The sequencing of the VP1 hypervariable region of the human enterovirus (HEV) genome has become the reference test for typing field isolates. This study describes a new strategy for typing HEV at the serotype level that uses a reverse transcription-PCR assay targeting the central part of the VP2 capsid protein. Two pairs of primers were used to amplify a fragment of 584 bp (with reference to the PV-1 sequence) or a part of it (368 bp) for typing. For a few strains not amplified by the first PCR, seminested primers enhanced the sensitivity (which was found to be approximately 10 ؊1 and 10 ؊4 50% tissue culture infective dose per reaction tube for the first and seminested assay, respectively). The typing method was then applied to 116 clinical and environmental strains of HEV. Sixty-one typeable isolates were correctly identified at the serotype level by comparison to seroneutralization. Forty-eight of 55 "untypeable" strains (87.3%) exhibited the same serotype using VP1 and VP2 sequencing methods. For six strains (four identified as EV-71, one as E-9, and one as E-30 by the VP2 method), no amplification was obtained by the VP1 method. The last strain, typed as CV-B4 by VP1 and CV-B3 by VP2 and monovalent antiserum, could exhibit recombination within the capsid region. Although the VP2 method was tested on only 36 of the 68 HEV serotypes, it appears to be a promising strategy for typing HEV strains isolated on a routine basis. The good sensitivity of the seminested technique could avoid cell culture and allow HEV typing directly from PCR products.Human enteroviruses (HEV) are among the most common of human viruses. Most infections are mild or asymptomatic, but some can lead to severe clinical presentations, especially in neonates and immunocompromised patients (32).The genus Enterovirus of the family Picornaviridae includes nonenveloped viruses comprising a 7,500-nucleotide singlestranded positive RNA genome protected by an icosahedral capsid. The genome encodes seven nonstructural proteins implicated in viral replication and maturation and four structural proteins, VP1 to VP4. VP1, VP2, and VP3 are located at the surface of the viral capsid and are exposed to immune pressure, whereas VP4 is located inside the capsid.The HEV serotypes were originally classified on the basis of antigenic properties and according to their natural and experimental pathogenesis: poliovirus (PV) infection in monkeys, coxsackievirus A (CV-A) and CV-B infection in suckling mice, and echovirus (E) infection in cell culture but not in mice (32). The molecular analysis of coding and noncoding regions (9) led to the classification of the 68 serotypes of HEV into five species (36): (i) HEV-A includes CV-A2, -3, -5 to -8, -10, -12, -14, and -16 and enterovirus 71 (EV-71) and -76; (ii) HEV-B includes CV-B1 to -6, CV-A9, and all Es, as well as EV-69, -73, -74, -75, -77, and -78; (iii) HEV-C includes CV-A1, -11, -13, -17, -20 to -22, and -24; (iv) EV-68 and -70 form the HEV-D group; and (v) the three serotypes of PV are still grouped into a separ...

Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to four species designed Human enterovirus A to D. The antigens of the structural proteins support the subdivision of enteroviruses into multiple serotypes. Comparative phylogeny based on molecular typing methods has been of great help to classify former and new types of enterovirus, and to investigate the diversity of enteroviruses and the evolutionary mechanisms involved in their diversity. By now, molecular typing methods of enterovirus rely mainly on the sequencing of an amplicon targeting a variable part of the region coding for the capsid proteins (VP1 and, alternatively, VP2 or VP4), either from a strain recovered by cell culture or, more recently, by direct amplification of a clinical or environmental specimen. In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.

Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence analysis of the whole structural genomic region showed the occurrence of a recombination event at position 1950 within the VP3 capsid gene, in a region coding for the 2b antigenic site previously described for CV-B3. This observation evidences for the first time the occurrence of an interserotypic recombination within the VP2-VP3-VP1 capsid region between two nonpoliovirus enterovirus strains. The neutralization pattern suggests that the major antigenic site is located within the VP2 protein.Enteroviruses are small, nonenveloped, positive singlestranded RNA viruses belonging to the family Picornaviridae. Human enteroviruses (HEV) comprise 68 serotypes, subdivided into five species on the basis of genetic properties: HEV-A to HEV-D and poliovirus (PV) (38). These viruses are responsible for a wide range of acute and chronic clinical manifestations (32).The RNA genome, 7.5 kb long, is constituted by a single open reading frame flanked by 5Ј and 3Ј untranslated regions. The coding region is translated into a single polyprotein of 2,200 amino acids and is then processed to generate four structural proteins (VP1 to VP4) and seven nonstructural proteins (2A to 2C and 3A to 3D). The four structural proteins constitute the icosahedral capsid that contains the major antigenic determinants (22) and the principal attachment sites to the cellular receptors (for a review, see reference 10).Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. Although recombination has long been recognized as an important property of PV (9, 17), several recent publications have demonstrated that it is also extremely frequent in non-PV enteroviruses (2, 6-8, 15, 17-20, 24, 27, 28, 31, 35). Despite this high level of genetic instability, the occurrence of intra-and interserotypic recombination events in the VP2-VP3-VP1 structural coding region has been shown to be a rare phenomenon and up to now restricted to PV strains (5,14,21,40). Structural requirements of the virion shell or of receptor binding were thought to be involved in the restriction of recombination within this region (17,30,36).In this paper, we describe an interserotypic recombination event occurring in the VP3 coding region of a clinical strain of HEV-B and leading to a chimeric coxsackievirus B3 (CV-B3)/ CV-B4 type. MATERIALS AND METHODSVirus isolation and identification. The analyzed virus strain (SE-03-78616) was isolated from the KB cell line in 2003 from stools of a patient admitted for meningitis in the pediatric unit of Toulon Hospital (France). The strain was purified by the limiting dilution method in ce...

BackgroundDecay Accelerating Factor (DAF) and Coxsackievirus-Adenovirus Receptor (CAR) have been identified as cellular receptors for Coxsackie B viruses (CV-B). The aim of this study is to elucidate the different binding properties of CV-B serotypes and to find out if there are any amino acid changes that could be associated to the different phenotypes.Twenty clinical CV-B isolates were tested on CaCo-2 cell line using anti-DAF (BRIC216) and anti-CAR (RmcB) antibodies. CV-B3 Nancy prototype strain and a recombinant strain (Rec, CV-B3/B4) were tested in parallel. The P1 genomic region of 12 CV-B isolates from different serotypes was sequenced and the Trans-Epithelial Electrical Resistance (TEER) along with the virus growth cycle was measured.ResultsInfectivity assays revealed clear differences between CV-B isolates with regard to their interactions with DAF and CAR. All tested CV-B isolates showed an absolute requirement for CAR but varied in their binding to DAF. We also reported that for some isolates of CV-B, DAF attachment was not adapted. Genetic analysis of the P1 region detected multiple differences in the deduced amino acid sequences.ConclusionWithin a given serotype, variations exist in the capacity of virus isolates to bind to specific receptors, and variants with different additional ligands may arise during infection in humans as well as in tissue culture.

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