated with a range of malignancies, including prostate cancer, suggesting that they may serve as useful diagnostic or prognostic cancer biomarkers. We analysed the expression of HERV-K (Gag and Env/Np9 regions), HERV-E 4.1 (Pol and Env regions), HERV-H (Pol) and HERV-W (Gag) sequences in prostate cancer cells lines and normal prostate epithelial cells using qRT-PCR. HERV expression was also analysed in matched malignant and benign prostate tissue samples from men with prostate cancer (n = 27, median age 65.2 years (range 47-70)) and compared to prostate cancer-free male controls (n = 11). Prostate cancer epithelial cell lines exhibited a signature of HERV RNA overexpression, with all HERVs analysed, except HERV-E Pol, showing heightened expression in at least two, but more commonly all, cell lines analysed. Analysis of primary prostate material indicated increased expression of HERV-E Pol but decreased expression of HERV-E Env in both malignant and benign regions of the prostate in men with prostate cancer as compared to those without. Expression of HERV-K Gag was significantly higher in malignant regions of the prostate in men with prostate cancer as compared to matched benign regions and prostate cancer-free men (p < 0.001 for both), with 85.2% of prostate cancers donors showing malignancy-associated upregulation of HERV-K Gag RNA. HERV-K Gag protein was detected in 12/18 (66.7%) malignant tissues using immunohistochemistry, but only 1/18 (5.6%) benign tissue sections. Heightened expression of HERV-K Gag RNA and protein appears to be a sensitive and specific biomarker of prostate malignancy in this cohort of men with prostate carcinoma, supporting its potential utility as a non-invasive, adjunct clinical biomarker.

An evolutionary arms race has been ongoing between retroviruses and their primate hosts for millions of years. Within the last century, a zoonotic transmission introduced the Human Immunodeficiency Virus (HIV-1), a retrovirus, to the human population that has claimed the lives of millions of individuals and is still infecting over a million people every year. To counteract retroviruses such as this, primates including humans have evolved an innate immune sensor for the retroviral capsid lattice known as TRIM5α. Although the molecular basis for its ability to restrict retroviruses is debated, it is currently accepted that TRIM5α forms higher-order assemblies around the incoming retroviral capsid that are not only disruptive for the virus lifecycle, but also trigger the activation of an antiviral state. More recently, it was discovered that TRIM5α restriction is broader than previously thought because it restricts not only the human retroelement LINE-1, but also the tick-borne flaviviruses, an emergent group of RNA viruses that have vastly different strategies for replication compared to retroviruses. This review focuses on the underlying mechanisms of TRIM5α-mediated restriction of retroelements and flaviviruses and how they differ from the more widely known ability of TRIM5α to restrict retroviruses.

BACKGROUND: Both SARS-CoV-2 and influenza virus share similarities such as clinical features and outcome, laboratory, and radiological findings. METHODS: Literature search was done using PubMed to find MEDLINE indexed articles relevant to this study. As of 25 November 2020, the search has been conducted by combining the MeSH words "COVID-19" and "Influenza". RESULTS: Eighteen articles were finally selected in adult patients. Comorbidities such as cardiovascular diseases, diabetes, and obesity were significantly higher in COVID-19 patients, while pulmonary diseases and immunocompromised conditions were significantly more common in influenza patients. The incidence rates of fever, vomiting, ocular and otorhinolaryngological symptoms were found to be significantly higher in influenza patients when compared with COVID-19 patients. However, neurologic symptoms and diarrhea were statistically more frequent in COVID-19 patients. The level of white cell count and procalcitonin was significantly higher in influenza patients, whereas thrombopenia and elevated transaminases were significantly more common in COVID-19 patients. Ground-grass opacities, interlobular septal thickening, and a peripheral distribution were more common in COVID-19 patients than in influenza patients where consolidations and linear opacities were described instead. COVID-19 patients were significantly more often transferred to intensive care unit with a higher rate of mortality. CONCLUSIONS: This study estimated differences of COVID-19 and influenza patients which can help clinicians during the co-circulation of the two viruses.

The concerning increase in HIV-1 resistance argues for prioritizing the development of host-targeting antiviral drugs because such drugs can offer high genetic barriers to the selection of drug-resistant viral variants. Targeting host proteins could also yield drugs that act on viral life cycle events that have proven elusive to inhibition, such as intracellular events of HIV-1 immature capsid assembly. Here, we review small molecule inhibitors identified primarily through HIV-1 self-assembly screens and describe how all act either narrowly post-entry or broadly on early and late events of the HIV-1 life cycle. We propose that a different screening approach could identify compounds that specifically inhibit HIV-1 Gag assembly, as was observed when a potent rabies virus inhibitor was identified using a host-catalyzed rabies assembly screen. As an example of this possibility, we discuss an antiretroviral small molecule recently identified using a screen that recapitulates the host-catalyzed HIV-1 capsid assembly pathway. This chemotype potently blocks HIV-1 replication in T cells by specifically inhibiting immature HIV-1 capsid assembly but fails to select for resistant viral variants over 37 passages, suggesting a host protein target. Development of such small molecules could yield novel host-targeting antiretroviral drugs and provide insight into chronic diseases resulting from dysregulation of host machinery targeted by these drugs.

In New York State, domestic animals are no longer considered rabies vector species, but given their ubiquity with humans, rabies cases in dogs and cats often result in multiple individuals requiring post-exposure prophylaxis. For over a decade, the New York State rabies laboratory has variant-typed these domestic animals to aid in epidemiological investigations, determine exposures, and generate demographic data. We produced a data set that outlined vaccination status, ownership, and rabies results. Our data demonstrate that a large percentage of felines submitted for rabies testing were not vaccinated or did not have a current rabies vaccination, while canines were largely vaccinated. Despite massive vaccination campaigns, free clinics, and education, these companion animals still occasionally contract rabies. Barring translocation events, we note that rabies-positive cats and dogs in New York State have exclusively contracted a raccoon variant. While the United States has made tremendous strides in reducing its rabies burden, we hope these data will encourage responsible pet ownership including rabies vaccinations to reduce unnecessary animal mortality, long quarantines, and post-exposure prophylaxis in humans.

The pestivirus envelope protein Erns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the Erns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the Erns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an Erns-only expression construct, these mutations enhanced the naturally occurring Erns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth.

Diarrhoeal disease is considered an important cause of morbidity and mortality in developing areas, and a large contributor to the burden of disease in children younger than five years of age. This study investigated the prevalence and genogroups of human sapovirus (SV) in children ≤5 years of age in rural communities of Vhembe district, South Africa. Between 2017 and 2020, a total of 284 stool samples were collected from children suffering with diarrhoea (n = 228) and from children without diarrhoea (n = 56). RNA extraction using Boom extraction method, and screening for SV using real-time PCR were done in the lab. Positive samples were subjected to conventional RT-PCR targeting the capsid fragment. Positive sample isolates were genotyped using Sanger sequencing. Overall SV were detected in 14.1% (40/284) of the stool samples (16.7% (38/228) of diarrhoeal and 3.6% (2/56) of non-diarrhoeal samples). Significant correlation between SV positive cases and water sources was noted. Genogroup-I was identified as the most prevalent strain comprising 81.3% (13/16), followed by SV-GII 12.5% (2/16) and SV-GIV 6.2% (1/16). This study provides valuable data on prevalence of SV amongst outpatients in rural and underdeveloped communities, and highlights the necessity for further monitoring of SV circulating strains as potential emerging strains.

The novel SARS-CoV-2 Variant of Concern (VOC)-202012/01 (also known as B.1.1.7), first collected in United Kingdom on 20 September 2020, is a rapidly growing lineage that in January 2021 constituted 86% of all SARS-CoV-2 genomes sequenced in England. The VOC has been detected in 40 out of 46 countries that reported at least 50 genomes in January 2021. We have estimated that the replicative advantage of the VOC is in the range 1.83-2.18 [95% CI: 1.71-2.40] with respect to the 20A.EU1 variant that dominated in England in November 2020, and in range 1.65-1.72 [95% CI: 1.46-2.04] in Wales, Scotland, Denmark, and USA. As the VOC strain will likely spread globally towards fixation, it is important to monitor its molecular evolution. We have estimated growth rates of expanding mutations acquired by the VOC lineage to find that the L18F substitution in spike has initiated a fast growing VOC substrain. The L18F substitution is of significance because it has been found to compromise binding of neutralizing antibodies. Of concern are immune escape mutations acquired by the VOC: E484K, F490S, S494P (in the receptor binding motif of spike) and Q677H, Q675H (in the proximity of the polybasic cleavage site at the S1/S2 boundary). These mutants may hinder efficiency of existing vaccines and expand in response to the increasing after-infection or vaccine-induced seroprevalence.

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.