C-terminus Of VP1 Research Articles

Modular Display of Plasmodium yoelii Circumsporozoite Surface Protein and Merozoite Surface Protein-1 on Norovirus-like Particles.

Recently, virus-like particles have been regarded as a promising platform for displaying foreign peptides or proteins on their surface. In this study, a dual-protein-displaying platform based on the norovirus-like particle (NoV-LP) was developed using SpyTag (SpT)/SpyCatcher (SpC) protein bioconjugation. A short 14-amino-acid SpT peptide was added to the C-terminus of VP1, with a rigid "EAAAK" spacer in between. Antigenic proteins from a rodent malaria parasite, Plasmodium yoelii, specifically the circumsporozoite protein (PyCSP) and the 19 kDa C-terminal region of merozoite surface protein 1 (PyMSP119), were displayed on the surface of NoV-LPs in both monovalent and bivalent formats. The immunogenicity of these VLP-based vaccines was assessed, and they were found to induce antigen-specific IgG responses against both PyCSP and PyMSP119 in BALB/c mice in the absence of an adjuvant, at levels comparable to those induced by subunit antigenic proteins with an alum adjuvant added. Interestingly, the bivalent vaccine raised IgG responses at a similar titer to the monovalent vaccine. This finding hints that the NoV-LP possesses an inherent adjuvanted property in the presence of a foreign antigen. The measured anti-PyCSP and anti-PyMSP119 antibodies through ELISA indicate that surface display of PyCSP and PyMSP119 on SpTagged-NoV-LP has the potential for further development as a bivalent vaccine against two different life-cycle stages of malaria.

Identification of four neutralizing antigenic sites on the enterovirus D68 capsid.

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 βB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Reporter Coxsackievirus A5 Expressing iLOV Fluorescent Protein or Luciferase Used for Rapid Neutralizing Assay in Cells and Living Imaging in Mice.

Coxsackievirus A5 (CV-A5) is a re-emerging enterovirus that causes hand, foot, and mouth disease in children under five years of age. CV-A5-M14-611 is a mouse-adapted strain that can infect orally and lead to the death of 14-day-old mice. Here, recombinants based on CV-A5-M14-611 were constructed carrying three reporter genes in different lengths. Smaller fluorescent marker proteins, light, oxygen, voltage sensing (iLOV), and nano luciferase (Nluc) were proven to be able to express efficiently in vitro. However, the recombinant with the largest insertion of the red fluorescence protein gene (DsRed) was not rescued. The construction strategy of reporter viruses was to insert the foreign genes between the C-terminus of VP1 and the N-terminus of 2A genes and to add a 2A protease cleavage domain at both ends of the insertions. The iLOV-tagged or Nluc-tagged recombinants, CV-A5-iLOV or CV-A5-Nluc, exhibited a high capacity for viral replication, genetic stability in cells and pathogenicity in mice. They were used to establish a rapid, inexpensive and convenient neutralizing antibody assay and greatly facilitated virus neutralizing antibody titration. Living imaging was performed on mice with CV-A5-Nluc, which exhibited specific bioluminescence in virus-disseminated organs, while fluorescence induced by CV-A5-iLOV was weakly detected. The reporter-gene-tagged CV-A5 can be used to study the infection and mechanisms of CV-A5 pathogenicity in a mouse model. They can also be used to establish rapid and sensitive assays for detecting neutralizing antibodies.

In silico epitope prediction and evolutionary analysis reveals capsid mutation patterns for enterovirus B.

Enterovirus B (EVB) is a common species of enterovirus, mainly consisting of Echovirus (Echo) and Coxsackievirus B (CVB). The population is generally susceptible to EVB, especially among children. Since the 21st century, EVB has been widely prevalent worldwide, and can cause serious diseases, such as viral meningitis, myocarditis, and neonatal sepsis. By using cryo-electron microscopy, the three-dimensional (3D) structures of EVB and their uncoating receptors (FcRn and CAR) have been determined, laying the foundation for the study of viral pathogenesis and therapeutic antibodies. A limited number of epitopes bound to neutralizing antibodies have also been determined. It is unclear whether additional epitopes are present or whether epitope mutations play a key role in molecular evolutionary history and epidemics, as in influenza and SARS-CoV-2. In the current study, the conformational epitopes of six representative EVB serotypes (E6, E11, E30, CVB1, CVB3 and CVB5) were systematically predicted by bioinformatics-based epitope prediction algorithm. We found that their epitopes were distributed into three clusters, where the VP1 BC loop, C-terminus and VP2 EF loop were the main regions of EVB epitopes. Among them, the VP1 BC loop and VP2 EF loop may be the key epitope regions that determined the use of the uncoating receptors. Further molecular evolution analysis based on the VP1 and genome sequences showed that the VP1 C-terminus and VP2 EF loop, as well as a potential "breathing epitope" VP1 N-terminus, were common mutation hotspot regions, suggesting that the emergence of evolutionary clades was driven by epitope mutations. Finally, footprints showed mutations were located on or near epitopes, while mutations on the receptor binding sites were rare. This suggested that EVB promotes viral epidemics by breaking the immune barrier through epitope mutations, but the mutations avoided the receptor binding sites. The bioinformatics study of EVB epitopes may provide important information for the monitoring and early warning of EVB epidemics and developing therapeutic antibodies.

Structural Basis for the Immunogenicity of the C-Terminus of VP1 of Echovirus 3 Revealed by the Binding of a Neutralizing Antibody.

Echovirus 3 (E3), a serotype of human enterovirus B (HEV-B), causes severe diseases in infants. Here, we determined the structures of E3 with a monoclonal antibody (MAb) 6D10 by cryo-EM to comprehensively understand the specificities and the immunological characteristic of this serotype. The solved cryo-EM structures of the F-, A-, and E-particles of E3 bound with 6D10 revealed the structural features of the virus-antibody interface. Importantly, the structures of E-particles bound with 6D10 revealed for the first time the nature of the C-terminus of VP1 for HEV-Bs at the structural level. The highly immunogenic nature of this region in the E-particles provides new strategies for vaccine development for HEV-Bs.

Sequence addition to the N- or C-terminus of the major capsid protein VP1 of norovirus affects its cleavage and assembly into virus-like particles

Norovirus (NoV) infection is a leading cause of non-bacterial gastroenteritis worldwide and there are currently no effective therapeutics available to target the virus. The norovirus major capsid protein VP1 is a potential candidate for the development of vaccines due to the similar morphology and immunogenicity of its assembled virus-like particles (VLPs) compared to native virions. In this study, we explored the effects of N- and C-terminal sequence additions to the VP1 of a GII.4 NoV during its assembly into VLPs. A series of sequences of different lengths derived from the minor capsid protein VP2 of the GII.4 NoV were added to the N- and C-terminus of VP1. The fusion proteins were expressed using a recombinant baculovirus expression system and the assembly of the expressed fusion proteins was subsequently observed under electron microscopy (EM). Our results indicated that all constructed fusion proteins were successfully expressed with different degrees of enzyme cleavage at the N-terminus. Electron microscopy revealed the successful assembly of VLPs of different sizes for all fusion proteins. An in vitro binding assay for VLP-histo-blood group antigens (HBGAs) indicated that all fusion proteins exhibited similar binding patterns compared with their wild-type VP1. Our results demonstrate that (Xi et al., 1990) [1] NoV VP1 can tolerate foreign sequences at its N- or C-terminus without affecting its ability to assemble into VLPs, and (Jiang et al., 1992) [2] that the cleavage pattern and effects of foreign sequences on the sizes of assembled VLPs observed in this study might represent important experimental data that can be used to elucidate VP1 self-assembly.

Identification of Epitopes on Rhinovirus 89 Capsid Proteins Capable of Inducing Neutralizing Antibodies.

Rhinoviruses (RVs) are major causes of the common cold, but they can also trigger exacerbations of asthma. More than 160 different RV strains exist and can be classified into three genetic species (RV-A, RV-B and RV-C) which bind to different receptors on human cells including intracellular adhesion molecule 1 (ICAM-1), the low-density lipoprotein receptor (LDLR) or the cadherin-related family member 3 (CDHR3). Epitopes located in the RV capsid have mainly been determined for RV2, a minor-group RV-A strain binding to LDLR, and for RV14, a major-group RV-B strain binding to ICAM-1. In order to study epitopes involved in the neutralization of RV89, an ICAM-1-binding RV-A strain which is highly different from RV2 and RV14 in terms of receptor specificity and sequence, respectively, we analyzed the specificity and epitopes of a highly neutralizing antiserum using recombinantly produced RV89 capsid proteins (VP1, VP2, VP3 and VP4), recombinant fragments and synthetic overlapping peptides thereof. We found that the antiserum which neutralized in vitro RV89 infection up to a dilution of 1:24,000 reacted with the capsid proteins VP1 and VP2 but not with VP3 and VP4. The neutralizing antibodies recognized recombinant fragments comprising approximately 100 amino acids of the N- and C-terminus of VP1 and the middle part of VP2, in particular, three peptides which, according to molecular modeling based on the three-dimensional structure of RV16, were surface-exposed on the viral capsid. Two recombinant fusion proteins containing the identified peptides fused to hepatitis B (HBV)-derived preS as a carrier protein induced upon immunization of rabbits antibodies capable of neutralizing in vitro RV89 infections. Interestingly, the virus-neutralizing epitopes determined for RV89 corresponded to those determined for minor-group RV2 binding to LDL and major-group RV14 belonging to the RV-B species, which are highly different from RV89. Our results indicate that highly different RV strains, even when reacting with different receptors, seem to engage similar parts of their capsid in the infection process. These results may be important for the design of active and passive immunization strategies for RV.

The N-terminal region (VP4) of the foot-and-mouth disease capsid precursor (P1-2A) is not required during its synthesis to allow subsequent processing by the 3C protease

The capsid precursor (P1-2A) of foot-and-mouth disease virus is processed by the 3C protease (3Cpro) to VP0, VP3 and VP1 plus 2A. During capsid assembly, the VP0 is cleaved to VP4 plus VP2. Single amino acid changes in a conserved motif (YCPRP) near the C-terminus of VP1 can block processing of the capsid precursor by the 3Cpro, although the cleavage sites are located hundreds of amino acids distant from this motif, presumably due to misfolding. In contrast, we show here that the absence of the VP4 sequence during the synthesis of the capsid precursor does not affect its subsequent processing. Cleavage of this truncated precursor by 3Cpro at the VP3/VP1 and VP2/VP3 junctions occurred efficiently. Thus, in contrast to the presence of the YCPRP motif in VP1, there are no critical motifs near the N-terminus of the precursor, within VP4, required for correct cleavage by 3Cpro.

Replication Kinetics for a Reporter Merkel Cell Polyomavirus.

Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, β-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.

A Novel Core Effector Vp1 Promotes Fungal Colonization and Virulence of Ustilago maydis

The biotrophic fungus Ustilago maydis secretes a plethora of uncharacterized effector proteins and causes smut disease in maize. Among the effector genes that are up-regulated during the biotrophic growth in maize, we identified vp1 (virulence promoting 1), which has an expression that was up-regulated and maintained at a high level throughout the life cycle of the fungus. We characterized Vp1 by applying in silico analysis, reverse genetics, phenotypic assessment, microscopy, and protein localization and provided a fundamental understanding of the Vp1 protein in U. maydis. The reduction in fungal virulence and colonization in the vp1 mutant suggests the virulence-promoting function of Vp1. The deletion studies on the NLS (nuclear localization signal) sequence and the protein localization study revealed that the C-terminus of Vp1 is processed after secretion in plant apoplast and could localize to the plant nucleus. The Ustilago hordei ortholog UhVp1 lacks NLS localized in the plant cytoplasm, suggesting that the orthologs might have a distinct subcellular localization. Further complementation studies of the Vp1 orthologs in related smut fungi revealed that none of them could complement the virulence function of U. maydis Vp1, suggesting that UmVp1 could acquire a specialized function via sequence divergence.

Comparative Pathogenicity of Duck Hepatitis A Virus-1 Isolates in Experimentally Infected Pekin and Muscovy Ducklings

Duck hepatitis virus (DHV) has always been considered one of the threats endangering duck farming in Egypt since the 1960s. In the current study, suspected DHV samples (n = 30) were obtained from commercial Pekin, Mulard (hybrid), and Muscovy duck farms and backyards in Beheira, Alexandria, Gharbia, Kafr El-Sheikh, and Giza provinces between 2012 and 2017. Diseased 3–21-day-old ducklings showed a clinical history of high mortality rates and nervous signs. Samples were screened by RT-PCR targeting the 5′UTR region and VP1 gene. The PCR-confirmed samples (n = 7) were isolated via allantoic route inoculation onto 9-day-old specific-pathogen-free embryonated chicken eggs. Embryos showed stunting, subcutaneous hemorrhages, and liver necrotic greenish-yellow foci. Duck hepatitis A virus-1 (DHAV-1) isolates were genetically analyzed in comparison to other field and vaccine strains. Phylogenetic analyses of the full-length VP1 gene sequences revealed that the obtained DHAV-1 field isolates clustered into genetic group 4 alongside other Egyptian strains isolated during the same period (95.9–99.72% similarity). Amino acid substitutions in the carboxyl-terminal of VP1 (I180T, G184E, D193N, and M213I) were identified in two strains. Also, deletion mutation at I189 was detected in three DHAV-1 strains. Additionally, the two amino acid residues E205 and N235 were common among the isolated strains and other virulent DHAV-1 strains. Two DHAV-1 isolates originated from Pekin source were selected for conducting the comparative pathogenicity testing based on detected point mutations at C-terminus of VP1. We evaluated the pathogenicity of these isolates by investigating clinical signs, mortality rates, and gross pathological and microscopic lesions. The study revealed that experimentally infected Pekin and Muscovy ducklings showed similar clinical signs including squatting down, lateral recumbency, and spasmodic kicking. Muscovy showed milder pathological changes in the liver compared to Pekin ducklings. Histopathological findings supported the gross pathological lesions detected in both breeds. In conclusion, these data provide updated information on the genetic diversity and pathotyping of Egyptian DHAV-1 strains. To the best of our knowledge, this is the first report of comparative pathogenicity of recent DHAV-1 strains in Pekin and Muscovy ducklings in Egypt and the Middle East region.

Complete genome sequences of the first parechoviruses A associated with sporadic pediatric acute gastroenteritis in Russia

Parechovirus (the Picornaviridae family), recently classified as Parechovirus A and formerly known as Human parechovirus (HPeV), can cause a wide range of human diseases. Over the past decade, several studies have reported HPeV epidemiology in different regions; however, information from Russia is limited. A total of 632 stool samples collected in Novosibirsk, Russia during January–March 2012 were screened for HPeV by RT-PCR. The study cohort comprised 572 patients with acute gastroenteritis and 60 healthy children. Seven of 572 (1.2%) gastroenteritis cases were HPeV-positive, including one co-infection with rotavirus and astrovirus. All positive patients were ≤1 year old, and five of them were younger than 3 months. None of the healthy controls provided an HPeV-positive sample. Six HPeV isolates were classified as HPeV-1 and one as HPeV-5 using phylogenetic analysis. Two complete genome sequences of HPeV-1 and one of HPeV-5 were determined and analyzed. Phylogenetic analysis showed that the studied Russian strains are probably recombinants. P1 region sequences of two Russian HPeV-1 strains clustered with rare contemporary HPeV-1A strains, whereas their P3 regions were phylogenetically closer to the archival Harris strain. The Russian HPeV-5 strain formed a common cluster with other HPeV-5 strains only for the P1 region, while the P3 region grouped with the German HPeV-2 strain. In the Russian HPeV-5 strain, the lack of the arginine-glycine-aspartic acid (RGD) motif at the C-terminus of VP1 was observed. This is the first complete genome characterization of the Russian HPeV strains detected in sporadic cases of pediatric acute gastroenteritis.

Identification of a short, highly conserved, motif required for picornavirus capsid precursor processing at distal sites.

Many picornaviruses cause important diseases in humans and other animals including poliovirus, rhinoviruses (causing the common cold) and foot-and-mouth disease virus (FMDV). These small, non-enveloped viruses comprise a positive-stranded RNA genome (ca. 7–9 kb) enclosed within a protein shell composed of 60 copies of three or four different capsid proteins. For the aphthoviruses (e.g. FMDV) and cardioviruses, the capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to generate VP0, VP3 and VP1 plus 2A. For enteroviruses, e.g. poliovirus, the capsid precursor is P1 alone, which is cleaved by the 3CD protease to generate just VP0, VP3 and VP1. The sequences required for correct processing of the FMDV capsid protein precursor in mammalian cells were analyzed. Truncation of the P1-2A precursor from its C-terminus showed that loss of the 2A peptide (18 residues long) and 27 residues from the C-terminus of VP1 (211 residues long) resulted in a precursor that cannot be processed by 3Cpro although it still contained two unmodified internal cleavage sites (VP0/VP3 and VP3/VP1 junctions). Furthermore, introduction of small deletions within P1-2A identified residues 185–190 within VP1 as being required for 3Cpro-mediated processing and for optimal accumulation of the precursor. Within this C-terminal region of VP1, five of these residues (YCPRP), are very highly conserved in all FMDVs and are also conserved amongst other picornaviruses. Mutant FMDV P1-2A precursors with single amino acid substitutions within this motif were highly resistant to cleavage at internal junctions. Such substitutions also abrogated virus infectivity. These results can explain earlier observations that loss of the C-terminus (including the conserved motif) from the poliovirus capsid precursor conferred resistance to processing. Thus, this motif seems essential for maintaining the correct structure of picornavirus capsid precursors prior to processing and subsequent capsid assembly; it may represent a site that interacts with cellular chaperones.

Novel duck hepatitis A virus type 1 isolates from adult ducks showing egg drop syndrome

Generally, duck hepatitis A virus type 1 (DHAV-1) only infects young ducklings. Since December 2016, severe outbreaks of duck viral infection with egg drop, feed consumption decline, and ovary-oviduct disease have occurred in some laying duck flocks in Shandong Province of China. DHAV-1 isolated from the affected ducks was confirmed as the causative pathogen of the egg drop. Compared with other DHAV-1 strains, the novel isolate has three special amino acid mutation points in the most variable regions at the C-terminus of VP1. The experimental infection in laying ducks indicated that successful immunization with DHAV-1 vaccine could protect laying duck from infection. To the best of our knowledge, this is the first reported incidence of a severe duck disease outbreak involving egg drop syndrome caused by DHAV-1.

Evolution of foot-and-mouth disease virus serotype A capsid coding (P1) region on a timescale of three decades in an endemic context

Three decades-long (1977–2013) evolutionary trend of the capsid coding (P1) region of foot-and-mouth disease virus (FMDV) serotype A isolated in India was analysed. The exclusive presence of genotype 18 since 2001 and the dominance of the VP359-deletion group of genotype 18 was evident in the recent years. Clade 18c was found to be currently the only active one among the three clades (18a, 18b and 18c) identified in the deletion group. The rate of evolution of the Indian isolates at the capsid region was found to be 4.96×10−3substitutions/site/year. The timescale analysis predicted the most recent common ancestor to have existed during 1962 for Indian FMDV serotype A and around 1998 for the deletion group. The evolutionary pattern of serotype A in India appears to be homogeneous as no spatial or temporal structure was observed. Bayesian skyline plots indicate a sharp decline in the effective number of infections after 2008, which might be a result of mass vaccination or inherent loss of virus fitness. Analyses of variability at 38 known antigenically critical positions in a countrywide longitudinal data set suggested that the substitutions neither followed any specific trend nor remained fixed for a long period since frequent reversions and convergence was noticed. A maximum of 6 different amino acid residues was seen in the gene pool at any antigenically critical site over the decades, suggesting a limited combination of residues being responsible for the observed antigenic variation. Evidence of positive selection at some of the antigenically critical residues and the structurally proximal positions suggest a possible role of pre-existing immunity in the host population in driving evolution. The VP1 C-terminus neither revealed variability nor positive selection, suggesting the possibility that this stretch does not contribute to the antigenic variation and adaptation under immune selection.

Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions.

In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 βE-βF loop, VP1 C-terminus and VP3 β-B knob) surrounding the fivefold symmetry axis (VP1 βF-βG loop) and at other discrete sites on the capsid (VP3 βG-βH loop, VP1 C-terminus, VP2 βC strand and VP1 βG-βH loop). A lysine insertion in the VP1 βF-βG loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 βG-βH loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 βB-βC loop) suggest their possible role in modulating the efficiency and specificity of interaction of the 'RGD' motif with αv-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.

An influenza A virus hemagglutinin (HA) epitope inserted in and expressed from several loci of the infectious bursal disease virus genome induces HA-specific antibodies

The N-terminus of infectious bursal disease virus (IBDV) VP5 has been shown to be capable of tolerating the insertion of small epitopes. The objective of the present study was to determine if IBDV genomic sites, including the 5' end of vp5, could carry an influenza A virus hemagglutinin (HA) epitope. HA-expressing IBDVs were generated when the HA epitope was fused to the N-terminus of VP5 (HA5-IBDV) or VP4 (HA4-IBDV) or the C-terminus of VP1 (1HA-IBDV). Viral titers obtained after co-transfection with cDNA from the ha-containing segment and the complementary genomic segment were 1.3 × 10(4), 3.7 × 10(3) and 3.8 × 10(4) pfu/ml for HA5-IBDV, HA4-IBDV and 1HA-IBDV, respectively. The HA tag expression remained stable after 10 passages when the tag gene was inserted into the vp4 and vp1 genes. HA-IBDVs did not cause pathogenicity in specific-pathogen-free (SPF) chickens. However, only HA4-IBDV and 1HA-IBDV induced HA-specific antibodies, which were measured by ELISA with a maximum optical density (OD) value of 0.701 and 0.769, respectively, at 24 days after infection. Thus, IBDV can potentially be employed as a bivalent viral vector when the epitope is fused with VP4 or VP1.

Mapping B-cell epitopes in equine rhinitis B viruses and identification of a neutralising site in the VP1 C-terminus

Erbovirus is a genus of the family Picornaviridae and equine rhinitis B virus (ERBV) is the sole species. Erboviruses infect horses causing acute respiratory disease and sub-clinical and persistent infections. Despite the high seroprevalence and worldwide distribution of these viruses, the pathogenesis and antigenic structure of the three ERBV serotypes (ERBV1, 2 and 3) is poorly understood. To characterise linear epitopes on ERBV structural proteins, a set of fusion proteins were expressed in Escherichia coli. These proteins were tested in Western blot and ELISA and reactive proteins were also used to identify neutralisation epitopes. VP1 contained serotype specific epitopes whereas VP2 was highly cross-reactive across the serotypes. The C-terminus of VP1 accounted for most of the reactivity of full-length VP1 and was also the location of a neutralising site in each serotype.

Identification of mixed equine rhinitis B virus infections leading to further insight on the relationship between genotype, serotype and acid stability phenotype

Equine rhinitis B virus (ERBV) is the single species in the genus Erbovirus, family Picornaviridae. Equine rhinitis B viruses exist in three serotypes and are associated with respiratory disease in horses. Members of the species vary in stability at acid pH. To date there has been discordance in genotype, serotype and acid stability phenotype groupings. To identify capsid regions associated with acid stability, two viruses were serially treated at pH 3.3 to isolate acid-stable mutants. An acid-stable mutant of the prototype acid-labile serotype 1 virus contained a single amino acid change in the C-terminus of VP1. Similar treatment with a separate isolate identified a multiple ERBV serotype infection with acid-labile serotype 1 and acid-stable serotype 3. Dual infections were subsequently identified in original swabs taken from the infected horse and from two further cell culture passaged viruses originally isolated in Switzerland. Serotype specific rat antisera were produced and used to examine a collection of isolates from a range of genotypes, acid stability phenotypes and serotypes. In contrast to previous reports, we showed viruses previously classified as acid-stable serotype 1 are in fact serotype 3 and that there is a clear association of serotype with genotype and acid stability phenotype in ERBVs. Additionally, we have shown that ERBV capsids dissociate into pentamers in acidic conditions below their threshold of stability, similarly to closely related viruses in the same family.

The unique role of domain 2A of the hepatitis A virus precursor polypeptide P1-2A in viral morphogenesis

The initial step during assembly of the hepatitis A virus particle is driven by domain 2A of P1-2A, which is the precursor of the structural proteins. The proteolytic removal of 2A from particulate VP1-2A by an as yet unknown host enzyme presumably terminates viral morphogenesis. Using a genetic approach, we show that a basic amino acid residue at the C-terminus of VP1 is required for efficient particle assembly and that host proteases trypsin and cathepsin L remove 2A from hepatitis A virus particles in vitro. Analyses of insertion mutants in the C-terminus of 2A reveal that this part of 2A is important for liberation of P1-2A from the polyprotein. The data provide the first evidence that the VP1/2A junction is involved in both viral particle assembly and maturation and, therefore, seems to coordinate the first and last steps in viral morphogenesis.