Despite four decades of research into the human immunodeficiency virus (HIV-1), a successful strategy to eradicate the virus post-infection is lacking. The major reason for this is the persistence of the virus in certain anatomical reservoirs where it can become latent and remain quiescent for as long as the cellular reservoir is alive. The Central Nervous System (CNS), in particular, is an intriguing anatomical compartment that is tightly regulated by the blood-brain barrier. Targeting the CNS viral reservoir is a major challenge owing to the decreased permeability of drugs into the CNS and the cellular microenvironment that facilitates the compartmentalization and evolution of the virus. Therefore, despite effective antiretroviral (ARV) treatment, virus persists in the CNS, and leads to neurological and neurocognitive deficits. To date, viral eradication strategies fail to eliminate the virus from the CNS. To facilitate the improvement of the existing elimination strategies, as well as the development of potential therapeutic targets, the aim of this review is to provide an in-depth understanding of HIV latency in CNS and the onset of HIV-1 associated neurological disorders.

Lychnis mottle virus (LycMoV), family Secoviridae, is one of several viruses recently detected in peony. Given the high prevalence of the virus in the more than 300 samples tested, the population structure of the virus was studied using 48 isolates representing at least 20 cultivars and collected from major producing and propagating states in the United States. The homogeneity of the United States population, based on data from the RNA2 coding region, along with phylogenetic analyses of all publicly available sequences point to the dissemination of the virus through propagation material rather that active vector-mediated transmission.

The B.1.1.7 SARS-CoV-2 strain that has emerged in the UK in early December presents seven mutations and three deletions on S-protein structure that could lead to a more infective strain. The P681H mutation in the "PRRAR" furin cleavage site might affect the binding affinity to furin enzyme and hence its infectivity. Therefore, in this study, various structural bioinformatics approaches were used to model the S-protein structure with the B.1.1.7 variant amino acid substitutions and deletions. In addition to modelling the binding of furin to the cleavage site of the wild-type and the B.1.1.7 variant. Conclusively the B.1.1.7 variant resulted in dynamic stability, conformational changes and variations in binding energies in the S-protein structure, resulting in a more favourable binding of furin enzyme to the SARS-CoV-2 S-protein.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). SP40 peptide was previously identified to inhibit EV-A71 strains from genotypes A, B and C. However, the stability and antiviral activity of SP40 peptide in human serum are yet to be established. To address this, we evaluated the stability and anti-EV-A71 activity of SP40 peptide after incubation in 25 % human serum. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC/MS) were utilized to evaluate serum stability and cleavage patterns of SP40 peptide after incubation in human serum. Cell protection assay was used to evaluate the anti-EV-A71 activity of SP40 peptide after incubation in human serum and to identify the minimal active sequence of SP40 peptide that retained antiviral activity. The results showed that the SP40 peptide was stable in human serum with 56 % of the full-length SP40 peptide being detected after 48 h incubation in human serum. The SP40 peptide was mainly cleaved by exopeptidases and no endoprotease recognition sites were identified within the SP40 peptide. Cell protection assays revealed that the SP40 peptide retained substantial activity after 24 and 48 h incubation in human serum. Furthermore, the data revealed that three amino acids at the N-terminus and one amino acid at the C-terminus of the SP40 peptide were dispensable for its antiviral activity. Importantly, the four truncated peptides displayed better potency than the full-length SP40 peptide. Overall, this study provided insights into the stability and activity of SP40 peptide in human serum and will facilitate the development of SP40 peptide as an anti-EV-A71 agent.

The genus Flavivirus incorporates bona fide arboviruses, as well as others viruses with restricted replication in insect cells. Among the latter, a large monophyletic cluster of viruses, known as cISF (classical insect-specific flaviviruses), has been sampled in many species of mosquitoes collected over a large geographic range. In this study, we investigated nucleotide and protein sequences with a suite of molecular characterization approaches including genetic distance, Shannon entropy, selective pressure analysis, polymorphism identification, principal coordinate analysis, likelihood mapping, phylodynamic reconstruction, and spatiotemporal dispersal, to further characterize this diverse group of insect-viruses. The different lineages and sub-lineages of viral sequences presented low sequence diversity and entropy (though some displayed lineage-specific polymorphisms), did not show evidence of frequent recombination and evolved under strong purifying selection. Moreover, the reconstruction of the evolutionary history and spatiotemporal dispersal was highly impacted by overall low signals of sequence divergence throughout time but suggested that cISF distribution in space and time is dynamic and may be dependent on human activities, including commercial trading and traveling.

Following Kobayashi and colleagues' seminal paper in 20171, in the past four years the rotavirus (RV) field has witnessed a burst in research and publications based on the use of a fully plasmid-based RV reverse genetics systems and subsequent modifications2,3. However, in most cases, the rotaviral strain under interrogation has been the prototypic simian RV SA11-L2 strain (G3P[2]). Of note, a variety of other weakly-to-modestly replication-competent animal and human RV strains, bioluminescent and fluorescent reporter viruses, and clinical isolates of human RVs have proved hard or impossible to rescue using the original reverse genetics system2,4, highlighting a critical need to further enhance the recovery efficiency and expand the rescue tool kit. A number of further modifications of the initial reverse genetics system have enabled the rescue of other RV strains such as the human RV KU and CDC-9 strains, and a murine RV D6/2-like strain4,5. Here, we discuss future possible modifications of existing RV reverse genetics systems to further increase efficiency based on past experience with the improvement of influenza A virus recovery. The development of RV to accommodate the insertion and expression of heterologous sequences has substantial potential in the design of next-generation RV vaccine candidates and enteric viral vectors.

Human immunodeficiency virus type one (HIV-1) infection is one of the major public health problems worldwide. Effective control of HIV-1 epidemic relies on early diagnosis of HIV-1 infection by using simple, rapid point-of-care test (POCT). An integrated assay was developed and evaluated in this study to combine a real-time isothermal reverse-transcription recombinase-aided amplification (rRT-RAA) and CRISPR Cas12a-mediated detection for HIV-1. The testing results could be directly observed with naked eye using a blue light imager, making it a suitable on-site testing assay. Our preliminary data indicated that the assay was capable of detecting 20 copies of purified HIV-1 DNA or RNA per reaction or as low as 123 copies/ml of HIV-1 viral load in clinical samples. When screening 155 clinical samples with or without HIV-1 infection, the sensitivity and specificity of the rRT-RAA assay were 98.95% (94/95) and 100% (60/60), respectively. The coefficient value was 0.986 when compared with the Chinese FDA approved HIV-1 RT-qPCR assay. Furthermore, the newly developed HIV-1 rRT-RAA assay could detect the major HIV-1 genotypes CRF01_AE, CRF07_BC, CRF08_BC, CRF08_BC and subtype B in China. Our preliminary results indicated that the rRT-RAA assay or its combination with CRISPR Cas12a-mediated detection could serve as a rapid, convenient, and robust assay for HIV-1 detection.

Long non-coding RNA-ATB (LncRNA-ATB) which is activated by transforming growth factor-β (TGF-β), is a key regulator of TGF-β signaling pathway. TGF-β plays an important role in various pathogenic processes, from inflammation and fibrosis to cirrhosis and cancer. In this study, we evaluated the plasma levels of lncRNA-ATB in patients with hepatitis B virus (HBV)-related cirrhosis and non-cirrhotic patients with chronic hepatitis B (CHB) and investigated the clinical values. Plasma samples were collected from 44 HBV-related cirrhosis patients, 45 non-cirrhotic CHB and 75 healthy controls. Briefly, after total RNA extraction and cDNA synthesis, quantitative real-time PCR (qPCR) was performed to detect plasma lncRNA-ATB levels. Results show the plasma levels of lncRNA-ATB in HBV-related cirrhosis patients were significantly higher in comparison to healthy controls (Fold change=2.60, p value=0.04). Also, we determined plasma levels of lncRNA-ATB as a specific biomarker of HBV-related cirrhosis (AUC=0.65, p value=0.03, Sensitivity 61.36%; Specificity 70.00%). In addition to, we investigated the plasma levels of lncRNA-ATB in non-cirrhotic CHB patients were significantly lower than healthy controls (Fold change= 0.33, p value=0.01). We also indicated plasma lncRNA-ATB levels were as a sensitive biomarker for diagnosis of non-cirrhotic CHB patients compared with healthy (AUC=0.66, p value=0.00, Sensitivity 71.11%; Specificity 57.78%). According to our results, circulating lncRNA-ATB has good specificity for diagnosing hepatitis B virus (HBV)-related cirrhosis and good sensitivity for diagnosis of non-cirrhotic chronic hepatitis B (CHB) patients.

Specific miRNA families are involved in susceptibility or antiviral immunity in plants. Manihot esculenta Crantz (cassava) is a perennial plant that is an important food security crop in sub-Saharan Africa. Cassava is susceptible to several begomoviruses that cause cassava mosaic disease (CMD). In this study, we investigated the leaf miRNAome response in a tolerant (TME3) and susceptible (T200) cassava landrace challenged with South African cassava mosaic virus. RNAseq was performed on leaf samples at 12, 32 and 67 days post infection (dpi), representing early, symptomatic and late persistent stages of CMD infection. Significantly, distinct profiles of conserved miRNA family expression between the T200 and TME3 landraces at the three infection stages were observed. Notably at 12 days post SACMV infection, TME3 exhibited significant downregulation (log2fold<2.0) of 42 %, compared to 9% in T200, of the conserved miRNA families. This demonstrates an overall early response to SACMV in TME3 prior to symptom appearance not observed in T200, and expression of a large cohort of miRNA-regulated genes. Notably, at early infection, downregulation of mes-miR162 and 168 that target antiviral posttransriptional gene silencing (PTGS) regulators DCL1 and AGO1, respectively, was observed in TME3, and AGO1 and DCL1 expression was higher compared to T200 post infection. Early rapid responses prior to symptom development, including RNA silencing, may be key to establishing the tolerance/recovery phenotype exhibited by TME3 landrace later on at 67 dpi. At recovery, TME3 was hallmarked by a highly significant down-regulation of mes-miR167. MiR167 targets an auxin responsive factor which plays a role in auxin signaling and adaptive responses to stress, suggesting the importance of the auxin signaling in recovery of SACMV-induced symptoms. The gene targets of these miRNAs and their associated networks may provide clues to the molecular basis of CMD tolerance in perennial hosts such as cassava.

Enterovirus A71 (EV-A71) is an important emerging virus posing a threat to children under five years old. Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recognized to play important roles in the onset and development of viral diseases. However, it has not been determined which specific circRNAs are involved in the pathological mechanisms of EV-A71 infection. In this study, RNA-sequencing (RNA-seq) was conducted to characterize differentially expressed circRNAs during the process of EV-A71 infection. Overall, 8726, 10405 and 4710 circRNAs were detected in the control, EV-A71-12 h and EV-A71-24 h groups, respectively, of which 1851 and 951 circRNAs were differentially expressed in the EV-A71-12 h and EV-A71-24 h groups versus the control group. The overlapping circRNAs in the EV-A71-infected groups were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, which further demonstrated that the host genes of these circRNAs were principally implicated in activities associated with the progression of viral infection, such as immune system process, Wnt signaling pathway, etc. Additionally, qRT-PCR detection showed that six selected circRNAs were identical to the sequencing data. To excavate the key circRNAs in EV-A71 infection, we comprehensively evaluated and integrated the relationship of circRNA/miRNA/mRNA, and eventually screened 2 key circRNA regulatory axes, namely hsa_circ_0017115/hsa-miR-150-5p/EGR1 and hsa_circ_0005060/hsa-miR-4685-5p/MMP2. In summary, our findings not only provide the first comprehensive expression and functional profile of circRNAs in response to EV-A71 infection, but also offer a novel direction to elucidate the molecular mechanism underlying viral pathogenesis and the cellular immune response in host-EV-A71 interactions.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) have spread globally and led to the limited choice of antimicrobial treatment of K. pneumoniae-induced infections. Bacteriophages are considered as an effective strategy against bacterial infections. In this study, we isolated a novel Klebsiella phage BUCT556A with lytic activity against KPC-producing K. pneumoniae, which was a multi-drug resistant isolate. Phage BUCT556A had a symmetrical head and a long, non-contractile tail, belonging to the family Siphoviridae, order Caudoviridae. Phage BUCT556A had a relatively narrow host range, and a medium burst size of 91 PFU/cell. It was stable at broad temperature/pH range, and exhibited good tolerance to chloroform. The genome of phage BUCT556A was a 49, 376-bp linear double-stranded DNA molecule with average G + C content of 50.2%, and contained 75 open reading frames. There was no tRNA, antibiotic resistance, toxin, virulence related genes or lysogen-formation gene clusters detected in the genome of phage BUCT556A. Phylogenetic analyses based on the major capsid protein Mcp suggested that this phage had a close relationship with Klebsiella phage KLPN1. Together, through phenotypic combined with genomic DNA sequencing and analyses, our study suggests that phage BUCT556A has the potential to be used as a bacterial treatment tool for multidrug-resistant strains K. pneumoniae.

OBJECTIVE: Association of SARS-CoV2 burden in the aerodigestive tract with the disease is sparsely understood. We propose to elucidate the implications of SARS-CoV2 copies in concurrent nasopharyngeal swab (NPS), whole mouth fluid (WMF) and respiratory droplet (RD) samples on disease pathogenesis/transmission. METHODS: SARS-CoV2 copies quantified by RT-PCR in concurrent NPS, WMF and RD samples from 80 suspected COVID-19 patients were analysed with demographics, immune response and disease severity. RESULTS: Among the 55/80 (69 %) NPS-positive patients, SARS-CoV2 was detected in 44/55 (80 %) WMF (concordance with NPS-84 %; p = 0.02) and 17/55 (31 %) RD samples. SARS-CoV2 copies were similar in NPS (median:8.74 × 10^5) and WMF (median:3.07 × 10^4), but lower in RD (median:3.60 × 10^2). The 25-75 % interquartile range of SARS-CoV2 copies in the NPS was significantly higher in patients who shed the virus in WMF (p = 0.0001) and RD (p = 0.01). Multivariate analyses showed that hospitalized patients shed significantly higher virus copies in the WMF (p = 0.01). Hospitalized patients with more severe disease (p = 0.03) and higher IL-6 values (p = 0.001) shed more SARS-CoV2 virus in the RD. CONCLUSIONS: WMF may be used reliably as a surrogate for diagnosis. High copy numbers in the NPS probably imply early disease onset, while in the WMF and RD may imply more severe disease and increased inflammation.

Severe leaf curl disease of tomato (ToLCD) was noticed recently in the central parts of India and is an emerging threat to the cultivation of tomato. The genomic components of the begomovirus isolate, DNA A and betasatellite associated with ToLCD were cloned by rolling circle amplification method and sequenced. The sequence analysis revealed that the DNA A (2766 nt) of this isolate had the nucleotide identity of >91% with other strains of Tomato leaf curl Karnataka virus (ToLCKV), hence this isolate is proposed as a strain of ToLCKV, named as ToLCKV-Raipur. Similarly, the betasatellite molecule (1355 nt) had the highest identity of 91.1% with Corchorus yellow vein mosaic betasatellite (CoYVMB) and named as CoYVMB-Raipur. The full-length dimerized clones of these two genomic components were agroinoculated on natural (tomato), experimental (Nicotiana benthamiana) hosts and other 20 plant species belong to six different families. The severe leaf curl symptoms appeared only in the hosts, N. benthamiana, and in tomato inoculated with ToLCKV-Raipur alone and ToLCKV-Raipur with CoYVMB-Raipur after 8 and 16-18 days inoculation, respectively. This isolate was also transmissible to healthy tomato plants by whitefly from the tomato plant agroinoculated with ToLCKV-Raipur alone and with CoYVMB-Raipur and produced symptoms within 14-16 days after inoculation. Interestingly, this isolate infects horse gram and chilli by whitefly transmission and both the hosts showed positive for DNA A alone but not for betasatellite. Quantification of the genomic components of this isolate with the agroinoculated N. benthamiana samples by qRT-PCR results showed that the quantity of ToLCKV-Raipur was enhanced by three-fold while inoculated with CoYVMB-Raipur compared to ToLCKV-Raipur alone inoculated plants. However, CoYVMB-Raipur did not enhance the levels of ToLCKV-Raipur in the agroinoculated tomato plants. This is the first evidence of the natural co-occurrence of ToLCKV with betasatellite, CoYVMB causing ToLCD.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) assembles its nucleocapsids and occlusion-derived virions (ODVs) in the nucleus, which requires AcMNPV regulation for viral structural proteins to accumulate in the nucleus during its replication in cells. It is generally accepted that the nuclear import receptor plays a predominant role in this process. CRM1 is a nuclear export receptor that forms an export complex with its cargo protein to exit the nucleus. We previously discovered that AcMNPV inhibited CRM1-dependent nuclear export by the viral protein Ac34. This finding suggested that Ac34 could sequester CRM1-dependent proteins in the nucleus and play a novel role in the nuclear accumulation of viral structural proteins. Using the CRM1 inhibitor leptomycin B (LMB), we demonstrated that CRM1 inhibition promoted AcMNPV replication, as LMB treatment readily increased the virus titer, and even functionally surrogate Ac34 to rescue the infectivity of an ac34-knockout virus. To elucidate whether CRM1 inhibition contributes to the nuclear accumulation of viral structural proteins, we systematically analyzed the impact of CRM1 inhibition on viral protein spatial distribution patterns. We found that the nucleocapsid protein Ac102 and ODV envelope protein E26 were retained in the nucleus in response to CRM1 inhibition by Ac34. This finding indicates that AcMNPV is evolving to simultaneously exploit bidirectional nucleocytoplasmic trafficking to assist in viral replication.

Posa-like viruses have been detected in the fecal samples of several host species and are considered unclassified members of Picornavirales. Here, we identified genomic fragments of novel posa-like viruses (monsaviruses) in monkey specimens through next generation sequencing and obtained 11 full-length genomes. This monsavirus shared 88.5-89.2% nucleotide similarity with the Tottori-HG1 strain (GenBank accession LC123275). In total, 713 nucleotide polymorphism sites were identified, indicating their persistent evolution during circulation. The genomic organization and phylogenetic relationship of monsavirus were determined. Subsequent phylogenetic analysis of the conserved replication block of Hel-Pro-RdRp and core RNA-dependent RNA polymerase domain-based analysis of posa-like viruses showed significant separation compared with other known families. Further, posa-like virus genomes possessed the classical replication block of picornavirus in the 5' part of genome and picorna-like capsid domains at the structural coding region of 3' part of genome. Based on these results, we proposed the new family Posaliviridae, within Picornavirales. Four genera, which showed 68.6-75.5% amino acid distances but similar genomic organization including the conserved replication block of Hel-Pro-RdRp, the same order of the genomic coding region, and picorna-like capsid domains, were identified. The flexible genomic organization strategy and a large evolutionary scale of Posaliviridae was explicit. This study provides novel information on monsaviruses and important taxonomic data for the family Posaliviridae.

Major themes in pathogen evolution are emergence, evolution of virulence, host adaptation and the processes that underlie them. RNA viruses are of particular interest due to their rapid evolution. The in vivo molecular evolution of an RNA plant virus was demonstrated here using a necrotic isolate of cowpea mild mottle virus (CPMMV) and a susceptible soybean genotype submitted to serial inoculations. We show that the virus lost the capacity to cause necrosis after six passages through the host plant. When a severe bottleneck was imposed, virulence reduction occurred in the second passage. The change to milder symptoms had fitness benefits for the virus (higher RNA accumulation) and for its vector, the whitefly Bemisia tabaci. Genetic polymorphisms were highest in ORF1 (viral replicase) and were independent of the symptom pattern. Recombination was a major contributor to this diversity - even with the strong genetic bottleneck, recombination events and hot spots were detected within ORF1. Virulence reduction was associated with different sites in ORF1 associated to recombination events in both experiments. Overall, the results demonstrate that the reduction in virulence was a consequence of the emergence of new variants, driven by recombination. Besides providing details of the evolutionary mechanisms behind a reduction in virulence and its effect under viral and vector fitness, we propose that this recombination-driven switch in virulence allows the pathogen to rapidly adapt to a new host and, potentially, switch back.

Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5-6 years rotavirus research has benefitted in a major way from the establishment of plasmid only-based reverse genetics systems, the creation of human and other mammalian intestinal enteroids, and from the wide application of structural biology (cryo-electron microscopy, cryo-EM tomography) and complementary biophysical approaches. All of these have permitted to gain new insights into structure-function relationships of rotaviruses and their interactions with the host. This review follows different stages of the viral replication cycle and summarizes highlights of structure-function studies of rotavirus-encoded proteins (both structural and non-structural), molecular mechanisms of viral replication including involvement of cellular proteins and lipids, the spectrum of viral genomic and antigenic diversity, progress in understanding of innate and acquired immune responses, and further developments of prevention of rotavirus-associated disease.

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to public health. An explicit investigation of COVID-19 immune responses, particularly the host immunity in recovered subjects, will lay a foundation for the rational design of therapeutics and/or vaccines against future coronaviral outbreaks. Here, we examined virus-specific T cell responses and identified T cell epitopes using peptides spanning SARS-CoV-2 structural proteins. These peptides were used to stimulate peripheral blood mononuclear cells (PBMCs) derived from COVID-19-recovered subjects, followed by an analysis of IFN-γ-secreting T cells by enzyme-linked immunosorbent spot (ELISpot). We also evaluated virus-specific CD4 or CD8 T cell activation by flow cytometry assay. By screening 52 matrix pools (comprised of 315 peptides) of the spike (S) glycoprotein and 21 matrix pools (comprised of 102 peptides) spanning the nucleocapsid (N) protein, we identified 28 peptides from S protein and 5 peptides from N protein as immunodominant epitopes. The immunogenicity of these epitopes was confirmed by a second ELISpot using single peptide stimulation in memory T cells, and they were mapped by HLA restrictions. Notably, SARS-CoV-2 specific T cell responses positively correlated with B cell IgG and neutralizing antibody responses to the receptor-binding domain (RBD) of the S protein. Our results demonstrate that defined levels of SARS-CoV-2 specific T cell responses are generated in some, but not all, COVID-19-recovered subjects, fostering hope for the protection of a proportion of COVID-19-exposed individuals against reinfection. These results also suggest that these virus-specific T cell responses may induce protective immunity in unexposed individuals upon vaccination, using vaccines generated based on the immune epitopes identified in this study. However, SARS-CoV-2 S and N peptides are not potently immunogenic, and none of the single peptides could universally induce robust T cell responses, suggesting the necessity of using a multi-epitope strategy for COVID-19 vaccine design.

Novirhabdoviruses belong to the Rhabdoviridae family of RNA viruses. All of the four members are pathogenic for bony fish. Particularly, Infectious hematopoietic necrosis virus (IHNV) and Viral hemorrhagic septicemia virus (VHSV) often cause mass animal deaths and huge economic losses, representing major obstacles to fish farming industry worldwide. The interactions between fish and novirhabdoviruses are becoming better understood. In this review, we will present our current knowledge of fish innate immunity, particularly type I interferon (IFN-I) response, against novirhabdoviral infection, and the evasion strategies exploited by novirhabdoviruses. Members of Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) appear to be involved in novirhabdovirus surveillance. NF-κB activation and IFN-I induction are primarily triggered for antiviral defense. Autophagy can also be induced by viral glycoprotein (G). Although sensitive to IFN-I, novirhabdoviruses have nucleoprotein (N), matrix protein (M), and non-virion protein (NV) to interfere with host signal transduction and gene expression steps toward antiviral state establishment. Moreover, novirhabdoviruses may exploit some microRNAs for immunosuppression.

Parvovirus is a common element of the feline virus group and usually causes gastroenteritis and leukopenia in cats. In this study, we identified a novel protoparvovirus from the Chinese domestic cats, which is genetically similar to canine bufavirus (98.0%-99.8%), but sharing low amino acid identities in the viral structural proteins 2 (VP2) (36.1-37.2%) to the well-known canine parvovirus type 2 and feline panleukopenia virus. This virus was provisionally designated as feline bufavirus (FBuV). Screening of fecal samples revealed a prevalence of 7.4% (19/257) in domestic cats. Diarrhea was present in 52.6% (10/19) of cats positive for FBuV. However, statistical analysis showed no association between FBuV and clinical signs. VP2 gene of the 19 field FBuV was sequenced and phylogenetic analysis demonstrated that FBuV determined from China had a genetic diversity. This study will strengthen the understanding of the epidemiology and genetic diversity of bufavirus and provide a foundation for further studies.

A novel virus with a quadruple genome of negative-sense, single-stranded RNA was identified by high-throughput sequencing (HTS) in a grass sample from Saxony-Anhalt, Germany, and tentatively called Festuca stripe-associated virus (FSaV). The genome of FSaV consists of four segments and a total of 16,535 nucleotides (nt) which encode seven open reading frames (ORF). FSaV shares highest nt identity (between 72.84% to 80.74%) to Iranian wheat stripe virus (IWSV) and rice hoja blanca virus (RHBV). Additionally, pairwise comparisons between the amino acid sequences of the ORFs on the genome of FSaV and the corresponding ones on the genomes of the members of the Tenuvirus genus showed that FSaV shared 83.17% and 90.85% (amino acid) aa identity to IWSV. Moreover, the non-coding intergenic regions (ncIR) shared only between 49.5% to 60.87% nt identity to the corresponding regions on the IWSV genome. Based on the ICTV species demarcation, the results suggest that FSaV may represent a new species of the genus Tenuivirus. Plastid sequence analysis of the HTS data showed that the original host is a member of the genus Festuca most likely the species Festuca pratensis.

The influenza A virus genome contains 8 gene segments encoding 10 commonly recognized proteins. Additional protein products have been identified, including PB1-F2 and PA-X. We report the in-silico identification of novel isoforms of PB1-F2 and PA-X in influenza virus genomes sequenced from avian samples. The isoform observed in PA-X includes a mutated stop codon that should extend the protein product by 8 amino acids. The isoform observed in PB1-F2 includes two nonsense mutations that should truncate the N-terminal region of the protein product and remove the entire mitochondrial targeting domain. Both isoforms were uncovered during automatic annotation of CEIRS sequence data. Nominally termed PA-X8 and PB1-F2-Cterm, both predicted isoforms were subsequently found in other annotated influenza genomes previously deposited in GenBank. Both isoforms were noticed due to discrepant annotations output by two annotation engines, indicating a benefit of incorporating multiple algorithms during gene annotation.

Respiratory syncytial virus can cause severe illness and even death, particularly in infants. The increased severity of disease in young children is thought to be due to a lack of previous exposure to the virus as well as the limited immune response in infants. While studies have examined the clinical differences in disease between infants and adults, there has been limited examination of how the viral dynamics differ as infants develop. In this study, we apply a mathematical model to data from cotton rats and ferrets of different ages to assess how viral kinetics parameters change as the animals age. We find no clear trend in the viral decay rate, infecting time, and basic reproduction number as the animals age. We discuss possible reasons for the null result including the limited data, lack of detail of the mathematical model, and the limitations of animal models.

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is characterized by the clonal growth of EBV-infected stomach epithelial cells. It has been reported that N6-methyladenosine (m6A) methylation can regulate the splicing, expression, decay and translation of mRNAs. Wilms' tumor 1-associating protein (WTAP) is an m6A "writer" with methyltransferase activity. An m6A RNA methylation quantification kit and immunofluorescence (IF) showed that the m6A total RNA methylation level of the Epstein-Barr virus-negative gastric carcinoma (EBVnGC) cell line (SGC7901) was higher than that in the EBVaGC cell line (GT38). To investigate the underlying mechanism of the downregulated expression of m6A RNA methylation, we analyzed the expression of WTAP. The results showed that the expression of WTAP protein in EBVaGC cell lines was significantly lower than that in EBVnGC cell lines according to western blotting and IF. Through plasmid overexpression and RNA interference technology, we further found that EBV-encoded small RNA1 (EBER1) could downregulate WTAP expression by activating the NF-κB signaling pathway. In addition, WTAP could increase proliferation and inhibit migration in gastric carcinoma cell lines. In summary, EBER1 of EBV potentially regulated WTAP by affecting the NF-κB signaling pathway and WTAP further affected cell proliferation and migration.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses innumerous challenges, like understanding what triggered the emergence of this new human virus, how this RNA virus is evolving or how the variability of viral genome may impact the primary structure of proteins that are targets for vaccine. We analyzed 19471 SARS-CoV-2 genomes available at the GISAID database from all over the world and 3335 genomes of other Coronoviridae family members available at GenBank, collecting SARS-CoV-2 high-quality genomes and distinct Coronoviridae family genomes. Additionally, we analyzed 199,984 spike glycoprotein sequences. Here, we identify a SARS-CoV-2 emerging cluster containing 13 closely related genomes isolated from bat and pangolin that showed evidence of recombination, which may have contributed to the emergence of SARS-CoV-2. The analyzed SARS-CoV-2 genomes presented 9632 single nucleotide variants (SNVs) corresponding to a variant density of 0.3 over the genome, and a clear geographic distribution. SNVs are unevenly distributed throughout the genome and hotspots for mutations were found for the spike gene and ORF 1ab. We describe a set of predicted spike protein epitopes whose variability is negligible. Additionally, all predicted epitopes for the structural E, M and N proteins are highly conserved. The amino acid changes present in the spike glycoprotein of variables of concern (VOCs) comprise between 3.4% and 20.7% of the predicted epitopes of this protein. These results favors the continuous efficacy of the available vaccines targeting the spike protein, and other structural proteins. Multiple epitopes vaccines should sustain vaccine efficacy since at least some of the epitopes present in variability regions of VOCs are conserved and thus recognizable by antibodies.

Fast evolving of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has caused the spreading of COVID-19 disease rapidly around the globe. The mutation, especially in the gene encoding spike protein has helped the virus adapt and evade human immune system, as well as affect the efficacy of the immunizations and treatments. SARS-CoV-2 variant carrying D614G amino acid change at the spike protein is the most dominant strain in the pandemic. Therefore, efficient detection of the SARS-CoV-2 variants including D614G mutation is critical to control the COVID-19 pandemic. Herein, we report a dual synthetic mismatches CRISPR/Cas12a (dsmCRISPR) method to detect the SARS-CoV-2 D614G mutation with high sensitivity and specificity. By targeting SARS-CoV-2 D614G mutation, synthetic mismatch crRNAs were designed from -3 to +3 position around the mutation site. To improve the sensitivity and specificity, a synthetic mismatch primer with a 3'-terminal base complementary to the D614G point mutation and a mismatch next to 3'-terminal base was used to specifically amplify the D614G mutation site with higher annealing temperature. Using synthetic mismatch crRNA-(-1), a higher ratio (13.45) of the fluorescence between G614 and D614 was observed. When combined with mismatch primer to amplify D614G mutation, the fluorescence ratio of G614/D164 template detected was increased by 73.53% to 23.12. This method can detect the SARS-CoV-2 D614G mutation nucleic acid with high sensitivity, which was validated with synthetic SARS-CoV-2 D614G RNA. Therefore, the dsmCRIPSR method has significant potential to serve as a sensitive and specific assay for SARS-CoV-2 D614G detection and could be further extended for the detection of other SARS-CoV-2 variants of interest.

Broad bean wilt virus 2 (BBWV2) is an evolutionarily successful RNA virus with an extensive host range and worldwide distribution that causes severe damage to crops. While numerous BBWV2 isolates from various plant species have been identified and their genome sequences determined, little information is available on the virulence and symptomatic characteristics corresponding to the genomic sequences. In this study, we provide integrated information on the molecular and pathogenic characteristics of three genetically distant BBWV2 isolates: BBWV2-PC, -LS2, and P3 obtained from Gentiana scabra, Leonurus sibiricus, and Pisum sativum, respectively. Phylogenetic and diversity analyses of the BBWV2 population included 42 isolates from various host species and revealed that RNA2 has higher genetic plasticity than RNA1 and may have evolved under host-imposed constraints. In addition, we generated an infectious cDNA clone of BBWV2-PC RNA2 (pBBWV2-PC-R2). Pseudo-recombination analysis of pBBWV2-PC-R2 further demonstrated that RNA2 determines the pathogenic characteristics of the PC isolate.

Enterovirus D68 is an emerging respiratory disease pathogen causing multiple outbreaks worldwide. Enterovirus D68 strain US/KY/14-18953 was adapted to propagate in Vero cells resulting alteration of seven amino acids. The Vero cell adapted virus was inactivated with Formalin and immunized in mice. Formalin inactivated vaccine elicited high virus specific IgG antibody titer and neutralization titer. Avidity of the IgG antibodies elicited by two different doses of formalin inactivated vaccine is moderately high which got augmented by alum adjuvanted formulations. Formalin inactivated unadjuvanted vaccine elicited a balanced IgG1 type and IgG2a type antibody indicating a more balanced Th2/Th1 type immune response while alum formulated formalin inactivated antigen elicited significantly high IgG1 antibody in immunized sera and Th2 cytokines in mice splenocytes denoting Th2 type T cell immune response. Additionally, the formalin inactivated vaccine formulations has displayed excellent serum mediated invivo protective efficacy. These data suggested that formalin inactivated Enterovirus D68 is a promising vaccine candidate.

The COVID-19 pandemic has already reached more than 110 million people and is associated with 2.5 million deaths worldwide. Brazil is the third worst-hit country, with approximately 10.2 million cases and 250 thousand deaths. International efforts have been established to share information about Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemiology and evolution to support the development of effective strategies for public health and disease management. We aimed to analyze the high-quality genome sequences from Brazil from February 2020-2021 to identify mutation hotspots, geographical and temporal distribution of SARS-CoV-2 lineages by using phylogenetics and phylodynamics analyses. We describe heterogeneous sequencing efforts, the progression of the different lineages along time, evaluating mutational spectra and frequency oscillations derived from the prevalence of specific lineages across different Brazilian regions. We found at least seven major (1-7) and two minor clades related to the six most prevalent lineages in the country and described its spatial distribution and dynamics. The emergence and recent frequency shift of lineages (P.1 and P.2) carrying mutations of concern in the spike protein (e. g., E484K, N501Y) draws attention due to their association with immune evasion and enhanced receptor binding affinity. Improvements in genomic surveillance are of paramount importance and should be extended in Brazil to better inform policy makers about better decisions to fight the COVID-19 pandemic.

Since its first detection in the UK in September 2020, a highly contagious version of the coronavirus, the alpha or British variant a.k.a. B.1.1.7 SARS-CoV-2 virus lineage, rapidly spread across several countries and became the dominant strain in the outbreak. Here it is shown that a very simple evolutionary model can fit the observed change in frequency of B.1.1.7 for several countries, regions of countries and the whole world with a single parameter, its relative fitness f, which is almost universal f ≈ 1.5. This is consistent with a 50% higher transmissibility than the local wild type and with the fact that the period in which this variant takes over has been in all the studied cases around 22 weeks.

Zika Virus (ZIKV) is an RNA virus that belongs to the Flavivirus (FV) genus. In the last years, several unique characteristics of ZIKV among FV have been revealed, as the multiple routes of transmission and its ability to reach different human tissues, including the central nervous system. Thus, one of the most intriguing features of ZIKV biology is its ability to cross diverse complex biological barriers. The main aim of this study is to contribute to the understanding of the still unclear mechanisms behind this viral activity. We investigated an African strain and two South American ZIKV isolates belonging to the Asian lineage, in order to characterize possible differences regarding their ability to disturb intercellular junctions. The Asian isolates correspond to an imported (Venezuelan) and an autochthonous (Argentinian) ZIKV strain for which there is still no data available. We focused on occludin and DLG1 expression as markers of tight and adherent junctions, respectively. For this, we applied a quantitative immunofluorescence assay that can ascertain alterations in the cell junction proteins expression in the infected cells. Our findings indicated that the different ZIKV strains were able to reduce the levels of both polarity proteins without altering their overall cell distribution. Moreover, the grade of this effect was strain-dependent, being the DLG1 reduction higher for the African and Asian Venezuelan isolates and, on the contrary, occludin down-regulation was more noticeable for the Argentinian strain. Interestingly, among both junction proteins the viral infection caused a relative larger reduction in DLG1 expression for all viruses, suggesting DLG1 may be of particular relevance for ZIKV infections. Taken together, this study contributes to the knowledge of the biological mechanisms involved in ZIKV cytopathogenesis, with a special focus on regional isolates.

Enterovirus D68 (EV-D68) belongs to the Picornaviridae family and can lead to severe clinical manifestations in the respiratory system. The 3D-polymerase (3Dpoly) is an important nonstructural protein during EV-D68 replication, but few studies have addressed its interaction with the host antiviral response during EV-D68 infection. Here, we used human bronchial epithelial cells to investigate the impact of the 3Dpoly on the mitochondrial dynamics and innate immune response. The results showed that the number and morphology of the mitochondria in 16HBE cells was affected during the early stage of infection, and these effects included the cellular apoptosis. Moreover, we found that the 3Dpoly of EV-D68 can interact with PGAM5 and promote mitofusin 2 protein upregulation, and subsequently, 3Dpoly impairs IFN-β expression by impacting the activation of the RIG-I receptor signaling pathway. Our findings suggest that during EV-D68 replication, the 3Dpoly, via its interaction with PGAM5, can affect the mitochondrial dynamics and suppress the expression of IFN-β by impacting the RIG-I-like receptor signal pathway.

Dengue, a mosquito-borne viral disease, caused by any of four serotypes of dengue viruses (DENV-1, -2, -3 and -4), is estimated to affect >1 million of the world's population daily. We showed earlier that a recombinant human adenovirus type 5 (HuAd5) vector, encoding a short hairpin RNA (shRNA), targeting a conserved sequence in the DENV genome, could effectively suppress pre-established DENV-2 infection in Vero cells. In this study, we identified an additional conserved shRNA target in the DENV genome, developed a HuAd5 vector to target this site, and evaluated if HuAd5-delivered shRNAs suppress pre-established infection by the remaining three DENV serotypes, not only in Vero cells, but also in macrophages, the in vivo sites of DENV replication in infected individuals. We also assessed the effect of anti-HuAd5 antibodies on shRNA delivery. We show that recombinant HuAd5 vectors, encoding shRNAs targeting conserved DENV genomic sequences, in the 5' non-translated region and capsid gene, can suppress ongoing replication of all four prototypic DENV serotypes in Vero cells and in a HuAd5-refractory human macrophage cell line expressing a DENV attachment factor. DENV suppression was assessed on the basis of inhibition of viral antigen secretion, viral RNA replication and progeny virus generation. Interestingly, HuAd5 vector-mediated DENV suppression in the macrophage cell line was dependent on the presence of anti-HuAd5 antibody. This suggests that HuAd5 vector complexed to its antibody enters these cells through the Fc receptor pathway. This may have implications for specific targeting of HuAd5 vector-mediated antiviral RNA interference therapy to macrophages.

Rhinovirus species C (RV-C) causes more severe asthma attacks than other rhinovirus species. However, the modulation of RV-C replication by drugs has not been well studied. Primary human nasal epithelial (HNE) cells cultured on filter membranes with air-liquid interface methods were infected with RV-C03, and the levels of RV-C03 RNA collected from the airway surface liquid (ASL) of HNE cells were measured with a SYBR Green assay. Pretreatment of HNE cells with the specific vacuolar H+-ATPase inhibitor bafilomycin A1 reduced the RV-C03 RNA levels in the ASL; inflammatory cytokines, including interleukin (IL)-1β, IL-6 and IL-8, in the supernatant; the mRNA expression of the RV-C receptor cadherin-related family member 3 (CDHR3) in the cells; and the number of acidic endosomes where RV-B RNA enters the cytoplasm. The levels of RV-C03 RNA in the ASL obtained from HNE cells with the CDHR3 rs6967,330 G/A genotype tended to be higher than those obtained from HNE cells with the G/G genotype. Pretreatment with the Na+/H+ exchanger inhibitor ethyl-isopropyl amiloride or either of the macrolides clarithromycin or EM900 also reduced RV-C03 RNA levels in the ASL and the number of acidic endosomes in HNE cells. In addition, significant levels of RV-A16, RV-B14 and RV-C25 RNA were detected in the ASL, and bafilomycin A1 also decreased the RV-C25 RNA levels. These findings suggest that bafilomycin A1 may reduce the release of RV-Cs and inflammatory cytokines from human airway epithelial cells. RV-Cs may be sensitive to drugs, including bafilomycin A1, that increase endosomal pH, and CDHR3 may mediate virus entry through receptor-mediated endocytosis in human airway epithelial cells.