VP1 is the primary determinant of neuropathogenesis in a mouse model of enterovirus D68 acute flaccid myelitis.

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory and neurologic disease [acute flaccid myelitis (AFM)]. Intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with US/IL/14-18952 (IL52), a clinical isolate from the 2014 EV-D68 epidemic, results in many of the pathogenic features of human AFM, including viral infection of the spinal cord, death of motor neurons, and resultant progressive paralysis. In distinction, CA/14-4231 (CA4231), another clinical isolate from the 2014 EV-D68 outbreak, does not cause paralysis in mice, does not grow in the spinal cord, and does not cause motor neuron loss following IM injection. A panel of chimeric viruses containing sequences from IL52 and CA4231 was used to demonstrate that VP1 is the main determinant of EV-D68 neurovirulence following IM injection of neonatal SW mice. VP1 contains four amino acid differences between IL52 and CA4231. Mutations resulting in substituting these four amino acids (CA4231 residues into the IL52 polyprotein) completely abolished neurovirulence. Conversely, mutations resulting in substituting VP1 IL52 amino acid residues into the CA4231 polyprotein created a virus that induced paralysis to the same degree as IL52. Neurovirulence following infection of neonatal SW mice with parental and chimeric viruses was associated with viral growth in the spinal cord. IMPORTANCE: Emerging viruses allow us to investigate mutations leading to increased disease severity. Enterovirus D68 (EV-D68), once the cause of rare cases of respiratory illness, recently acquired the ability to cause severe respiratory and neurologic disease. Chimeric viruses were used to demonstrate that viral structural protein VP1 determines growth in the spinal cord, motor neuron loss, and paralysis following intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with EV-D68. These results have relevance for predicting the clinical outcome of future EV-D68 epidemics as well as targeting retrograde transport as a potential strategy for treating virus-induced neurologic disease.

Adeno-associated virus 9 (AAV9) viral proteins VP1, VP2, and membrane-associated accessory protein (MAAP) differentially influence in vivo transgene expression.

Adeno-associated virus (AAV) is a Dependoparvovirus with a ssDNA ~4.7 kb genome in a ~25 nm icosahedral capsid structure. AAV genomes encode nine known functional proteins from two open reading frames between two inverted terminal repeats (ITRs). In recombinant AAV vectors for gene therapy use, the AAV genome is replaced with a transgene of interest flanked by ITRs and subsequently packaged within an AAV capsid made up of three viral structural proteins (VP1, VP2, and VP3) in an approximate 1:1:10 ratio, respectively. The AAV capsid, particularly VP3, has traditionally been ascribed to capsid-cellular receptor binding. However, AAV9 VP1/VP2 exhibits a capsid-promoter interaction that can alter neuronal cellular tropism in the rat and non-human primate central nervous system. This capsid-promoter interaction is altered by AAV9EU (AAV9 with six glutamates inserted at aa139) which exhibits a significant reduction in nuclear transgene DNA, a decrease in neuronal transduction, and a reduction in vivo relative transgene mRNA levels. AAV9EU has six amino acid insertions in VP1, VP2, and MAAP (membrane-associated accessory protein), but no combination of VP with MAAP recapitulated the AAV9EU in vivo phenotype. Surprisingly, AAV9 produced in the absence of MAAP9 exhibits an increase in relative transgene levels. While co-infusing two AAV9 vectors, differing only in transgene and MAAP9 presence during production, exhibit a significantly increased in vivo transgene fluorescence intensity by fivefold of both transgenes. Together, an MAAP9-related activity acts both in cis and in trans to increase AAV9 transgene mRNA levels and AAV9 transgene protein levels in vivo. IMPORTANCE: Recombinant adeno-associated viruses (AAVs) are used extensively in clinical gene therapy for treating a range of tissues and pathologies in humans. In particular, AAV9 occupies a prominent position in central nervous system (CNS) gene therapy given its central role in ongoing clinical trials and an FDA-approved therapeutic. Despite its widespread use, recent studies have identified unique roles for the AAV capsid in in vivo transgene expression; for example, interior-facing capsid residues of AAV VP1 and VP2 modulate cellular transgene expression in vivo. The following experiments identified that the AAV9 MAAP protein exerts a significant influence on in vivo transgene expression. This finding could further explain how AAV can remain latent after infection in vivo. Together, these studies provide novel functional insights that highlight the importance of further understanding basic AAV biology.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Genomic epidemiology and evolution of rhinovirus in western Washington State, 2021-22.

BACKGROUND: Human rhinoviruses (RV) primarily cause the common cold, but infection outcomes vary from subclinical to severe cases, including asthma exacerbations and fatal pneumonia in immunocompromised individuals. To date, therapeutic strategies have been hindered by the high diversity of serotypes. Global surveillance efforts have traditionally focused on sequencing VP1 or VP2/VP4 genetic regions, leaving gaps in our understanding of RV genomic diversity. METHODS: We sequenced 1,078 RV genomes from nasal swabs of symptomatic and asymptomatic individuals to explore viral evolution during two epidemiologically distinct periods in Washington State: when the COVID-19 pandemic affected the circulation of other seasonal respiratory viruses except for RV (February - July 2021), and when the seasonal viruses reemerged with the severe RSV and influenza outbreak (November-December 2022). We constructed maximum likelihood and BEAST-phylodynamic trees to characterize intra-genotype evolution. RESULTS: We detected 99 of 168 known genotypes and observed inter-genotypic recombination and genotype cluster swapping from 2021 to 2022. We found a significant association between the presence of symptoms and viral load, but not with RV species or genotype. Phylodynamic trees, polyprotein selection pressure, and Shannon entropy revealed co-circulation of divergent clades within genotypes with high amino acid constraints throughout polyprotein. DISCUSSION: Our study underscores the dynamic nature of RV genomic epidemiology within a localized geographic region, as more than 20% of existing genotypes within each RV species co-circulated each studied month. Our findings also emphasize the importance of investigating correlations between rhinovirus genotypes and serotypes to understand long-term immunity and cross-protection.

Acute Gastroenteritis Associated with Norovirus GII.8[P8], Thailand, 2023.

Acute gastroenteritis associated with human norovirus infection was reported in Phuket, Thailand, in June 2023. We amplified GII.8[P8] from the outbreak stool specimens. Retrospective sample analysis identified infrequent GII.8[P8] in the country beginning in 2018. In all, the 10 whole-genome GII.8[P8] sequences from Thailand we examined had no evidence of genotypic recombination.

The Feline calicivirus capsid protein VP1 is a client of the molecular chaperone Hsp90.

Feline calicivirus (FCV) icosahedral viral capsids are composed of dozens of structural subunits that rely on cellular chaperones to self-assemble in an orderly fashion. Here, we report that the heat shock protein 90 (Hsp90) inhibition significantly reduced FCV particle production, suggesting a role in the replicative cycle. We found that Hsp90 inhibition was not related to the synthesis or stability of the early proteins that translate from the gRNA nor to the minor capsid protein VP2 but with a reduction in the major capsid protein VP1 levels, both translated late in infection from the subgenomic RNAs. Reduction in VP1 levels was observed despite an augment of the leader of the capsid (LC)-VP1 precursor levels, from which the LC and VP1 proteins are produced after proteolytic processing by NS6/7. The direct interaction of VP1 with Hsp90 was observed in infected cells. These results suggest that upon release from the polyprotein precursor, VP1 becomes a client of Hsp90 and that this interaction is required for an efficient FCV replicative cycle.

Rotavirus core shell protein sites that regulate intra-particle polymerase activity.

IMPORTANCE: Rotaviruses are important causes of severe gastroenteritis in young children. A characteristic feature of rotaviruses is that they copy ribonucleic acid (RNA) inside of the viral particle. In fact, the viral polymerase (VP1) only functions when it is connected to the viral inner core shell protein (VP2). Here, we employed a biochemical assay to identify which sites of VP2 are critical for regulating VP1 activity. Specifically, we engineered VP2 proteins to contain amino acid changes at structurally defined sites and assayed them for their capacity to support VP1 function in a test tube. Through this work, we were able to identify several VP2 residues that appeared to regulate the activity of the polymerase, positively and negatively. These results are important because they help explain how rotavirus synthesizes its RNA while inside of particles and they identify targets for the future rational design of drugs to prevent rotavirus disease.

PRMT5 Facilitates Infectious Bursal Disease Virus Replication through Arginine Methylation of VP1.

The infectious bursal diseases virus (IBDV) polymerase, VP1 protein, is responsible for transcription, initial translation and viral genomic replication. Knowledge about the new kind of post-translational modification of VP1 supports identification of novel drugs against the virus. Because the arginine residue is known to be methylated by protein arginine methyltransferase (PRMT) enzyme, we investigated whether IBDV VP1 is a substrate for known PRMTs. In this study, we show that VP1 is specifically associated with and methylated by PRMT5 at the arginine 426 (R426) residue. IBDV infection causes the accumulation of PRMT5 in the cytoplasm, which colocalizes with VP1 as a punctate structure. In addition, ectopic expression of PRMT5 significantly enhances the viral replication. In the presence of PMRT5, enzyme inhibitor and knockout of PRMT5 remarkably decreased viral replication. The polymerase activity of VP1 was severely damaged when R426 mutated to alanine, resulting in impaired viral replication. Our study reports a novel form of post-translational modification of VP1, which supports its polymerase function to facilitate the viral replication. IMPORTANCE Post-translational modification of infectious bursal disease virus (IBDV) VP1 is important for the regulation of its polymerase activity. Investigation of the significance of specific modification of VP1 can lead to better understanding of viral replication and can probably also help in identifying novel targets for antiviral compounds. Our work demonstrates the molecular mechanism of VP1 methylation mediated by PRMT5, which is critical for viral polymerase activity, as well as viral replication. Our study expands a novel insight into the function of arginine methylation of VP1, which might be useful for limiting the replication of IBDV.

Phosphorylation of VP1 Mediated by CDK1-Cyclin B1 Facilitates Infectious Bursal Disease Virus Replication.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Avibirnavirus in the family Birnaviridae. It can cause serious failure of vaccination in young poultry birds with impaired immune systems. Post-translational modifications of the VP1 protein are essential for viral RNA transcription, genome replication, and viral multiplication. Little information is available so far regarding the exact mechanism of phosphorylation of IBDV VP1 and its significance in the viral life cycle. Here, we provide several lines of evidence that the cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex phosphorylates VP1, which facilitates viral replication. We show that the CDK1-cyclin B1 specifically interacts with VP1 and phosphorylates VP1 on the serine 7 residue, located in the N-terminal 7SPAQ10 region, which follows the optimal phosphorylation motif of CDK1, p-S/T-P. Additionally, IBDV infection drives the cytoplasmic accumulation of CDK1-cyclin B1, which co-localizes with VP1, supporting the kinase activity of CDK1-cyclin B1. Treatment with CDK1 inhibitor RO3306 and knockdown of CDK1-cyclin B1 severely disrupts the polymerase activity of VP1, resulting in diminished viral replication. Moreover, the replication of S7A mutant recombinant IBDV was significantly decreased compared to that of wild-type (WT) IBDV. Thus, CDK1-cyclin B1 is a crucial enzyme which phosphorylates IBDV VP1 on serine 7, which is necessary both for the polymerase activity of VP1 and for viral replication. IMPORTANCE Infectious bursal disease virus still poses a great economic threat to the global poultry farming industry. Detailed information on the steps of viral genome replication is essential for the development of antiviral therapeutics. Phosphorylation is a common post-translational modification in several viral proteins. There is a lack of information regarding the significance of VP1 phosphorylation and its role in modulating the viral life cycle. In this study, we found that CDK1-cyclin B1 accumulates in the cytoplasm and phosphorylates VP1 on serine 7. The presence of a CDK1 inhibitor and the silencing of CDK1-cyclin B1 decrease IBDV replication. The mutation of VP1 serine 7 to alanine reduces VP1 polymerase activity, disrupting the viral life cycle, which suggests that this residue serves an essential function. Our study offers novel insights into the regulatory mechanism of VP1 phosphorylation.

Detection of wildtype Merkel cell polyomavirus genomic sequence and VP1 transcription in a subset of Merkel cell carcinoma.

AIMS: Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV). Characteristic for these virus-positive (VP) MCC is MCPyV integration into the host genome and truncation of the viral oncogene Large T antigen (LT), with full-length LT expression considered as incompatible with MCC growth. Genetic analysis of a VP-MCC/trichoblastoma combined tumour demonstrated that virus-driven MCC can arise from an epithelial cell. Here we describe two further cases of VP-MCC combined with an adnexal tumour, i.e. one trichoblastoma and one poroma. METHODS AND RESULTS: Whole-genome sequencing of MCC/trichoblastoma again provided evidence of a trichoblastoma-derived MCC. Although an MCC-typical LT-truncating mutation was detected, we could not determine an integration site and we additionally detected a wildtype sequence encoding full-length LT. Similarly, Sanger sequencing of the combined MCC/poroma revealed coding sequences for both truncated and full-length LT. Moreover, in situ RNA hybridization demonstrated expression of a late region mRNA encoding the viral capsid protein VP1 in both combined as well as in a few cases of pure MCC. CONCLUSION: The data presented here suggest the presence of wildtype MCPyV genomes and VP1 transcription in a subset of MCC.

Stability and integrity of self-assembled bovine parvovirus virus­like particles (BPV­VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment.

BACKGROUND: Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences. METHODS: In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism. RESULTS: Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs. CONCLUSIONS: In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

Rapid detection of four major HFMD-associated enteroviruses by multiplex HiFi-LAMP assays.

Hand, foot, and mouth disease (HFMD) caused by various enteroviruses is a major public health concern globally. Human enterovirus 71(EVA71), coxsackievirus A16 (CVA16), coxsackievirus A6 (CVA6), and coxsackievirus A10 (CVA10) are four major enteroviruses responsible for HFMD. Rapid, accurate, and specific point-of-care (POC) detection of the four enteroviruses is crucial for the prevention and control of HFMD. Here, we developed two multiplex high-fidelity DNA polymerase loop-mediated isothermal amplification (mHiFi-LAMP) assays for simultaneous detection of EVA71, CVA16, CVA6, and CVA10. The assays have good specificity and exhibit high sensitivity, with limits of detection (LOD) of 11.2, 49.6, 11.4, and 20.5 copies per 25 µL reaction for EVA71, CVA16, CVA6, and CVA10, respectively. The mHiFi-LAMP assays showed an excellent clinical performance (sensitivity 100.0%, specificity 83.3%, n = 47) when compared with four singleplex RT-qPCR assays (sensitivity 93.1%, specificity 100%). In particular, the HiFi-LAMP assays exhibited better performance (sensitivity 100.0%, specificity 100%) for CVA16 and CVA6 than the RT-qPCR assays (sensitivity 75.0-92.3%, specificity 100%). Furthermore, the mHiFi-LAMP assays detected all clinical samples positive for the four enteroviruses within 30 min, obviously shorter than about 1-1.5 h by the RT-qPCR assays. The new mHiFi-LAMP assays can be used as a robust point-of-care testing (POCT) tool to facilitate surveillance of HFMD at rural and remote communities and resource-limited settings.

Environmental surveillance reveals co-circulation of distinctive lineages of enteroviruses in southwest China's border cities, 2020-2022.

AIMS: Enteroviruses are significant human pathogens associated with a range of mild to severe diseases. This study aims to understand the diversity and genetic characterization of enteroviruses circulated in southwest China's border cities by using environmental surveillance. METHODS AND RESULTS: A total of 96 sewage samples were collected in three border cities and a port located in Yunnan Province, China from July 2020 to June 2022. After cell culture and VP1 sequencing, a total of 590 enterovirus isolates were identified, belonging to 21 types. All PV strains were Sabin-like with ≤6 nucleotide mutations in the VP1 coding region. Echovirus 6, echovirus 21 (a rare serotype in previous studies), and coxsackievirus B5 were the predominant serotypes, which accounted for 21.19%, 18.31%, and 13.39% of the total isolates, respectively. The prevalence of the common serotypes varied across different border cities and periods. Phylogenetic analysis revealed the presence of multiple evolutionary lineages for E21, E6, and E30, some of which formed distinct branches. CONCLUSIONS: High diversity of enteroviruses and distinct lineages of predominant serotypes circulated in southwest China's border cities.

Parvovirus B19 Infection Is Associated with the Formation of Neutrophil Extracellular Traps and Thrombosis: A Possible Linkage of the VP1 Unique Region.

Neutrophil extracellular traps (NETs) formation, namely NETosis, is implicated in antiphospholipid syndrome (APS)-related thrombosis in various autoimmune disorders such as systemic lupus erythematosus (SLE) and APS. Human parvovirus B19 (B19V) infection is closely associated with SLE and APS and causes various clinical manifestations such as blood disorders, joint pain, fever, pregnancy complications, and thrombosis. Additionally, B19V may trigger the production of autoantibodies, including those against nuclear and phospholipid components. Thus, exploring the connection between B19V, NETosis, and thrombosis is highly relevant. An in vitro NETosis model using differentiated HL-60 neutrophil-like cells (dHL-60) was employed to investigate the effect of B19V-VP1u IgG on NETs formation. A venous stenosis mouse model was used to test how B19V-VP1u IgG-mediated NETs affect thrombosis in vivo. The NETosis was observed in the dHL-60 cells treated with rabbit anti-B19V-VP1u IgG and was inhibited in the presence of either 8-Br-cAMP or CGS216800 but not GSK484. Significantly elevated reactive oxygen species (ROS), myeloperoxidase (MPO), and citrullinated histone (Cit-H3) levels were detected in the dHL60 treated with phorbol myristate acetate (PMA), human aPLs IgG and rabbit anti-B19V-VP1u IgG, respectively. Accordingly, a significantly larger thrombus was observed in a venous stenosis-induced thrombosis mouse model treated with PMA, human aPLs IgG, rabbit anti-B19V-VP1u IgG, and human anti-B19V-VP1u IgG, respectively, along with significantly increased amounts of Cit-H3-, MPO- and CRAMP-positive infiltrated neutrophils in the thrombin sections. This research highlights that anti-B19V-VP1u antibodies may enhance the formation of NETosis and thrombosis and implies that managing and treating B19V infection could lower the risk of thrombosis.

Shedding Light on Viral Shedding: Novel Insights into Nuclear Assembly, Cytoplasmic Transformation and Extracellular Vesicle Release of the BK Virus.

Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

Molecular Characterization of BK Polyomavirus Replication in Allogeneic Hematopoietic Cell Transplantation Patients.

BACKGROUND: High-level BK polyomavirus (BKPyV) replication in allogeneic hematopoietic cell transplantation (HCT) predicts failing immune control and BKPyV-associated hemorrhagic cystitis. METHODS: To identify molecular markers of BKPyV replication and disease, we scrutinized BKPyV DNA-loads in longitudinal urine and plasma pairs from 20 HCT patients using quantitative nucleic acid testing (QNAT), DNase-I treatment prior to QNAT, next-generation sequencing (NGS), and tested cell-mediated immunity. RESULTS: We found that larger QNAT amplicons led to under-quantification and false-negatives results (P < .001). DNase-I reduced urine and plasma BKPyV-loads by >90% (P < .001), indicating non-encapsidated BKPyV genomes. DNase-resistant urine BKPyV-loads remained infectious in cell culture. BKPyV genome fragmentation of ≤250 bp impaired NGS coverage of genetic variation using 1000-bp and 5000-bp amplicons. Conversely, 250-bp amplicons captured viral minority variants. We identified genotype-specific and genotype-independent changes in capsid Vp1 or T-antigen predicted to escape from antibody neutralization or cytotoxic CD8 T-cells, respectively. Genotype-specific changes in immunodominant 9mers were associated with reduced or absent CD8 T-cell responses. Thus, failure to control BKPyV replication in HCT Patients may involve insufficient genotype-specific cytotoxic CD8 T-cell responses, potentially predictable by low neutralizing antibodies as well as genotype-independent immune escape. CONCLUSIONS: Our results provide new insights for patient evaluation and for designing immune protection through neutralizing antibodies, adoptive T-cell therapy, or vaccines.

Chemical Evolution of Rhinovirus Identifies Capsid-Destabilizing Mutations Driving Low-pH-Independent Genome Uncoating.

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

Genetic characterizations and molecular evolution of human norovirus GII.6 genotype during the past five decades.

The norovirus genotype GII.6 is circulating in the population with a relatively high prevalence, but in-depth molecular characterization studies of GII.6 are needed. In this study, norovirus GII.6 sequences were retrieved and analyzed to demonstrate the molecular characterizations of norovirus GII.6. The results show that GII.6 VP1 gene can be divided into three variants and all variants cocirculated in humans in the past decades. The intragenotypic showed no growth trend over time. Since the overall evolutionary rate was 3.432 × 10-3 substitutions/site/year, the infer most recent common ancestor was estimated as 1913. Only a few amino acid sites were recognized to be under positive selection pressure. The mean effective population size was stable in the recent years. The variant C (especially 87 GII.P7-GII.6 strains) had a higher evolutionary rate and more sites under positive selection pressure compared to other variants. NS4 protein had the higher diversity than other nonstructural proteins, and VP1 and VP2 genes had the same phylogenetic relationships. This study provides a systematic description of genetic characterizations and molecular evolution of GII.6. Research on norovirus molecular epidemiology should be pursued to enrich the genomic data of the diverse genotypes and improve their analysis.

The analysis of the genotype of Sapovirus outbreaks in Zhejiang Province.

BACKGROUND: Sapovirus (SaV) infection is increasing globally. Concurrently, several SaV-outbreaks were observed in children of Zhejiang province, China, in recent years, In this study, the genotypes of Sapovirus from seven outbreaks in the Zhejiang province were analysed. METHODS: A total of 105 faecal samples were collected from children aged between 4 and 17 years from the Zhejiang Provincial Center for Disease Control and Prevention between October 2021 and February 2023. Genotypes were processed using reverse transcription polymerase chain reaction and Sanger sequencing, while next-generation sequencing was used to generate a complete viral genome. Deduced amino acid sequences were analysed to detect VP1 gene mutations. RESULTS: In total, 60 SaV-positive patients were detected at a 57.14% (60/105) positivity rate. Positive rates in the seven outbreaks were: 22.22% (2/9), 15.00% (3/20), 93.10% (27/29), 84.21% (16/19), 28.57% (2/7), 53.33% (8/15) and 33.33% (2/6), respectively. Four genotypes were identified in the seven outbreaks, of which, GI.1 accounted for 14.29% (1/7), GI.2 accounted for 14.29% (1/7), GI.6 and GII.5 accounted for 14.29% (1/7), and GI.6 accounted for 57.14% (4/7). All patients were children and outbreaks predominantly occurred in primary schools and during cold seasons. Additionally, the complete sequence from the GI.6 outbreak strain showed high homology (identity: 99.99%) with few common substitutions (Y300S, N302S and L8M) in VP1 protein. CONCLUSIONS: SaV genotype diversity was observed in the seven outbreaks, with GI.6 being the main SaV genotype in Zhejiang province. It demonstrated high homology and may provide a platform for SaV prevention and control measures.