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We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5′-cap structure is essential for the virus' generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly. [ABSTRACT FROM AUTHOR]

Senecavirus A (SVA) is an RNA virus in the family Picornaviridae that has been detected in swine-production systems and is associated with vesicular disease and neonate mortality. The viral capsid is composed of four structural proteins: VP1-VP4. Although the VP1 protein has been reported to be the most immunogenic protein in vivo, no information on the immunodominant regions of the SVA polyprotein is available. The objective of this study was to identify the immunodominant regions of SVA polyprotein using an enzyme-linked immunosorbent assay (ELISA) epitope-mapping approach. The binding effect of SVA polyclonal antibody (SVA-pAb), SVA-VP1 monoclonal antibodies (SVA-mAb), and SVA-positive sera from clinically affected animals were characterized using a set of 18 overlapping SVA VP1-derived peptides by indirect and blocking ELISAs. All VP1 peptides yielded significant signal against SVA-pAb and SVA-VP1-mAb upon indirect ELISA. One peptide (aa 1-20) showed significantly high optical density on SVA recombinant VP1 protein (rVP1) and whole-virus-based indirect ELISAs. The blocking ELISA results demonstrated that peptides spanning aa 165-185 and 225-245 had a 50 % or greater inhibitory effect on SVA-pAb, while six groups of overlapping peptides spanning aa 1-35, 45-80, 90-140, 150-170, 195-230, and 240-264 and two groups of overlapping peptides spanning aa 1-50 and 60-264 showed a 50 % inhibitory effect or greater on swine VP1-mAb and SVA-seropositive swine serum, respectively, against SVA rVP1. Three-dimensional protein homology modeling showed that the peptides binding SVA-pAb are located on the outer surface of the viral capsid, while SVA mAbs and swine-positive sere can bind to epitopes located in both the inner and outer surfaces of the capsid. These linear epitopes showed differential binding and inhibitory activity on mAb and pAb; however, further studies will be necessary to evaluate whether they can act as decoy or neutralizing epitopes. Because mAb antibodies demonstrated a high binding affinity for this set of peptides, this information could lay the foundation for generating and screening specific antibodies for therapeutic potential., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pablo Pineyro reports was provided by Iowa State University of Science and Technology College of Veterinary Medicine. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

Coxsackievirus A2 (CVA2) is associated with multiple diseases in children. Currently, there is limited research on immunological detection methods for CVA2. Herein, the VP1 gene of CVA2 strain 201711, belonging to cluster 2 within genotype D, was analyzed. The structures of VP1 from CVA2 strains 201711, 7-1 and 12-1, enterovirus A71 (EV-A71) strain 201713, coxsackievirus A16 (CVA16) strain 201717, and coxsackievirus A6 (CVA6) strain JLS10 were compared. The Escherichia coli BL21(DE3)/pET vector system was employed to express the recombinant protein containing the entire VP1 of CVA2 strain 201711. Mice were immunized with the purified protein, and the sera were collected and used to specifically identify the VP1 in CVA2-infected RD cells by Western blot and immunofluorescence assay. There was no evident cross-reactivity of the sera with the VP1 of EV-A71, CVA16, and CVA6 strains mentioned above. Therefore, this study provided mouse-specific anti-CVA2 VP1 polyclonal antibodies for CVA2 detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pyrazolo[3,4-d]pyrimidines represent one potent class of well tolerated and highly active rhinovirus (RV) inhibitors that act as capsid binders. The lead compound OBR-5-340 inhibits a broad-spectrum of RVs. Aiming to improve lead activity, we evaluated the impact of structural modifications in the 3-phenyl ring of OBR-5-340 on its potency and spectrum of anti-RV activity vitro. Our results demonstrate the crucial role of substitution at position 4 for strong, broad-spectrum anti-RV activity. The 4-methyl (RCB23137) and 4-chloro (RCB23138) derivatives outperformed OBR-5-340 in terms of potency and anti-RV activity spectrum. Based on these findings, the compounds were selected for computational binding studies. Molecular dynamic simulations with six RVs differing in OBR-5-340, RCB23137, and RCB23138 sensitivity proved the impact of dynamic features of two VP1 loops enveloping these inhibitors on antiviral potency., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Grants/funding This work was supported by the Russian Science Foundation (project number 24-15-00066) and the Dritte Patentportfolio Beteiligungsgesellschaft mbH & Co. KG (Germany). Patents: 50 2007 006 042.9. 50 2012 017 045.1., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6–32% of VP1+ cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68–94% of them losing parts of their DNA and exhibiting membrane damage, appearing as “ghost cells” but still VP1+. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.

Burundi is a small, densely populated country in the African Great Lakes region. In March 2016, several hundreds of cattle were reported with vesicular lesions, suggesting foot-and-mouth disease (FMD). Epithelial samples, saliva, and blood were collected in six of the affected provinces spread over the country. The overall seroprevalence of FMD virus (FMDV) in the affected herds, as determined by antibodies against FMDV non-structural proteins, was estimated at 87%. Antibodies against FMDV serotypes O (52%), A (44%), C (19%), SAT1 (36%), SAT2 (58%), and SAT3 (23%) were detected across the provinces. FMDV genome was detected in samples from five of the six provinces using rRT-PCR. FMDV was isolated from samples from three provinces: in Cibitoke province, serotypes A and SAT2 were isolated, while in Mwaro and Rutana provinces, only serotype SAT2 was isolated. In Bururi and Cankuzo provinces, the serological profile suggested a recent incursion with serotype SAT2, while in Bubanza province, the serological profile suggested past incursions with serotype O and possibly serotype SAT1. The phylogenetic assessments showed the presence of topotypes A/Africa/G-I and SAT2/IV, similarly to previously characterized virus strains from other countries in the region, suggesting a transboundary origin and necessitating a regional approach for vaccination and control of FMD. [ABSTRACT FROM AUTHOR]

Infectious bursal disease (IBD) is caused by infectious bursal disease virus (IBDV), which has a genome consisting of two segments of double-stranded linear RNA. IBDVs have been traditionally divided into four phenotypes based on their pathogenicity and antigenicity, including classic, variant, very virulent, and attenuated IBDV. With the emergences of divergent molecular characteristics of novel strains produced by continuous mutations and recombination, it is increasingly difficult to define new IBDV strains using the traditional descriptive classification method. The most common classification scheme for IBDV with segmented genome is based solely on segment A, while the significance of segment B has been largely neglected. In this study, an improved scheme for IBDV genotype classification based on the molecular characteristics of both VP2 (a viral capsid protein encoded by segment A) and VP1 (an RNA-dependent RNA polymerase protein encoded by segment B) was proposed for the first time. In this scheme, IBDV was classified into nine genogroups of A and five genogroups of B, respectively; the genogroup A2 was further divided into four lineages. The commonly used phenotypic classifications of classic, variant, very virulent, and attenuated IBDVs correspond to the A1B1, A2B1, A3B2, and A8B1 genotypes of the proposed classification scheme. The novel variant IBDVs including the strains identified in this study were classified as belonging to genotype A2dB1. The flexibility and versatility of this improved classification scheme will allow the unambiguous identification of existing and emerging IBDV strains, which will greatly facilitate molecular epidemiology studies of IBDV.

Chicken anemia virus (CAV) is one of the primary causes of morbidity and mortality in young chickens. Given the importance of timely detection for maintaining livestock quality, there is a pressing need for rapid and field-deployable diagnostic tools. This study introduces a highly sensitive paper-based electrochemical immunosensor (PEI) for the detection of the 60 amino acid N-terminally truncated viral protein 1 (Δ60VP1), a derivative of the CAV capsid (VP1). A custom antibody was produced for precise immunoassay detection, with results obtainable within 30 min using Square Wave Voltammetry (SWV). The underlying mechanism involves an immunocomplex in the sample zone that hinders the electron transfer of redox species, thereby reducing the current signal in proportion to the Δ60VP1 concentration. Under optimal conditions, the detection linearity for Δ60VP1 ranged from 80 to 2500 ng/mL, with a limit of detection (LoD) of 25 ng/mL. This device was then successfully applied to detect VP1 in 29 chicken serum samples, achieving 91.6% sensitivity and 94.1% selectivity. In conclusion, the PEI device presents a promising solution for rapid, sensitive, and disposable detection of chicken pathogens, potentially revolutionizing productivity and quality assurance in chicken farming. [Display omitted] • The paper-based electrochemical immunosensor is a novel tool for detecting CAV. • This method provides rapid and high-sensitivity CAV detection. • A detection limit for CAV is established at 25 ng/mL. • This device demonstrates excellent specificity in chicken serum tests. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) has long been recognized as a highly contagious, transboundary disease of livestock incurring substantial losses and burdens to animal production and trade across Africa, the Middle East, and Asia. Due to the recent emergence of the O/ME-SA/Ind-2001 lineage globally contributing to the expansion of FMD, molecular epidemiological investigations help in tracing the evolution of foot-and-mouth disease virus (FMDV) across endemic and newly affected regions. In this work, our phylogenetic analysis reveals that the recent FMDV incursions in Russia, Mongolia, and Kazakhstan in 2021–2022 were due to the virus belonging to the O/ME-SA/Ind-2001e sublineage, belonging to the cluster from Cambodian FMDV isolates. The studied isolates varied by 1.0–4.0% at the VP1 nucleotide level. Vaccine matching tests indicated that the vaccination policy in the subregion should be tailored according to the peculiarities of the ongoing epidemiologic situation. The current vaccination should change from such vaccine strains as O1 Manisa (ME–SA), O no 2102/Zabaikalsky/2010 (O/ME-SA/Mya-98) (r1 = 0.05–0.28) to strains that most closely antigenically match the dominant lineage O No. 2212/Primorsky/2014 (O O/ME-SA//Mya-98) and O No. 2311/Zabaikalsky/2016 (O ME-SA/Ind-2001) (r1 = 0.66–1.0).

Background: The quasi-enveloped picornavirus, Hepatitis A Virus (HAV), causes acute hepatitis in humans and infects approximately 1.5 million individuals a year, which does not include the asymptomatically infected population. Several severe outbreaks in developing nations in recent years have highlighted the reduction in HAV endemicity, which increases the risk of infections in the vulnerable population. The current HAV vaccines are based on growing wildtype or attenuated virus in cell culture, which raises the cost of production. For generation of cheaper, subunit vaccines or strategies for antibody-based diagnostics, production of viral structural proteins in recombinant form in easily accessible expression systems is a priority. Results: We attempted several strategies for recombinant production of one of the major capsid proteins VP1, from HAV, in the E. coli expression system. Several efforts resulted in the formation of soluble aggregates or tight association of VP1 with the bacterial chaperone GroEL. Correctly folded VP1 was eventually generated in a discrete oligomeric form upon purification of the protein from inclusion bodies and refolding. The oligomers resemble oligomers of capsid proteins from other picornaviruses and appear to have the correct secondary and antigenic surface structure. Conclusions: VP1 oligomers generated in the bacterial expression system can be utilized for understanding the molecular pathway of HAV capsid assembly and may also have potential biomedical usages in prevention and diagnostics of HAV infections. [ABSTRACT FROM AUTHOR]

In this study, we sought to develop a subunit vaccine against the increasingly prevalent Duck hepatitis A virus serotype 3 (DHAV-3). The VP1 protein of DHAV-3 and a truncated version containing the C-terminal region of VP1, termed VP1-C, were expressed recombinantly in Escherichia coli as vaccine antigens. For enhanced immune response, a truncated version of flagellin, nFliC, was included as vaccine adjuvant. Ducklings were vaccinated once for immune response analysis and challenge test. Results showed that VP1-C elicited a higher level of virus-specific antibody response and neutralization titer than VP1. The addition of nFliC further enhanced the antibody response. In terms of cellular immune response, the VP1-C + nFliC vaccine elicited the highest level of T cell proliferation among the vaccine formulations tested. Examination of the cytokine expression profile showed that peripheral blood mononuclear cells from the VP1-C + nFliC vaccine group expressed the highest levels of pro-inflammatory (IL-6) and TH-1 type (IL-12 and IFN-γ) cytokines. Finally, in a DHAV-3 challenge test, the VP1-C + nFliC vaccine provided a 75% protection rate (n = 8), in contrast to 25% for the VP1 vaccine. In conclusion, E. coli-expressed VP1-C has been shown to be a promising antigen when combined with nFliC and may be further developed as a single-dose subunit vaccine against DHAV-3. [ABSTRACT FROM AUTHOR]

Three strains of the FMD virus (A, O, and SAT 2) were recognised as causes of the FMD circulating in Egypt. The aims of this study were to trace the FMDV isolates from outbreaks in Egypt to understand their epidemiology and evolution and to understand the situation of the vaccine strains compared with the circulating serotypes.

Enterovirus 71 (EV-A71) infection causes severe hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children under 5 years of age. Although there are available vaccines for EV-A71 C4, however, there are no efficient drugs for severe cases. Thus, there is an urgent need to find new direct-antiviral agents (DAAs) to control EV-A71 infection. In this study, we report our discovery of the EV-A71 capsid inhibitor PTC-209HBr, a small-molecule Bmi-1 inhibitor and an anticancer agent, and its derivatives that inhibit multiple enteroviruses with an EC50 at a submicromolar efficacy. The mechanism of action of PTC-209HBr was confirmed by time-of-addition, resistance selection and reverse genetics experiments, microscale thermophoresis (MST), viral binding and entry assays, coimmunoprecipitation (Co-IP) and immunofluorescence experiments (IF). Mechanistic studies indicated that PTC-209HBr inhibited EV-A71 infection by impeding the binding between VP1 and the receptor hSCARB2 during the early stage of EV-A71 infection through hindering viral entry into host cells. Collectively, these findings indicated that PCT-209HBr is a novel inhibitor of enteroviruses with a confirmed mechanism of action that can be further developed into EV-A71 DAAs.

Abstract Background Enterovirus 71 (EV71) is the most commonly implicated causative agent of severe outbreaks of paediatric hand, foot, and mouth disease (HFMD).VP1 protein, a capsid protein of EV71, is responsible for the genotype of the virus and is essential for vaccine development and effectiveness. However, the genotypes of EV71 isolates in China are still not completely clear. Methods The VP1 gene sequences of 3712 EV71 virus strains from China, excluding repetitive sequences and 30 known EV71 genotypes as reference strains, between 1986 and 2019 were obtained from GenBank. Phylogenetic tree, amino acid homology, genetic variation and genotype analyses of the EV71VP1 protein were performed with MEGA 6.0 software. Results The amino acid identity was found to be 88.33%–100% among the 3712 EV71 strains, 93.47%–100% compared with vaccine strain H07, and 93.04%–100% compared with vaccine strains FY7VP5 or FY-23 K-B. Since 2000, the prevalent strains of EV71 were mainly of the C4 genotype. Among these, the C4a subgenotype was predominant, followed by the C4b subgenotype; other subgenotypes appeared sporadically between 2005 and 2018 in mainland China. The B4 genotype was the main genotype in Taiwan, and the epidemic strains were constantly changing. Some amino acid variations in VP1 of EV71 occurred with high frequencies, including A289T (20.99%), H22Q (16.49%), A293S (15.95%), S283T (15.11%), V249I (7.76%), N31D (7.25%), and E98K (6.65%). Conclusion The C4 genotype of EV71 in China matches the vaccine and should effectively control EV71. However, the efficacy of the vaccine is partially affected by the continuous change in epidemic strains in Taiwan. These results suggest that the genetic characteristics of the EV71-VP1 region should be continuously monitored, which is critical for epidemic control and vaccine design to prevent EV71 infection in children.

Abstract The seven serotypes of foot-and-mouth disease virus (FMDV) differ on the surface exposed regions on the VP1, 2 and 3 proteins. Amongst the three, the VP1 protein has been produced the most for use in serotyping assays for some of the Euro-Asian serotypes. In this study the VP1 protein of the FMDV SAT2/ZIM/7/83 was expressed in Escherichia coli BL21 cells in Luria broth and EnPresso® B media in shake flasks. Production was further developed and the VP1 protein was produced at 2.15 g L−1 in fed-batch fermentations at 2 L scale. The protein formed insoluble inclusion bodies that were isolated, denatured and refolded. When tested in ELISA, the protein was found to be highly reactive with serum from a SAT2 vaccinated guinea pig, and not reactive to SAT1 and SAT3 antisera. These results open avenues to evaluate recombinantly expressed VP1 proteins for differentiation of the three Southern African Territories serotypes of FMDV that co-occur in Southern and East Africa. In addition, this could mitigate the need for employing virus as reagent, or having to raise reagent antibodies.

Introduction: Coxsackievirus A10 (CVA10) is a non-enveloped, positive-sense single-stranded RNA virus classified within the Enterovirus genus in the Picornaviridae family. It is among the pathogens that can cause hand, foot and mouth disease. This study aimed to analyze the temporal and spatial distribution of CVA10 in China to understand its epidemiological characteristics of CVA10., Methodology: We collected the VP1 sequences of CVA10 from January 1, 2004, to December 31, 2019, from the GenBank database and created the global map using MapChart. We selected 56 known CVA10 genotype sequences. Then, MEGA6.06 was used to construct a phylogenetic tree with the collected gene sequences and the known reference sequences for comparative analysis to assess the distribution of CVA10 genotypes in different countries between 2004 and 2019., Results: CVA10 has been widely detected or reported globally. In China, the prevalent genotype of CVA10 was mainly genotype B before 2008 and genotype C after 2009. In other countries, the prevalence of genotype D was dominant, followed by genotypes C and F, and the prevalence of CVA10 varied from continent to continent., Conclusions: Monitoring CVA10 genotypes or evolutionary branches should be strengthened, and the study of epidemic genotype characteristics should be enhanced. This will serve as a basis for further research and development of monovalent CVA10 or polyvalent vaccines designed for effective disease prevention., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Hua Wang, Wenhong Wang, Guoqing Chen, Chuanjie Hu, Shengjie Chen, Lingxiang Mao, Hongxing Shen.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., Competing Interests: The authors declare no conflict of interest.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) possesses multiple biological activities such as virus restriction, innate immunity regulation, and autoimmunity. Our previous study demonstrated that SAMHD1 potently inhibits the replication of enterovirus 71 (EV71). In this study, we observed that SAMHD1 also restricts multiple enteroviruses (EVs), including coxsackievirus A16 (CA16) and enterovirus D68 (EVD68), but not coxsackievirus A6 (CA6). Mechanistically, SAMHD1 competitively interacted with the same domain in VP1 that binds to VP2 of EV71 and EVD68, thereby interfering with the interaction between VP1 and VP2, and therefore viral assembly. Moreover, we showed that the SAMHD1 T592A mutant maintained the EV71 inhibitory effect by attenuating the interaction between VP1 and VP2, whereas the T592D mutant failed to. We also demonstrated that SAMHD1 could not inhibit CA6 because a different binding site is required for the SAMHD1 and VP1 interaction. Our findings reveal the mechanism of SAMHD1 inhibition of multiple EVs, and this could potentially be important for developing drugs against a broad range of EVs. IMPORTANCE Enterovirus causes a wide variety of diseases, such as hand, foot, and mouth disease (HFMD), which is a severe public problem threatening children under 5 years. Therefore, identifying essential genes which restrict EV infection and exploring the underlying mechanisms are necessary to develop an effective strategy to inhibit EV infection. In this study, we report that host restrictive factor SAMHD1 has broad-spectrum antiviral activity against EV71, CA16, and EVD68 independent of its well-known deoxynucleoside triphosphate triphosphohydrolase (dNTPase) or RNase activity. Mechanistically, SAMHD1 restricts EVs by competitively interacting with the same domain in VP1 that binds to VP2 of EVs, thereby interfering with the interaction between VP1 and VP2, and therefore viral assembly. In contrast, we also demonstrated that SAMHD1 could not inhibit CA6 because a different binding site is required for the SAMHD1 and CA6 VP1 interaction. Our study reveals a novel mechanism for the SAMHD1 anti-EV replication activity. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of South China Agricultural University is the property of Gai Kan Bian Wei Hui and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Significant challenges to poultry health are posed by chicken anemia virus (CAV), which induces immunosuppression and causes increased susceptibility to secondary infections. The effective management and containment of CAV within poultry stocks require precise and prompt diagnosis. However, a deficiency persists in the availability of low-cost, rapid, and portable CAV detection devices. In this study, an immunochromatographic lateral-flow test strip-based assay was developed for CAV detection using in-house generated monoclonal antibodies (MABs) against CAV viral protein 1 (VP1). The recombinant truncated VP1 protein (Δ60VP1), with amino acid residues 1 to 60 of the native protein deleted, was produced via a prokaryotic expression system and utilized for immunizing BALB/c mice. Subsequently, high-affinity MABs against Δ60VP1 were generated and screened using conventional hybridoma technology combined with serial dilution assays. Two MABs, MAB1, and MAB3, both binding to distinct epitopes of Δ60VP1, were selected for the development of a lateral-flow assay. Sensitivity analysis demonstrated that the Δ60VP1 antigen could be detected by our homemade lateral-flow assay at concentrations as low as 625 ng/mL, and this sensitivity was maintained for at least 6 mo. The assay exhibited high specificity, as evidenced by its lack of reactivity with surrogate recombinant proteins and the absence of cross-reactivity with other chicken viruses and viral antigens. Comparative analysis with quantitative PCR data demonstrated substantial agreement, with a Kappa coefficient of 0.66, utilizing a sample set comprising 305 clinical chicken serum samples. In conclusion, the first lateral-flow assay for CAV detection was developed in this study, utilizing 2 specific anti-VP1 MABs. It is characterized by simplicity, rapidity, sensitivity, and specificity. [ABSTRACT FROM AUTHOR]

Gumboro virus is one of the most dangerous immunosuppressant viruses that infect chickens and causes massive financial losses worldwide. The current study aims to conduct a molecular characterization of chicken farms for the infectious bursal disease virus (IBDV). Based on postmortem (PM) lesions, 125 bursal samples from 25 farms were collected from clinically diseased commercial chicken farms with increased mortality and suspected Gumboro virus infection. Pooled bursal samples from suspected IBD-vaccinated flocks were tested for IBDV by reverse transcriptase polymerase chain reaction (RT-PCR). Fifteen out of 25 pooled specimens were found positive for IBDV, with a 60% detection rate, and confirmed positive for very virulent IBDV (vvIBDV) by sequence analysis. Nucleotide phylogenetic analysis of VP1 and VP2 genes was employed to compare the 5 chosen isolates with strains representing different governorates in Egypt during 2022. All strains were clustered with vvIBDV with no evidence of reassortment in the VP1 gene. The VP1 and VP2 genes are divided into groups (I, II). The strains in our study were related to group II, and it acquired a new mutation in the VP2 gene that clustered it into new subgroup B. By mutation analysis, the VP2 gene of all strains had a characteristic mutation to vvIBDV. It acquired new mutations in HVRs compared with HK46 in Y220F, A222T/V in all strains in our study, and Q221K that was found in IBD-EGY-AH5 and AH2 in the loop P BC in addition to G254S in all strains in our study and Q249k that found in IBD-EGY-AH1 and AH3 in the loop P DE. These mutations are important in the virulency and antigenicity of the virus. The VP1 had 242E, 390M, and 393D which were characteristic of vvIBDV and KpnI restriction enzyme (777GGTAC/C782) in addition to a new mutation (F243Y and N383H) in IBD-EGY-AH1 and AH4 strains. According to the current study, the strains were distinct from the vaccinal strain; they could be responsible for the most recent IBDV outbreaks observed in flocks instead of received vaccinations. The current study highlighted the importance of molecular monitoring to keep up to date on the circulating IBDV for regular evaluation of commercial vaccination programs against circulating field viruses. [ABSTRACT FROM AUTHOR]

Abstract Background VP1 of the chicken anemia virus (CAV) is a structural protein that is required for virus encapsulation. VP1 proteins are present both in the nucleus and cytoplasm; however, the functional nuclear localization signal (NLS) and nuclear export signal (NES) of VP1 are still unknown. This study aimed to characterize the NLS and NES motifs of VP1 using bioinformatics methods and multiple-site fragment deletions, and investigate shuttling of VP2 from nucleus to cytoplasm by co-transfection with VP1. Methods Two putative NLS motifs were predicted by the WoLF PSORT and NLStradamus programs from the amino acid sequence of VP1. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. All mutants were created by multiple-site fragment deletion mutagenesis. VP1 and VP2 were co-expressed in cells using plasmid transfection. Results A functional NLS motif was identified at amino acid residues 3 to 10 (RRARRPRG) of VP1. Critical amino acids 3 to 10 were significantly involved in nuclear import in cells and were evaluated using systematic deletion mutagenesis. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. A functional NES was identified at amino acid residues 375 to 388 (ELDTNFFTLYVAQ). Leptomycin B (LMB) treatment demonstrated that VP1 export from nucleus to cytoplasm occurred through a chromosome region maintenance 1 (CRM1)-dependent pathway. With co-expression of VP1 and VP2 in cells, we observed that VP1 may transport VP2 from nucleus to cytoplasm. Conclusion Our data showed that VP1 of CAV contained functional NLS and NES motifs that modulated nuclear import and export through a CRM1-dependent pathway. Further, VP1 may play a role in the transport of VP2 from nucleus to cytoplasm.

Foot and Mouth Disease (FMD) is the most economically important viral-induced livestock disease worldwide. From April to May of 2015, tongue epithelial tissue samples were collected from 36 cattle in six villages, which share the border with Iran. Samples were screened using RT-PCR to amplify a conserved region in the VP1 gene, and phylogenetic tree analysis was performed based on the VP1 nucleotide sequence results. Furthermore, the nucleotide sequence was converted to an amino acid sequence in order to detect similarities between the studied samples and those previously published in GenBank (NCBI). Epidemically, based on the amino acid residues, genetic similarity, and amino acid substitutions, the VP1 nucleotide sequences were determined to be close to a novel group, group VII, with 94% identity. The VP1 amino acid sequence analysis revealed a close relationship to the Asia/BAL/PAK/iso-2/2011 isolate (Accession no. JX435109), with 95.7% identity, which is not significantly different. Analysis of the studied samples revealed that the FMDV serotype Asia1 causing the outbreak in the Basne district belonged to group VII, which was introduced from the Balochistan province of Pakistan through illegal movement of animals from this region.

We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5′-cap structure is essential for the virus’ generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly.

Tumor progression locus 2 (TPL2) is a serine/threonine kinase that belongs to the mitogen-activated protein 3 kinase (MAP3K) family, and it plays an important role in pathogen infection. The trimer complex of TPL2, p105, and ABIN2 is essential for maintenance of TPL2 steady-state levels and host cell response to pathogens. Foot-andmouth disease virus (FMDV) is a positive-strand RNA virus of the family Picornaviridae that encodes proteins capable of antagonizing host immune responses to achieve infection. The VP1 protein of FMDV is a multifunctional protein that can bind host cells and induce an immune response as well as cell apoptosis. However, the role and mechanisms of TPL2 in FMDV infection remain unknown. Here, we determined that FMDV infection could inhibit TPL2, p105, and ABIN2 at the transcription and protein levels, while VP1 could only inhibit TPL2, p105, and ABIN2 at the protein level. TPL2 inhibited the replication of FMDV in vivo and in vitro, and the 268- to 283-amino-acid region in the TPL2 kinase domain was essential for interaction with VP1. Moreover, VP1 promoted K48-linked polyubiquitination of TPL2 and degraded TPL2 by the proteasome pathway. However, VP1-induced degradation of p105 and ABIN2 was independent of proteasome, autophagy, lysosome, and caspase-dependent pathways. Further studies showed that VP1 destroyed the stability of the TPL2-p105-ABIN2 complex. Taken together, these results revealed that VP1 antagonized TPL2-meditated antivirus activity by degrading TPL2 and destroying its complex. These findings may contribute to understanding FMDV-host interactions and improving the development of a novel vaccine to prevent FMDV infection. [ABSTRACT FROM AUTHOR]

Duck hepatitis A virus (DHAV) causes a highly contagious and acute disease in ducklings younger than 3 weeks of age and spreads rapidly by horizontal transmission to all susceptible ducklings in the flock. To date, there is no evidence of vertical transmission of DHAV‐1. In a previous study, we identified a novel DHAV type 1 (DHAV‐1) isolate that could infect adult ducks and induce laying drop. In this study, 30 non‐embryonated duck eggs and 60 17‐day‐old embryos were collected from three breeding duck flocks with egg drop syndrome caused by DHAV‐1 in China, and 30 17‐day‐old embryos were randomly selected from the 60 embryos and allowed to hatch. DHAV‐1 RNA was detected by RT‐PCR in 10 of 30 non‐embryonated eggs, 9 of 30 17‐day‐old embryos, 5 of 7 dead embryos and 5 of 23 newly hatched ducklings. Overall, 29 of 90 (32.2%) eggs and embryos were positive for DHAV‐1. Three DHAV‐1 strains were isolated from the dead duck embryos of the three breeding duck flocks, respectively. Pathogenicity studies showed that the three DHAV‐1 isolates had median embryo lethal doses but were highly pathogenic to healthy ducklings. Compared with the DHAV reference strains, there were two specific amino acid mutation sites (F169 and S220) in VP1 of the three isolates. To the best of our knowledge, this is the first report that DHAV‐1 is isolated from duck embryos. The findings provide evidence of possible vertical transmission of DHAV‐1 from breeding ducks to ducklings. [ABSTRACT FROM AUTHOR]

Infection with the re-emerging enterovirus 71 (EV-A71) is associated with a wide range of disease severity, including herpangina, encephalitis, and cardiopulmonary failure. At present, there is no FDA-approved therapeutics for EV-A71. Early diagnosis for the high-risk children is the key to successful patient care. We examined viral genome sequences at the 5′ untranslated region (UTR) and the capsid protein VP1 from 36 mild and 27 severe cases. We identified five EV-A71 mutations associated with severe diseases, including (1) the 5′ UTR mutations C580U, A707G, C709U; (2) a VP1 alanine-to-threonine mutation at position 280 (280T), and (3) a VP1 glutamic acid-to-(non-glutamic acid) at position 145 [145(non-E)]. SCARB2 is a known entry receptor for EV-A71. Based on a recent cryoEM structure of the EV-A71-SCARB2 binding complex, VP1-280T is near the binding interface between the VP1-VP2 complex and its entry receptor SCARB2. A de novo created hydrogen bonding between the mutant VP1-280T and the VP2-139T, could help strengthen a web-like interaction structure of the VP1-VP2 complex. A stabilized loop turn of VP2, once in contact with SCARB2, can enhance interaction with the host SCARB2 receptor for viral entry. Our findings here could facilitate early detection of severe cases infected with EV-A71 in clinical medicine. In addition, it opens up the opportunity of functional studies via infectious cDNA cloning, site-directed mutagenesis, and animal models in the future.

ABSTRACT Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro. Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates. IMPORTANCE Enterovirus D68 (EV-D68) causes mild to severe respiratory disease and is associated with acute flaccid myelitis since 2014. Currently, the understanding of the ability of EV-D68 to replicate in the central nervous system (CNS), and whether it is associated with a specific clade of EV-D68 viruses or specific viral factors, is lacking. Comparing different EV-D68 clades did not reveal clade-specific phenotypic characteristics. However, we did show that viruses which acquired a cell culture-adapted amino acid substitution in VP1 (E271K) recognized heparan sulfate as an additional receptor. Recognition of heparan sulfate resulted in an increase in attachment, infection, and replication in neuroblastoma cells compared with viruses without this specific amino acid substitution. The ability of EV-D68 viruses to acquire cell culture-adaptive substitutions which have a large effect in experimental settings emphasizes the need to sequence virus stocks.

In this study, we sought to develop a subunit vaccine against the increasingly prevalent Duck hepatitis A virus serotype 3 (DHAV-3). The VP1 protein of DHAV-3 and a truncated version containing the C-terminal region of VP1, termed VP1-C, were expressed recombinantly in Escherichia coli as vaccine antigens. For enhanced immune response, a truncated version of flagellin, nFliC, was included as vaccine adjuvant. Ducklings were vaccinated once for immune response analysis and challenge test. Results showed that VP1-C elicited a higher level of virus-specific antibody response and neutralization titer than VP1. The addition of nFliC further enhanced the antibody response. In terms of cellular immune response, the VP1-C + nFliC vaccine elicited the highest level of T cell proliferation among the vaccine formulations tested. Examination of the cytokine expression profile showed that peripheral blood mononuclear cells from the VP1-C + nFliC vaccine group expressed the highest levels of pro-inflammatory (IL-6) and TH-1 type (IL-12 and IFN-γ) cytokines. Finally, in a DHAV-3 challenge test, the VP1-C + nFliC vaccine provided a 75% protection rate (n = 8), in contrast to 25% for the VP1 vaccine. In conclusion, E. coli-expressed VP1-C has been shown to be a promising antigen when combined with nFliC and may be further developed as a single-dose subunit vaccine against DHAV-3.

Enterovirus genus has over one hundred genotypes and could cause several kinds of severe animal and human diseases. Understanding the role of conserved residues in the VP1 capsid protein among the enterovirus genus may lead to anti-enteroviral drug development. The highly conserved residues were found to be located at the loop and ß-barrel intersections. To elucidate the role of these VP1 residues among the enterovirus genus, alanine substitution reverse genetics (rg) variants were generated, and virus properties were investigated for their impact. Six highly conserved residues were identified as located near the inside of the canyon, and four of them were close to the ß-barrel and loop intersection. The variants rgVP1-R86A, rgVP1-P193A, rgVP1-G231A, and rgVP1-K256A were unable to be obtained, which may be due to disruption in the virus replication process. In contrast, rgVP1-E134A and rgVP1-P157A replicated well and rgVP1-P157A showed smaller plaque size, lower viral growth kinetics, and thermal instability at 39.5°C when compared to the rg wild type virus. These findings showed that the conserved residues located at the ß-barrel and loop junction play roles in modulating viral replication, which may provide a pivotal role for pan-enteroviral inhibitor candidate.

Infection with the re-emerging enterovirus 71 (EV-A71) is associated with a wide range of disease severity, including herpangina, encephalitis, and cardiopulmonary failure. At present, there is no FDA-approved therapeutics for EV-A71. Early diagnosis for the high-risk children is the key to successful patient care. We examined viral genome sequences at the 5′ untranslated region (UTR) and the capsid protein VP1 from 36 mild and 27 severe cases. We identified five EV-A71 mutations associated with severe diseases, including (1) the 5′ UTR mutations C580U, A707G, C709U; (2) a VP1 alanine-to-threonine mutation at position 280 (280T), and (3) a VP1 glutamic acid-to-(non-glutamic acid) at position 145 [145(non-E)]. SCARB2 is a known entry receptor for EV-A71. Based on a recent cryoEM structure of the EV-A71-SCARB2 binding complex, VP1-280T is near the binding interface between the VP1-VP2 complex and its entry receptor SCARB2. A de novo created hydrogen bonding between the mutant VP1-280T and the VP2-139T, could help strengthen a web-like interaction structure of the VP1-VP2 complex. A stabilized loop turn of VP2, once in contact with SCARB2, can enhance interaction with the host SCARB2 receptor for viral entry. Our findings here could facilitate early detection of severe cases infected with EV-A71 in clinical medicine. In addition, it opens up the opportunity of functional studies via infectious cDNA cloning, site-directed mutagenesis, and animal models in the future. [ABSTRACT FROM AUTHOR]

Objectives: The capsid protein (VP1) of the foot-and-mouth (FMD) AKT-III strain was expressed on the surface of the T7 phage capsid (AKT-T7 strain) and the potential of AKT-T7 strain as an FMD vaccine was evaluated. Results: The AKT-T7 strain was successfully constructed and was not cytotoxic to BHK-21, MDBK, or sheep kidney cells. The AKT-T7 strain was well phagocytosed by mouse macrophages. Immunization of BALB/c mice revealed that animals were quickly induced and produced high levels of FMDV antibodies. Monitoring data indicated that FMDV antibody levels could be maintained at higher levels for longer periods of time. The AKT-T7 strain induced high levels of IFN-γ levels in mice with little effect on IL-4. Conclusions: The AKT-T7 induced the mice to produce FMDV antibodies, which has the advantage of phage and FMDV, and is a potential candidate for an FMD vaccine. [ABSTRACT FROM AUTHOR]

As an immunosuppressive disease virus, chicken infectious anemia virus (CIAV) mainly infects chickens, causing aplastic anemia and systemic lymphoid tissue atrophy. In recent years, the prevalence of CIAV in the poultry industry globally has caused huge economic losses. In this study, a total of 223 clinical samples, including anal swabs, tissues, blood, and vaccines, were collected from 19 broiler farms or breeding companies in Jiangsu province, with symptoms of significant anemia and immunosuppression during 2020-2022. Among them, 75 samples (75/223, 33.6%) were positive for CIAV in polymerase chain reaction (PCR) test, and 20 CIAV strains were successfully isolated. The phylogenetic trees of the 20 isolates and 42 CIAV strains deposited in GenBank formed four distinct groups (A-D). And the isolates mainly belonged to Group A but with high genetic diversity. Analysis for VP1 indicated that these isolates possess key characteristics of highly pathogenic strains. Meanwhile, VP2 and VP3 were much conserved with much fewer mutations compare to VP1. The above epidemiological study of CIAV provides novel insights into molecular characterization of CIAV and lays the foundation for developing efficient strategies for control of CIAV in China., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Ma, Li, Xie, Wan, Qin, Ye, Shao and Wang.)

One of the constraints to controlling foot‐and‐mouth disease (FMD) in East Africa is the incomplete knowledge of the specific FMD virus (FMDV) strains circulating and the way in which these viruses move across countries in the region. This retrospective study focuses on Ethiopia, which has one of the largest FMD‐susceptible livestock populations in Africa. Analyses of FMDV positive samples collected between 2008 and 2019 demonstrate that serotypes O (n = 175), A (n = 51) and SAT 2 (n = 33) were present in the country. Phylogenetic analysis of the VP1 sequences for these viruses showed that there were at least seven different FMD viral clades circulating during this period: O/EA‐3, O/EA‐4, A/AFRICA/G‐I, A/AFRICA/G‐IV, A/AFRICA/G‐VII, SAT2/VII and SAT2/XIII. Although these results only represent a snapshot and might not reflect all FMDV lineages that were present, they highlight the importance of serotype O, as well as the complexity and co‐existence of FMDV serotypes in Ethiopia and surrounding countries. These sequence data also support the idea that there are two FMDV ecosystems existing in East Africa. Data from retrospective studies, such as these presented here, will be beneficial for vaccine selection and vaccination campaigns to control FMDV within Ethiopia. [ABSTRACT FROM AUTHOR]

p53 is implicated in several cellular pathways such as induction of cell-cycle arrest, differentiation, senescence, and apoptosis. p53 is activated by a broad range of stress signals, including viral infections. While some viruses activate p53, others induce its inactivation, and occasionally p53 is differentially modulated during the replicative cycle. During calicivirus infections, apoptosis is required for virus exit and spread into the host; yet, the role of p53 during infection is unknown. By confocal microscopy, we found that p53 associates with FCV VP1, the protease-polymerase NS6/7, and the dsRNA. This interaction was further confirmed by proximity ligation assays, suggesting that p53 participates in the FCV replication. Knocked-down of p53 expression in CrFK cells before infection, resulted in a strong reduction of the non-structural protein levels and a decrease of the viral progeny production. These results indicate that p53 is associated with the viral replication complex and is required for an efficient FCV replication. • Host cell p53 protein levels and subcellular localization do not change during FCV infection. • Host cell p53 associates with FCV major viral capsid protein VP1, protease-polymerase NS6/7, and the dsRNA in FCV infected cells. • Host cell p53 is required for a FCV replication. [ABSTRACT FROM AUTHOR]

Nigeria is a large densely populated country in West Africa. Most of its livestock is raised in a pastoralist production system with typical long distance migration in search of water and feed. As the demand for animal products largely exceeds the domestic production, large numbers of livestock are imported from neighboring countries without sanitary restrictions. In Nigeria, foot-and-mouth disease virus (FMDV) serotypes O, A, and Southern African Territories (SAT)2 are endemic for a long time. Clinical outbreaks of FMD due to serotype SAT1 are described again since 2015, after an absence of more than 30 years. Historically, outbreaks of FMD due to serotypes O, A, SAT1, and SAT2 were each time associated with trade of cattle entering Nigeria from neighboring countries. In the present study, tissue samples from 27 outbreaks of FMD were collected in Nigerian cattle from 2012 until 2017 in six different States and in the Federal Capital Territory. FMDV was isolated and serotyped and further characterized by VP1 sequencing and phylogenetic analysis to gain more knowledge on FMDV circulation in Nigeria. Half of the outbreaks were characterized as FMDV topotype O/EA-3, while outbreaks with other serotypes and topotypes were—in descending order—less prevalent: A/Africa/G-IV, SAT1/X, SAT2/VII, and O/WA. The high dynamics and omnipresence of FMD in Nigeria were illustrated in Plateau State where FMDV serotypes O, SAT1, and SAT2 were isolated during the course of the study, while at some point in the study, outbreaks due to FMDV serotype A were observed in three remote States. The genetic and phylogenetic analysis suggests a mixed origin of FMD outbreaks. Some outbreaks seem to be caused by sustained local transmission of FMDV strains present in Nigeria since a number of years, while other outbreaks seem to be related to recent incursions with new FMDV strains. The role of African buffaloes in the etiology of FMD in Nigeria is unclear, and sampling of wildlife is needed. The results of the present study suggest that systematic sample collection is essential to understand the complex concomitance of FMDV strains in Nigeria and essential to support the implementation of a vaccination-based control plan.

Abstract Background Chicken anaemia virus (CAV) is commonly found in poultry. VP1 is the sole structural protein of CAV, which is the major component responsible for capsid assembly. The CAV virion consists of the VP1 protein and a viral genome. However, there is currently no information on the protein-nucleic acid interactions between VP1 and DNA molecules. Results In this study, the recombinant VP1 protein of CAV was expressed and purified to characterize its DNA binding activity. When VP1 protein was incubated with a DNA molecule, the DNA molecule exhibited retarded migration on an agarose gel. Regardless of whether the sequence of the viral genome was involved in the DNA molecule, DNA retardation was not significantly influenced. This outcome indicated VP1 is a DNA binding protein with no sequence specificity. Various DNA molecules with different conformations, such as circular dsDNA, linear dsDNA, linear ssDNA and circular ssDNA, interacted with VP1 proteins according to the results of a DNA retardation assay. Further quantification of the amount of VP1 protein required for DNA binding, the circular ssDNA demonstrated a high affinity for the VP1 protein. The preferences arranged in the order of affinity for the VP1 protein with DNA are circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular DNA and linear dsDNA. Conclusions The results of this study demonstrated that the interaction between VP1 and DNA molecules exhibited various binding preferences that were dependent on the structural conformation of DNA. Taken together, the results of this report are the first to demonstrate that VP1 has no sequence-specific DNA binding activity. The particular binding preferences of VP1 might play multiple roles in DNA replication or encapsidation during the viral life cycle.

Abstract John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle. This short report is an important extension of our previous efforts in defining our reliable model culture system able to support a productive JCV infection. Supernatants collected from transfected cells have been used to infect freshly seeded COS-7 cell line. An infectious viral progeny was obtained as confirmed by Western blot and immunofluorescence assay. During infection, the archetype regulatory region was conserved. Importantly, in this study we developed an improved culture system to obtain a large scale production of JC virus in order to study the genetic features, the biology and the pathogenic mechanisms of JC virus that induce PML.

During June 2017–December 2018, norovirus was responsible for 10.9% of acute gastroenteritis cases in Thailand. Genogroup I (GI) was found in 14% of samples, of which 12 were co-infected with genogroup II (GII). In 35.8% of samples, GII.Pe-GII.4 Sydney predominated. Diverse recombinant strains of GI and GII norovirus co-circulated year-round.

Copyright of Avian Diseases is the property of American Association of Avian Pathologists, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Infectious bursal disease virus (IBDV) of the Birnaviridae family leads to immunosuppression of young chickens by destroying B cells in the bursa of Fabricius (BFs). Given the increasing number of variant IBDV strains, we urgently require a method to produce attenuated virus for vaccine development. To accomplish this goal, the dualpromoter plasmids in which the RNA polymerase II and RNA polymerase I (Pol I) promoters were placed upstream of the IBDV genomic sequence, which was followed by mouse Pol I terminator and a synthetic polyadenylation signal, were developed for rapid generation of IBDV. This approach did not require trans-supplementation of plasmids for the expression of VP1 and VP3, the main components of IBDV ribonucleoprotein (RNP). Based on the finding in this study that the IBDV RNP activity was partially retained by VP1-FLAG, we successfully rescued the replication-competent IBDV/1FLAG expressing VP1-FLAG. Compared with its parental counterpart, IBDV/1FLAG formed smaller size plaques in cultured cells and induced the same 100% immune protection in vivo. However, neither retarded development nor severe BFs lesion was observed in the IBDV/ 1FLAG-inoculated chickens. Collectively, this is the first report that viral RNP activity was affected by the addition of an epitope tag on the componential viral proteins. Furthermore, this work demonstrates the rapid generation of attenuated IBDV from dualpromoter plasmids via reducing viral RNP activity by a fused FLAG tag on the C terminus of VP1. This would be a convenient strategy to attenuate epidemic variant IBDV strains for rapid and efficient vaccine development. [ABSTRACT FROM AUTHOR]

Enterovirus A71 (EV-A71) is the major cause of severe hand-foot-and-mouth diseases (HFMD), especially encephalitis and other nervous system diseases. EV-A71 capsid protein VP1 mediates virus attachment and is the important virulence factor in the EV-A71pathogenesis. In this study, we explored the roles of VP1 in the permeability of blood–brain barrier (BBB). Sera albumin, Evans blue, and dextran leaked into brain parenchyma of the 1-week-old C57BL/6J mice intracranially injected with VP1 recombinant protein. VP1 also increased the permeability of the brain endothelial cells monolayer, an in vitro BBB model. Tight junction protein claudin-5 was reduced in the brain tissues or brain endothelial cells treated with VP1. In contrast, VP1 increased the expression of virus receptor vimentin, which could be blocked with VP1 neutralization antibody. Vimentin expression in the VP1-treated brain endothelial cells was regulated by TGF-β/Smad-3 and NF-κB signal pathways. Moreover, vimentin over-expression was accompanied with compromised BBB. From these studies, we conclude that EV-A71 virus capsid protein VP1 disrupted BBB and increased virus receptor vimentin, which both may contribute to the virus entrance into brain and EV-A71 CNS infection. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a highly contagious, economically important viral disease. The structural protein VP1 plays significant roles during FMDV infection. Here, we identified that VP1 interacted with host ribosomal protein SA (RPSA). RPSA is a viral receptor for dengue virus and classical swine fever virus infections. However, the incubation of susceptible cells using the anti-RPSA antibodies did not block the infection of FMDV. Overexpression of porcine RPSA in the insusceptible cells could not trigger FMDV infection, suggesting RPSA was not responsible for FMDV entry and infection. On the contrary, we found that overexpression of RPSA suppressed FMDV replication, and knockdown of RPSA enhanced FMDV replication. We further determined that FMDV infection activated mitogen-activated protein kinases (MAPKs) pathway, and demonstrated that MAPKs pathway activation was critically important for FMDV replication. RPSA negatively regulated MAPKs pathway activation during FMDV infection, showing an antiviral function. FMDV VP1 interacted with RPSA to abrogate RPSA-mediated suppressive role on MAPKs pathway activation. Together, our study indicated that the MAPKs pathway activation was required for FMDV replication, and host RPSA played a negatively regulatory role on MAPKs pathway activation to suppress FMDV replication. FMDV VP1 bound to RPSA to promote viral replication by repressing RPSA-mediated function and maintaining the activation of MAPKs signal pathway. [ABSTRACT FROM AUTHOR]

The seven serotypes of foot-and-mouth disease virus (FMDV) differ on the surface exposed regions on the VP1, 2 and 3 proteins. Amongst the three, the VP1 protein has been produced the most for use in serotyping assays for some of the Euro-Asian serotypes. In this study the VP1 protein of the FMDV SAT2/ZIM/7/83 was expressed in Escherichia coli BL21 cells in Luria broth and EnPresso® B media in shake flasks. Production was further developed and the VP1 protein was produced at 2.15 g L−1 in fed-batch fermentations at 2 L scale. The protein formed insoluble inclusion bodies that were isolated, denatured and refolded. When tested in ELISA, the protein was found to be highly reactive with serum from a SAT2 vaccinated guinea pig, and not reactive to SAT1 and SAT3 antisera. These results open avenues to evaluate recombinantly expressed VP1 proteins for differentiation of the three Southern African Territories serotypes of FMDV that co-occur in Southern and East Africa. In addition, this could mitigate the need for employing virus as reagent, or having to raise reagent antibodies. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated viral (rAAV) vectors have been widely used in human gene therapy. One major impediment to its broad application is the inability to produce high-quality vectors in mass quantity. Here, an efficient and scalable suspension cell culture system for the production of rAAV vectors is described. In this system, the AAV trans factors, Rep78, Rep52, VP1, VP2, and VP3, were stably integrated into a single vaccinia virus carrier by maximizing the use of alternative codons between genes with identical amino acids, and the cis rAAV genome was carried by an E1/E3 gene-deleted adenovirus. Infection of improved, E1 integrated, suspension-cultured cells with these two viral vectors resulted in the robust production of rAAV vectors. The newly enhanced system can consistently produce ∼1 × 1015 genome containing rAAV vectors per liter of suspension cells. Moreover, the capsid composition of rAAV vectors produced by this system is markedly different from those produced using the traditional system in that the VP1 protein is more abundant than the VP2 protein (19:1 versus 1:1). The unique VP1 superabundant rAAV vectors produced in this new system exhibited improved transduction in vivo after intravitreal injection.

The hand, food, and mouth disease (HFMD) is primarily caused by Enterovirus A71 (EV-A71). EV-A71 outbreaks in the Asia Pacific have been associated with severe neurological disease and high fatalities. Currently, there are no FDA-approved antivirals for the treatment of EV-A71 infections. In this study, the SP81 peptide, derived from the VP1 capsid protein of EV-A71 was shown to be a promising antiviral candidate for the treatment of EV-A71 infections. SP81 peptide was non-toxic to RD cells up to 45 μM, with a half-maximal cytotoxic concentration (CC 50) of 90.32 μM. SP81 peptide exerted antiviral effects during the pre- and post-infection stages with 50% inhibitory concentrations (IC 50) of 4.529 μM and 1.192 μM, respectively. Direct virus inactivation of EV-A71 by the SP81 peptide was also observed with an IC 50 of 8.076 μM. Additionally, the SP81 peptide exhibited direct virus inactivation of EV-A71 at 95% upon the addition of the SP81 peptide within 5 min. This study showed that the SP81 peptide exhibited significant inhibition of EV-A71 and could serve as a promising antiviral agent for further clinical development against EV-A71 infections. • The SP81 peptide derived from the VP1 capsid protein of EV-A71 is a promising antiviral candidate against EV-A71. • It was non-toxic to RD cells up to 45 μM. • SP81 demonstrated three modes of action: cell protection, direct virus inactivation, and inhibition during post-infection. • It was observed to exert direct virus inactivation against EV-A71 within a minute of co-incubation with EV-A71. [ABSTRACT FROM AUTHOR]