Viral VP1 Research Articles

Molecular epidemiology of noroviruses in Northern Greece, 2005-2006.

Noroviruses (NoVs) are important human pathogens associated with acute viral gastroenteritis worldwide, displaying significant genetic heterogeneity. Genotype GII.4 is responsible for the majority of outbreaks reported to date. A total of 460 faecal samples from sporadic gastroenteritis cases were screened for the presence of NoV RNA. Four additional human samples collected during a waterborne NoV gastroenteritis outbreak observed in 2005 in northern Greece, were also included in the study. All PCR-positive samples were tested further using a multiplex RT-PCR, which targets the viral capsid VP1 region D. PCR products from all outbreak samples and from 20 randomly selected samples were sequenced. Phylogenetic analysis revealed that GII.4 genotype predominated (70%), while genotypes GII.2 (10%), GII.7 (15%), and GI.1 (5%) were also detected. All the outbreak NoV strains belonged to the GI.1 genotype. The present study provides a first insight into the epidemiology and genetic diversity of NoVs in Greece and shows that various strains are circulating in the country and cause sporadic cases or outbreaks.

Factors that affect the intracellular localization and trafficking of African horse sickness virus core protein, VP7

African horse sickness virus (AHSV) VP7 is the major core protein of the virion. Apart from its role in virus assembly, VP7 forms crystalline-like particles during infection and when expressed in insect cells. The aim of this study was to investigate the process of VP7 crystalline-like particle formation. The intracellular distribution of VP7 was characterized in different systems and the association of VP7 with virus factories during AHSV infection was investigated. It was shown that the majority of VP7 is sequestered into these particles, and is therefore not available for new virion assembly. This is likely to have a negative impact on virus assembly and yield. By using specific markers and inhibitors of host trafficking pathways, VP7 localization was shown to be independent of host trafficking mechanisms and evaded host defenses against aggregation. Studying the process of VP7 crystalline-like particle formation will help us further understand AHSV replication and assembly.

Expression of a functional recombinant chimeric protein of human hepatitis A virus VP1 and an Fc antibody fragment in transgenic tomato plants

Transgenic tomato plants expressing the gene of a chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus (HAV) VP1 and an Fc antibody fragment have been obtained. Recombinant VP1-Fc protein with a molecular mass of approximately 68 kDa was purified from transgenic tomato plants using Protein A Sepharose affinity chromatography. The recombinant protein elicited production of specific IgG antibodies in the serum after intraperitoneal immunization of BALB/c mice. The antibodies produced by mice against transgenic plant-derived recombinant VP1-Fc most likely recognize epitopes in the HAV viral antigen. Following vaccination with recombinant VP1-Fc protein, expression of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Our findings indicate that transgenic tomato plants can provide a useful system for the production of HAV antigens.

Relationship between genotypes and serotypes of genogroup 1 recoviruses: a model for human norovirus antigenic diversity.

Human norovirus (NoV) research greatly relies on cell culture-propagable surrogate caliciviruses, including murine NoVs and the prototype 'recovirus' (ReCV), Tulane virus. However, the extreme biological diversity of human NoVs cannot be modelled by a uniform group of viruses or single isolate. Based on a diverse group of recently described ReCVs, a more advanced model reflecting human NoV biological diversity is currently under development. Here, we have reported the genotypic and serotypic relationships among 10 G1 ReCV isolates, including Tulane virus and nine other recent cell culture-adapted strains. Based on the amino acid sequences of virus capsid protein, VP1, and classification constraints established for NoVs, G1 ReCVs were separated into three genotypes, with variable organization of the three open reading frames. Interestingly, cross-neutralization plaque assays revealed the existence of four distinct serotypes, two of which were detected among the G1.2 strains. The amino acid (aa) difference between the two G1.2 ReCV serotypes (12%) was less than the minimum 13% difference established between NoV genotypes. Interestingly, one of the G1.3 ReCVs was equally neutralized by antisera raised against the G1.3 (6% aa difference) and G1.1 (25% aa difference) representative strains. These results imply the existence of a large number of human NoV serotypes, but also shared cross-neutralization epitopes between some strains of different genotypes. In conclusion, the newly developed ReCV surrogate model can be applied to address biologically relevant questions pertaining to enteric CV diversity.

133 Structural basis for ebolavirus matrix assembly and budding; protein plasticity allows multiple functions

Proteins, particularly viral proteins, can be multifunctional, but the mechanism(s) behind this trait are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry and cellular microscopy that the Ebola virus VP40 protein rearranges into different structures, each with a distinct and essential function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer trafficks to the cellular membrane. There, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multi-layered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly, but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle, and demonstrate how a single, wild-type, unmodified polypeptide can assemble into different structures for different functions.

In Silico Studies on Development of Novel Virostatic Agents against Bluetongue Virus

The core of BTV is organized into three concentric structures of which VP7 protein forms the major core protein. The subcore consists of VP3 protein and the innermost part of the core is made of three minor proteins: VP1, VP4, and VP6. Earlier it was reported that core-like particles (CLPs) composed of viral VP7 and VP3 proteins were produced in order to study role of VP7 protein in intermolecular interactions in the BTV assembly process. Site specific mutational studies revealed that substitution of the single lysine residue of VP7 (Lys-255) by leucine abrogated CLP formation, indicating a critical role for this lysine. In the present study, homology modeling, mutagenesis, and docking studies were carried out in order to design potent leads in modulation of VP7 protein in abrogating CLP formation.

Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus‐like particles produced in Escherichia coli

Protein nanoparticles such as virus-like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self-assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK(-) E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells.

Crystal structure of Marburg virus VP24.

The VP24 protein plays an essential, albeit poorly understood role in the filovirus life cycle. VP24 is only 30% identical between Marburg virus and the ebolaviruses. Furthermore, VP24 from the ebolaviruses is immunosuppressive, while that of Marburg virus is not. The crystal structure of Marburg virus VP24, presented here, reveals that although the core is similar between the viral genera, Marburg VP24 is distinguished by a projecting β-shelf and an alternate conformation of the N-terminal polypeptide.

The 2010 outbreak of poliomyelitis in Tajikistan: epidemiology and lessons learnt

A large outbreak of poliomyelitis, with 463 laboratory-confirmed and 47 polio-compatible cases, took place in 2010 in Tajikistan. Phylogenetic analysis of the viral VP1 gene suggested a single importation of wild poliovirus type 1 from India in late 2009, its further circulation in Tajikistan and expansion into neighbouring countries, namely Kazakhstan, Russia, Turkmenistan and Uzbekistan. Whole-genome sequencing of 14 isolates revealed recombination events with enterovirus C with cross-overs within the P2 region. Viruses with one class of recombinant genomes co-circulated with the parental virus, and representatives of both caused paralytic poliomyelitis. Serological analysis of 327 sera from acute flaccid paralysis cases as well as from patients with other diagnoses and from healthy people demonstrated inadequate immunity against polio in the years preceding the outbreak. Evidence was obtained suggesting that vaccination against poliomyelitis, in rare cases, may not prevent the disease. Factors contributing to the peculiarities of this outbreak are discussed. The outbreak emphasises the necessity of continued vaccination against polio and the need, at least in risk areas, of quality control of this vaccination through well planned serological surveillance.

Antiviral Activity of Hederasaponin B from Hedera helix against Enterovirus 71 Subgenotypes C3 and C4a

Enterovirus 71 (EV71) is the predominant cause of hand, foot and mouth disease (HFMD). The antiviral activity of hederasaponin B from Hedera helix against EV71 subgenotypes C3 and C4a was evaluated in vero cells. In the current study, the antiviral activity of hederasaponin B against EV71 C3 and C4a was determined by cytopathic effect (CPE) reduction method and western blot assay. Our results demonstrated that hederasaponin B and 30% ethanol extract of Hedera helix containing hederasaponin B showed significant antiviral activity against EV71 subgenotypes C3 and C4a by reducing the formation of a visible CPE. Hederasaponin B also inhibited the viral VP2 protein expression, suggesting the inhibition of viral capsid protein synthesis.These results suggest that hederasaponin B and Hedera helix extract containing hederasaponin B can be novel drug candidates with broad-spectrum antiviral activity against various subgenotypes of EV71.

Modulation of genes related to the recruitment of immune cells in the digestive tract of trout experimentally infected with infectious pancreatic necrosis virus (IPNV) or orally vaccinated

There are still many details of how intestinal immunity is regulated that remain unsolved in teleost. Although leukocytes are present all along the digestive tract, most immunological studies have focused on the posterior segments and the importance of each gut segment in terms of immunity has barely been addressed. In the current work, we have studied the regulation of several immune genes along five segments of the rainbow trout (Oncorhynchus mykiss) digestive tract, comparing the effects observed in response to an infectious pancreatic necrosis virus (IPNV) infection to those elicited by oral vaccination with a plasmid coding for viral VP2. We have focused on the regulation of several mucosal chemokines, chemokine receptors, the major histocompatibility complex II (MHC-II) and tumor necrosis factor α (TNF-α). Furthermore, the recruitment of IgM+ cells and CD3+ cells was evaluated along the different segments in response to IPNV by immunohistochemical techniques. Our results provide evidences that there is a differential regulation of these immune genes in response to both stimuli along the gut segments. Along with this chemokine and chemokine receptor induction, IPNV provoked a mobilization of IgM+ and IgT+ cells to the foregut and pyloric caeca region, and CD3+ cells to the pyloric caeca and midgut/hindgut regions. Our results will contribute to a better understanding of how mucosal immunity is orchestrated in the different gut segments of teleost.

Human polyoma JC virus minor capsid proteins, VP2 and VP3, enhance large T antigen binding to the origin of viral DNA replication: Evidence for their involvement in regulation of the viral DNA replication

JC virus (JCV) lytically infects the oligodendrocytes in the central nervous system in a subset of immunocompromized patients and causes the demyelinating disease, progressive multifocal leukoencephalopathy. JCV replicates and assembles into infectious virions in the nucleus. However, understanding the molecular mechanisms of its virion biogenesis remains elusive. In this report, we have attempted to shed more light on this process by investigating molecular interactions between large T antigen (LT-Ag), Hsp70 and minor capsid proteins, VP2/VP3. We demonstrated that Hsp70 interacts with VP2/VP3 and LT-Ag; and accumulates heavily in the nucleus of the infected cells. We also showed that VP2/VP3 associates with LT-Ag through their DNA binding domains resulting in enhancement in LT-Ag DNA binding to Ori and induction in viral DNA replication. Altogether, our results suggest that VP2/VP3 and Hsp70 actively participate in JCV DNA replication and may play critical roles in coupling of viral DNA replication to virion encapsidation.

Whole genome analysis of epizootic hemorrhagic disease virus identified limited genome constellations and preferential reassortment

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides transmitted orbivirus that causes haemorrhagic disease in wild and domestic ruminants. A collection of 44 EHDV isolated from 2008 to 2012 was fully sequenced and analysed phylogenetically. Serotype 2 viruses were the dominant serotype all years except 2012 when serotype 6 viruses represented 63 % of the isolates. High genetic similarity (>94 % identity) between serotype 1 and 2 virus VP1, VP3, VP4, VP6, NS1, NS2 and NS3 segments prevented identification of reassortment events for these segments. Additionally, there was little genetic diversity (>96 % identity) within serotypes for VP2, VP5 and VP7. Preferential reassortment within the homologous serotype was observed for VP2, VP5 and VP7 segments for type 1 and type 2 viruses. In contrast, type 6 viruses were all reassortants containing VP2 and VP5 derived from an exotic type 6 with the remaining segments most similar to type 2 viruses. These results suggest that reassortment between type 1 and type 2 viruses requires conservation of the VP2, VP5 and VP7 segment constellation while type 6 viruses only require VP2 and VP5 and are restricted to type 2-lineage VP7. As type 6 VP2 and VP5 segments were exclusively identified in viruses with type 2-derived VP7, these results suggest functional complementation between type 2 and type 6 VP7 proteins.

Clinical relevance and genotypes of circulating noroviruses in northern Taiwan, 2006-2011

The incidence of noroviral gastroenteritis has increased dramatically in recent years, and norovirus (NoV) genogroup II.4 (GII.4) is associated with outbreaks worldwide. The NoV genotypes and their clinical relevance in children hospitalized with acute gastroenteritis between 2006 and 2011 in northern Taiwan were evaluated in this study. NoV sequences were amplified from 47 clinical specimens and phylogenetic analysis was performed. Based on noroviral capsid protein (VP1) and RNA dependent RNA polymerase (RdRp) phylogeny, circulating NoV could be divided into GII.2, GII.3, GII.12, and GII.4 and GII.16, GII.12, GII.g, and GII.4; respectively. The GII.4 subtype was predominant and could be divided further into the 2004 (Hunter), 2006b, and 2010 (New Orleans) subtypes. Regarding clinical manifestations, convulsive disorder occurred only in cases caused by NoV GII.4 2006b. Patients affected by NoV GII.4 2006b presented with a higher frequency of diarrhea (P = 0.0204), longer duration of diarrhea (P = 0.0215), more frequent hypoglycemia (P = 0.038), and electrolyte imbalance (P = 0.0487) than acute gastroenteritis caused by NoV GII.4 2010. Structural analysis showed that the amino acid changes in viral VP1 between GII.4 2006b and 2010 subtype were located mainly in the protruding domain 2 (P2 domain). In conclusion, the NoV GII.4 variants 2006b and 2010 were the main causes of acute gastroenteritis in hospitalized children in northern Taiwan during 2006-2011. The clinical presentations and structural changes in VP1 of the two NoV GII.4 variants should be evaluated in the future.

Recent outbreak of aseptic meningitis in Italy due to Echovirus 30 and phylogenetic relationship with other European circulating strains

BackgroundEnteroviruses (EVs) are common human viral pathogens, causing a variety of diseases, including aseptic meningitis. Recently, EV aseptic meningitis outbreaks have been reported across Europe, but, in Italy, knowledge of recent EV molecular epidemiology is very limited. ObjectivesWe report an outbreak of EV aseptic meningitis in 10 adults in North-Western Italy, from October to November 2012. Patients were parents or close relatives of children <5 years old attending the same class of a nursery school, suffering from a mild febrile upper respiratory disease. Phylogenetic relationship with other European circulating strains was analyzed updating E30 circulation in Italy in recent years. Study designEVs were detected from cerebrospinal fluid (CSF) specimens with a real-time reverse transcription polymerase chain reaction and virus isolation was achieved from rectal and pharyngeal swabs. For cluster definition and phylogenetic studies, viral VP1 region was directly amplified and sequenced from CSF. ResultsEVs were identified in CSF from all patients and from rectal and pharyngeal swabs in 7 of them. Direct sequencing of CSF revealed the presence of the same Echovirus 30 (E30) in all patients and phylogenetic analysis identified it as a diverging clade within E30 genotype VII, the most recent strain circulating in UK, Finland and Denmark since 2006. ConclusionMolecular techniques allowed the rapid identification and typing of E30 from CSF. Phylogenetic analysis revealed that the cluster might be due to a new E30 variant within the genotype VII currently circulating in Europe, thus updating the epidemiology of EV circulation in Italy.

Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. colifor sero-diagnostic applications

BackgroundChicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention.ResultsThree CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other subunit proteins. Moreover, the VP2-396N and VP2-345 based ELISAs had better sensitivity (97.5%) and excellent specificity (100%) during serodiagnosis testing using a mean plus three standard deviations cut-off. The VP3-246M based ELISA showed a sensitivity of 85% and a specificity of 100% at the same cut-off value.ConclusionsThis is the first report to systematically assess the antigenic characteristics of CAV viral proteins for sero-diagnosis purposes. Purified recombinant VP2-396N and VP2-345N subunit proteins, which span defined regions of VP2, were demonstrated to have good antigenicity and higher sensitivities than VP3-246M and were able to recognize CAV-positive chicken serum using an ELISA assay. The defined antigenicity potential of these chimeric subunit proteins produced by expression in E. coli seem to have potential and could be useful in the future for the development of the CAV diagnostic tests based on a subunit protein ELISA system.

Multiple large foreign protein expression by a single recombinant baculovirus: A system for production of multivalent vaccines

Baculovirus expression system offers the advantage of expression of several large proteins simultaneously by a single recombinant virus. To date, expression of multiple large (>100kDa) proteins has been hampered by the need to generate large constructs and repeat use of homologous sequence and promoter. The development of multi-loci baculovirus expression system overcomes these issues by enabling the recombination of large foreign sequences into different regions of the genome. In this paper, we have examined the co-expression of African horse sickness virus (AHSV) VP2 proteins from multiple serotypes in a single recombinant baculovirus. To this end, recombinant baculoviruses expressing multiple AHSV VP2 proteins were generated and it was found that up to six different AHSV serotypes (serotype 1, 3, 4, 5, 7 and 8) VP2 proteins (∼120kDa) could be expressed simultaneously from different loci of baculovirus genome. The expression of VP2 of one serotype was not significantly hindered by the presence of other serotypes, although there were slight differences in expression level between different serotypes. The expression of VP2 of further serotypes from additional loci resulted in a lesser expression level of VP2 proteins. Based on these findings, three additional recombinant baculoviruses encompassing all nine AHSV serotypes were constructed (serotypes 1, 7, 8 or serotypes 2, 4, 5 or serotypes 3, 6, 9) and each of the triple recombinant viruses exhibited similar expression level of each VP2. This system allows for the expression of a number of large proteins that has the potential to be exploited for multivalent vaccines production.

The endosomal pathway and the Golgi complex are involved in the infectious bursal disease virus life cycle.

Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra- and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus.

Engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (FMDV) VP1 inhibits type I interferon response in cells

Foot-and-mouth disease (FMD) is an acute, highly contagious animal disease caused by FMD virus (FMDV). Although FMDV-induced immunosuppression in host has been well established, the exact molecular mechanism for such induction is not very clear. We report here the identification of FMDV VP1 as an interferon-suppressor by interacting with soluble resistance-related calcium binding protein (sorcin). We found that VP1 suppressed tumor necrosis factor (TNF)-α or Sendai virus (SeV)-induced type I interferon response in HEK293T cells, and that this suppression could be completely abolished by knockdown of sorcin by shRNA. Furthermore, overexpression of sorcin inhibited type I interferon response. Conversely, TNF- or SeV-induced type I interferon response increased when sorcin knocked down, leading to inhibition of vesicular stomatitis virus (VSV) replication. Thus, VP1-induced suppression of type I interferon is mediated by interacting with sorcin, a protein that appears to regulate cell response to viral infections.

Analysis and solution of false-positives when testing CVA16 sera using an antibody assay against the EV71 virus

Hand, foot and mouth disease (HFMD) in humans is caused mainly by Enterovirus 71(EV71) and Coxsackievirus A16 (CVA16). EV71 is associated with severe HFMD cases but not CVA16. Use of IgM-capture enzyme-linked immunosorbent assay (ELISA) is important for the early diagnosis of EV71 infection, but cross-reactivity of the anti-CVA16 IgM antibody with EV71 produces false-positive results. In this report, we designed a new EV71 IgM-capture ELISA method using the EV71 VP1 peptide instead of the EV71 virion as the detectable antigen, and tested sera from patients infected with EV71 or CVA16. The results showed that acute sera from 76 EV71-infected patients had similar sensitivity for virus detection (98.68%) or VP1 detection (97.37%). When acute sera from patients infected with CVA16 were used, significant differences between the two methods were observed. The cross-reactivity rate of the virus detection method was 29.4% (5/17), but no cross-reactivity was observed using the VP1 detection method. Western immunoblotting demonstrated that EV71 VP3 cross-reacted with part of the CVA16 IgM antibody. The results demonstrate that EV71 VP3 is the cross-reactive antigen in the EV71 IgM-capture ELISA when testing CVA16 sera using the virus-antibody detection method. The problem of false-positive results was resolved by using the VP1 peptide as the detectable antigen.