Protein VP1 Research Articles

Novel polyomavirus in the endangered garden dormouse Eliomys quercinus

BackgroundThe garden dormouse (Eliomys quercinus) has experienced a significant population decline across Europe in recent decades. While habitat loss and climate change are often cited as primary factors, pathogen exposure, either to novel or to previously known, may play a role in such a decline. This study aimed to investigate the presence of polyomaviruses in garden dormice, given that these viruses are highly prevalent and can cause disease, particularly in immunocompromised individuals.MethodsThe carcasses of garden dormice (n = 89) were collected throughout Germany. Kidney samples were tested for the presence of polyomavirus DNA using nested degenerate and specific diagnostic PCRs. Seroprevalence was assessed from chest cavity fluid samples through an enzyme-linked immunosorbent assay using polyomavirus VP1 virus-like particles produced in yeast.ResultsA new polyomavirus, related to chimpanzee (Pan troglodytes) polyomaviruses 4 and 5 and human Merkel cell polyomavirus, was identified in the garden dormouse. Two 5,380 bp-length complete viral genomes were sequenced from dormice kidney samples (sequences PQ246041 and PQ246042). Genes encoding the putative structural proteins VP1, VP2, and VP3, as well as the Large, Middle, and small T antigens, containing conserved functional domains were identified. Polyomavirus DNA was detected in 2 of 74 dormice (2.7%, 95% confidence interval: 0-6.4%) through PCR, while 12 of 69 animals (17.4%, 95% confidence interval: 8.4-26.3%) tested positive for polyomavirus-specific antibodies.ConclusionsIn conclusion, here we describe a novel polyomavirus in the garden dormouse with molecular and serological detection. Pairwise sequence comparison and phylogenetic analysis suggest that this novel virus may represent a novel species within the genus Alphapolyomavirus. Future work should examine if this virus is garden dormouse-specific and whether it is associated with disease in dormice.

Human norovirus disturbs intestinal motility and transit time through its capsid proteins.

Human norovirus (HuNoV) accounts for over 700 million cases of gastroenteritis annually. Episodes of HuNoV disease are characterized by vomiting and diarrhea as the two most prominent symptoms. Despite its prevalence, our understanding of the pathophysiological mechanisms triggered upon HuNoV infection is limited, mainly due to a lack of suitable animal models. Our aim was to use the recent HuNoV zebrafish larvae model to study the effect of HuNoV infection on intestinal motility and investigate whether one viral protein could act as an enterotoxin, as seen with rotavirus. We studied whether HuNoV infection affects the contraction frequency of the intestinal bulb and the posterior intestine as well as the transit time. Infection of larvae, following injection of a HuNoV GII.4-containing stool sample in the yolk, resulted in an increased contraction frequency in the intestinal bulb. A comparable effect was observed in serotonin-treated larvae, corresponding to the natural function of serotonin. The higher replication efficacy of HuNoV GII.4 likely explains why they have a more marked effect on gut motility, when compared to other genotypes. Additionally, transit time of fluorescent food was prolonged in HuNoV GII.4 infected larvae, suggesting a loss of coordination in bowel movements upon infection. To identify the proteins responsible for the effect, individual HuNoV non-structural proteins and virus-like particles (VLPs) were injected intraperitoneally (ip). VLPs carrying VP1/VP2, but not those with only VP1, induced increased contraction frequencies in the intestinal bulb in a dose-dependent manner. In conclusion, our findings suggest that the viral capsid and potentially the minor capsid protein VP2 play a crucial role in the aetiology of symptoms associated with HuNoV, potentially acting as a viral enterotoxin. This work contributes to the understanding of the pathophysiological mechanisms in HuNoV-induced disease and further attests zebrafish as a valuable HuNoV disease model.

Identification and Characterization of Linear Epitopes of Monoclonal Antibodies Against African Horse Sickness Virus Serotype 1 VP2 Protein.

African horse sickness (AHS) is an acute, fatal, contagious disease of animals of the family Equidae and is caused by infection with the African horse sickness virus (AHSV). Based on the outer capsid protein VP2, AHSV is classified into nine serotypes (AHSV-1 to -9) with little or no serological cross-reactivity between them. In 2020, AHS outbreaks caused by AHSV-1 were reported in Thailand and Malaysia, marking the first occurrences of AHS in Southeast Asia. However, little is known about the antigenic profile of AHSV-1 VP2. In this study, a recombinant VP2 protein was expressed in Escherichia coli and used as an immunogen, and three monoclonal antibodies (mAbs), designated 7D11, 10A9, and 9E7, against AHSV-1 VP2, were generated. These three mAbs were then successfully used in IFA, WB, and ELISA for the detection of AHSV-1 VP2. Two overlapping linear epitopes, 670NEFDFE675 (E670-675) recognized by 9E7 and 670NEFDF674 (E670-674) recognized by 7D11 and 10A9, were identified through truncation of GST-fused VP2. Amino acid sequence alignment shows that the 670NEFDFE675 motif is completely conserved within AHSV-1 but is highly divergent in other AHSV serotypes. Our studies provide an important tool for basic research into AHSV-1 and for the diagnosis of AHSV-1.

Vaccine candidates based on MVA viral vectors expressing VP2 or VP7 confer full protection against Epizootic hemorrhagic disease virus in IFNAR(-/-) mice.

Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an emerging and severe livestock disease. Recent incursion and distribution of EHDV in Europe have outlined the need for vaccine research against this viral disease. In this work, we report modified vaccinia virus Ankara (MVA)-vectored vaccines designed to express protein VP2 of EHDV-8 or protein VP7 of EHDV-2. Prime boost immunization of adult IFNAR(-/-) mice with the MVA-VP2 vaccine candidate induced high titers of EHDV-8-specific neutralizing antibodies (NAbs) and conferred full protection against homologous lethal challenge with EHDV-8. However, no heterologous protection was observed after lethal challenge with EHDV-6. In contrast, the MVA-VP7 vaccine candidate elicited strong cytotoxic CD8+ T-cell responses against VP7 and conferred complete protection against lethal challenge with either EHDV-8 or EHDV-6 in IFNAR(-/-) mice in the absence of NAbs, being the first multiserotype vaccine candidate against EHDV. Moreover, we expressed recombinant proteins VP2 and VP7 of EHDV in the baculovirus expression system, which were used to analyze the potential DIVA (differentiating infected from vaccinated animals) character of these vaccine candidates.IMPORTANCEEmergence and re-emergence of arthropod-borne viruses are major concerns for both human and animal health. The most recent example is the fast expansion of EHDV-8 through Europe. Besides, EHDV-8 relates with a high prevalence of pathologic cases in cattle populations. No vaccine is currently available in Europe, and vaccine research against this arboviral disease is negligible. In this work, we present novel DIVA vaccine candidates against EHDV, and most importantly, we identified the protein VP7 of EHDV as an antigen capable of inducing multiserotype protection, one of the major challenges in vaccine research against orbiviruses.

Molecular Analysis of Coxsackievirus B2 Associated With Severe Symptoms of the Central Nervous System.

Coxsackievirus B2 (CVB2) is a member of the enterovirus group known to induce a spectrum of illnesses, from mild to severe. In the summer of 2022, an unusual outbreak of enteroviral central nervous system (CNS) infections occurred that was attributed to CVB2. Cerebrospinal fluid (CSF) samples collected from patients in 2015-2022 were tested for enterovirus via RT-PCR, followed by Sanger sequencing for positive cases. CVB2 samples were further sequenced in the P1 region using NGS. A total of 30 CSF samples were identified as CVB2, with 60% of these cases occurring between June and August 2022. The 2022 CVB2 variants were associated with severe clinical symptoms, including encephalitis (50%) and ataxia (27.8%). Samples from 2015 to 2020 were also included due to the absence of these symptoms. Phylogenetic analysis revealed that CVB2 strains from 2019 to 2020 were also distinct from those obtained in 2022. Amino acid analysis of the capsid proteins VP1, VP2, and VP3 identified three unique substitutions with potential antigenic significance in the 2022 variant: S67A in VP2, and T93A and E274D in VP1. These substitutions were not present in earlier strains or reported in the literature, suggesting they may influence the virus's virulence. The clinical observations from this study highlight new patterns of CVB2 infection in the CNS that had not been previously observed. Additionally, it identifies unique genetic changes in the 2022 CVB2 variant that may account for the increased virulence seen in the 2022 outbreak. However, further analysis is required to validate this assumption.

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

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.

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.

Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis

Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2. Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene. Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks. Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.

Impact of VP2 structure on antigenicity: comparison of BTV1 and the highly virulent BTV8 serotype.

Bluetongue virus (BTV) is an agriculturally and economically significant insect-borne virus that causes serious illness and death in sheep and other domestic and wild ruminants in large areas of the world. Numerous BTV serotypes exist, and distant serotypes exhibit unique neutralizing antibody profiles, which target the outermost capsid protein VP2. The predominant serotype-specific nature of the antibody response to VP2 is a barrier to the development of broad-spectrum prophylactic BTV vaccine candidates. Although VP2 is the main serotype determinant of BTV, the structural basis of serotype specificity has not been investigated. In this study, we utilized the recently available atomic structure of VP2 with a modeled tip domain to carry out in silico structural comparisons between distant serotypes BTV1 and BTV8. These analyses identified structural differences in the tip domain, positioned at the apex of VP2, and informed the design of mutant VP2 constructs. Dissection of tip domain antigenicity demonstrated that the region of structural difference between BTV1 and highly virulent BTV8 was a target of BTV neutralizing antibodies and that mutation of this region resulted in a loss of neutralizing antibody recognition. This study has for the first time provided insights into the structural differences, which underpin the serotype-specific neutralizing antibody response to BTV.IMPORTANCEThe immune system can protect against virus infection by producing antibodies, which bind and inhibit the virus from infecting the susceptible host. These antibodies are termed neutralizing antibodies and generally target the viral receptor binding protein, such as the VP2 of bluetongue virus (BTV). This pressure from the immune system can drive mutation of the viral protein resulting in escape from antibody-mediated neutralization and the evolution of serotypes, as is the case for BTV. Understanding the structural differences, which underpin the different BTV serotypes, could help guide the design of a BTV vaccine that targets multiple serotypes. In this study, we have mapped the VP2 structural differences between distant serotypes, to a region targeted by neutralizing antibodies, and have demonstrated for the first time how VP2 structure is the fundamental basis of serotype specificity.

Molecular Characterization and Phylogenetic Analysis of Honeybee (Apis mellifera) Mite-Borne Pathogen DWV-A and DWV-B Isolated from Lithuania.

Deformed wing virus (DWV) is known as one of the main viruses that affect honeybees' health all around the world. The virus has two widespread genotypes, DWV-A and DWV-B (VDV-1), transmitted mainly by V. destructor mites. In this study, we collected honeycombs with covered broods from 73 apiaries in eight Lithuanian regions and initially investigated the prevalence of V. destructor mites. Mites were collected from May to the end of July in 2021 from 124 hives. The prevalence of V. destructor infestations in beehives reached 30% and 63% in investigated apiaries. The presence of DWV-A and DWV-B pathogens in mites and broods was examined by RT-qPCR targeting the CRPV-capsid region. The molecular characterization of the virus in mite samples was based on sequence analysis of the RNA-dependent RNA polymerase (RdRp) region. In addition, leader polypeptide (LP), structural protein (Vp3), Helicase, and RdRp genes were used for phylogenetic characterization of dual infection. The prevalences of DWV-B in mites and broods were 56.5% and 31.5%, respectively, while DWV-A was detected in 12.9% of mite samples and 24.7% of brood samples. Some of the examined mite samples harboured dual virus infections. Our findings showed that bee colonies from the same apiary were not always infected by the same viruses. Some bee colonies were virus-free, while others were highly infected. Phylogenetic analysis of 21 sequences demonstrated the presence of highly variable DWV-B and DWV-A genotypes in Lithuania and possible recombinant variants of the virus. This study represents the first molecular characterization of mite-borne pathogens hosted by honeybees (Apis mellifera) in Lithuania.

ANAX4 is a downstream molecule of LGP2 and promotes GCRV proliferation

This study explored the key molecules and signal pathways in the pathogenesis of grass carp reovirus (GCRV). Using immunoprecipitation mass spectrometry and Co-IP validation, the protein CiANXA4 was identified which interacts indirectly with CiLGP2. CiANXA4 encodes 321 amino acids, including 4 ANX domains. To explore the role of CiANXA4 in the anti-GCRV immune response, we used overexpression and siRNA knockdown in cells. The results showed that overexpression of the CiANXA4 gene significantly increased the mRNA content of vp2 and vp7 in GCRV-infected cells, and the virus titer greatly increased. Knockdown of CiANXA4 significantly inhibited the mRNA levels of vp2 and vp7, and the protein levels of viral protein VP7 also significantly decreased. This suggests that CiANXA4 promotes viral proliferation. Further, we demonstrate that the ANX3 and ANX4 domains are key domains that limit CiANXA4 function by constructing domain-deletion mutants. Finally, we investigated the relationship between CiLGP2 and CiANXA4. RT-PCR and Western blot results showed that CiLGP2 mRNA and protein expression levels were not affected by CiANXA4 overexpression. In contrast, overexpression of CiLGP2 resulted in significant reductions in CiANXA4 mRNA and protein levels. This suggests that the function of CiANXA4 is restricted by CiLGP2, and CiANXA4 is a downstream molecule of CiLGP2. These results reveal that CiANXA4 plays a critical role in the anti-GCRV innate immune response of grass carp, and provides new targets and strategies to develop antiviral drugs and improve disease resistance in grass carp.

TRIM103 activates the RLRs pathway to enhance antiviral response by targeting VP5 and VP7

Grass carp (Ctenopharyngodon idella), crucial to global inland aquaculture with a production of 5.8 million tones in 2020, faces significant challenges from hemorrhagic disease caused by grass carp reovirus (GCRV). Rapid mutations compromise current vaccines, underscoring the need for a deeper understanding of antiviral mechanisms to enhance molecular marker-assisted selection. This study investigates the role of Tripartite Motif (TRIM) family in the innate immune response of grass carp, focusing on TRIM103 from Ctenopharyngodon Idella (CiTRIM103), a member of the TRIM-B30.2 family, which includes proteins with the B30.2 domain at the N-terminus, known for antiviral properties in teleosts. CiTRIM103 bind to the outer coat proteins VP5 and VP7 of GCRV. This binding is theorized to strengthen the function of the RIG-I-like Receptor (RLR) signaling pathway, crucial for antiviral responses. Demonstrations using overexpression and RNA interference (RNAi) techniques have shown that CiTRIM103 effectively inhibits GCRV replication. Moreover, molecular docking and pulldown assays suggest potential binding interactions of CiTRIM103's B30.2 domain with GCRV outer coat proteins VP5 and VP7. These interactions impede viral replication, enhance RLR receptor expression, and activate key transcription factors to induce type I interferons (IFNs). These findings elucidate the antiviral mechanisms of CiTRIM103, provide a foundation for future Molecular genetic breeding in grass carp.

The Structure of Spiroplasma Virus 4: Exploring the Capsid Diversity of the Microviridae.

Spiroplasma virus 4 (SpV4) is a bacteriophage of the Microviridae, which packages circular ssDNA within non-enveloped T = 1 icosahedral capsids. It infects spiroplasmas, which are known pathogens of honeybees. Here, the structure of the SpV4 virion is determined using cryo-electron microscopy to a resolution of 2.5 Å. A striking feature of the SpV4 capsid is the mushroom-like protrusions at the 3-fold axes, which is common among all members of the subfamily Gokushovirinae. While the function of the protrusion is currently unknown, this feature varies widely in this subfamily and is therefore possibly an adaptation for host recognition. Furthermore, on the interior of the SpV4 capsid, the location of DNA-binding protein VP8 was identified and shown to have low structural conservation to the capsids of other viruses in the family. The structural characterization of SpV4 will aid future studies analyzing the virus-host interaction, to understand disease mechanisms at a molecular level. Furthermore, the structural comparisons in this study, including a low-resolution structure of the chlamydia phage 2, provide an overview of the structural repertoire of the viruses in this family that infect various bacterial hosts, which in turn infect a wide range of animals and plants.

Novel goose parvovirus VP1 targets IRF7 protein to block the type I interferon upstream signaling pathway

Outbreaks of short beak and dwarfism syndrome (SBDS), caused by a novel goose parvovirus (NGPV), have occurred in China since 2015. The NGPV, a single-stranded DNA virus, is thought to be vertically transmitted. However, the mechanism of NGPV immune evasion remains unclear. In this study, we investigated the impact of NGPV infection on the Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in duck embryonic fibroblast (DEF) cells. Our findings demonstrate that NGPV infection stimulates the mRNA expression of cGAS but results in weak IFN-β induction. NGPV impedes the expression of IFN-β and downstream interferon-stimulated genes, thereby reducing the secretion of IFN-β induced by interferon-stimulating DNA (ISD) and poly (I: C). RNA-seq results show that NGPV infection downregulates interferon mRNA expression while enhancing the mRNA expression of inflammatory factors. Additionally, the results of viral protein over-expression indicate that VP1 exhibits a remarkable ability to inhibit IFN-β expression compared to other viral proteins. Results indicated that only the intact VP1 protein could inhibit the expression of IFN-β, while the truncated proteins VP1U and VP2 do not possess such characteristics. The immunoprecipitation experiment showed that both VP1 and VP2 could interact with IRF7 protein, while VP1U does not. In summary, our findings indicate that NGPV infection impairs the host's innate immune response by potentially modulating the expression and secretion of interferons and interferon-stimulating factors via IRF7 molecules, which are regulated by the VP1 protein.

Genetic and recombination heterogeneity of canine bufaviruses detected in diarrheal dogs in China

Bufavirus (BuV) was first identified in feces from children with acute diarrhea, and a genetically related Canine bufavirus (CBuV) was first reported in Italy in 2018. In this study, through the investigation of CBuV in 622 anal swabs from dogs with diarrhea symptoms collected from various provinces in northern, central and eastern China during 2018–2022, 14 samples were detected to be positive. And 5 samples were from dogs co-infected with other canine diarrhea related viruses, which consist of CPV-2, CDV and CCoV. The complete genome sequences (4219 nt) of the fourteen strains were amplified and sequenced. Through comparative analysis with 51 reference BuV strains, six strains might recombinate from the CBuV strains (HUN/2012/22, CaBuV/9AS/2005/ITA and CaBuV/35/2016/ITA) in Hungary and Italy as the parents, and two genetic recombination events from various parents were predicted to occur on the BUV-422 strain. Combined analyzing the phylogenetic tree and sequence alignment, it was found that these CBuVs are highly conserved in the nonstructural protein NS1, but indeed various amino acid mutation sites in the capsid protein VP2, and even some amino acid sites coincide with putative protein plastic regions and potential epitopes. The BUV-422 and BUV-512 strains show sequential mutation sites identical to the divergent strains of CaBuV/9AS/2005/ITA and CaBuV/35/2016/ITA. This study would enrich the molecular data of CBuV in China and provide essential reference for the epidemiological research and vaccine development of CBuV in the future.

Poliovirus capsid protein VP3 can penetrate vascular endothelial cells.

The poliovirus (PV) enters the central nervous system (CNS) via the bloodstream, suggesting the existence of a mechanism to cross the blood-brain barrier. Here, we report that PV capsid proteins (VP1 and VP3) can penetrate cells, with VP3 being more invasive. Two independent parts of VP3 are responsible for this function. Both peptides can penetrate human umbilical cord vascular endothelial cells, and one peptide of VP3 could also penetrate peripheral blood mononuclear cells. In an in vitro blood-brain barrier model using rat-derived astrocytes, pericytes, and endothelial cells, both peptides were observed to traverse from the blood side to the brain side at 6 h after administration. These results provide insights into the molecular mechanisms underlying PV invasion into the CNS.

First identification of canine parvovirus -2a/2b variant in unvaccinated domestic dogs with gastrointestinal signs in Türkiye.

Canine parvovirus type 2 (CPV-2) is the most common enteric virus that infects canids. CPV is the causative agent of a contagious disease defined mostly by clinical gastrointestinal signs in dogs. During the late 1970s, CPV-2 emerged as a new virus capable of infecting domestic dogs and growing across the world. The VP2 gene stands out as a key determinant in the pathogenicity, antigenicity, and host interactions of CPV-2. The molecular characterization of the VP2 gene is crucial for understanding CPV evolution and epidemiology. Genes encoding the VP2 protein were sequenced and compared to reference strains worldwide. The maximum likelihood method was used to build a phylogenetic tree using CPV VP2 gene nucleotide sequences. Our phylogenetic analysis of the VP2 gene revealed that five strains were very similar and clustered together, and three strains were in the 2b clade, whereas the other two were in the 2a/2b clade. This paper reports the molecular characterization of two novel CPV-2a/2b subtypes in dogs with gastrointestinal symptoms. Genetic analysis was conducted on a CPV genomic region encompassing one of the open reading frames (ORFs) encoding the structural protein VP2. Sequence analysis indicates new and unreported sequence changes, mainly affecting the VP2 gene, which includes the mutations Ser297Ala and Leu87Met. This study represents the first evidence of a new CPV-2a/2b subtype in Türkiye. Due to VP2's crucial role in encoding the capsid protein of CPV-2 and its significant involvement in the host-virus interaction, it is critical to closely monitor its evolutionary changes and be cautious while searching for novel or pre-existing subtypes. This study highlights the significance of continuous molecular research for acquiring more insights on the circulation of novel CPV mutants.

Human Rotaviruses of Multiple Genotypes Acquire Conserved VP4 Mutations during Serial Passage.

Human rotaviruses exhibit limited tropism and replicate poorly in most cell lines. Attachment protein VP4 is a key rotavirus tropism determinant. Previous studies in which human rotaviruses were adapted to cultured cells identified mutations in VP4. However, most such studies were conducted using only a single human rotavirus genotype. In the current study, we serially passaged 50 human rotavirus clinical specimens representing five of the genotypes most frequently associated with severe human disease, each in triplicate, three to five times in primary monkey kidney cells then ten times in the MA104 monkey kidney cell line. From 13 of the 50 specimens, we obtained 25 rotavirus antigen-positive lineages representing all five genotypes, which tended to replicate more efficiently in MA104 cells at late versus early passage. We used Illumina next-generation sequencing and analysis to identify variants that arose during passage. In VP4, variants encoded 28 mutations that were conserved for all P[8] rotaviruses and 12 mutations that were conserved for all five genotypes. These findings suggest there may be a conserved mechanism of human rotavirus adaptation to MA104 cells. In the future, such a conserved adaptation mechanism could be exploited to study human rotavirus biology or efficiently manufacture vaccines.

Chicken PRMT3 facilitates IBDV replication

Protein arginine methyltransferases (PRMTs) in mammals play a role in various signaling pathways, such as virus infection, inflammasome responses, and cancer growth. While some PRMTs have been found to regulate interferon production in mammals, the mechanism in chickens remains to be fully understood. This study focused on investigating the function of chicken PRMTs. Our findings indicate that chicken PRMTs act as inhibitors of interferon production in response to dsRNA or MDA5 stimulation. Each PRMT is involved in different stages of interferon induction through the MDA5-MAVS-TBK1 pathway. Furthermore, we observed the colocalization of multiple PRMTs with the viral protein VP3 of infectious bursal disease virus (IBDV). Among the chicken PRMTs studied, PRMT3 was found to be widely expressed in various organs and its expression was upregulated during IBDV infection. Notably, PRMT3 supported IBDV replication, as demonstrated by ectopic expression and inhibition studies using SGC-707. Silencing of PRMT3 led to enhanced interferon production and inhibition of IBDV replication. This study provides novel insights into the role of chicken PRMTs, particularly PRMT3, in promoting IBDV replication by suppressing interferon signaling.

Hunnivirus structural protein VP2 inhibits beta interferon production by targeting the IRF3 essential modulator

Water buffalo Hunnivirus (BufHuV) belongs to the family Picornaviridae and is a newly discovered member of the Hunnivirus A genus. It causes intestinal diseases in cattle, mainly lead to subclinical infections, thereby seriously threatening the health of cattle herds. In addition, it can also bring about various clinical disease syndromes which results in severe economic losses to the cattle industry. To date, there have been no reports worldwide on the study of Hunnivirus virus infecting host cells and causing innate immune responses. In this study, we found that interferon treatment effectively blocked BufHuV replication and infection with the virus weakened the host antiviral responses. Inhibiting the transcription of IFN-β and ISGs induced by either Sendai virus (SeV) or poly(I:C) in MDBK and HCT-8 cells, were dependent on the IRF3 or NF-κB signaling pathways, and this inhibited the activation of IFN-β promoter by TBK1 and its upstream molecules, RIGI and MDA5. By constructing and screening five BufHuV proteins, we found that VP2, 2 C, 3 C and 3D inhibited the activation of IFN-β promoter induced by SeV. Subsequently, we showed that VP2 inhibited the activation of IRF3 induced by SeV or poly (I:C), and it inhibited IRF3 activation by inhibiting its phosphorylation and nuclear translocation. In addition, we confirmed that VP2 inhibited the activation of IFNβ induced by signaling molecules, MDA5 and TBKI. In summary, these findings provide new insights into the pathogenesis of Hunnivirus and its mechanisms involved in evading host immune responses.