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 RNA has been found previously in specimens of muscle biopsy from patients with idiopathic dilated cardiomyopathy, chronic inflammatory muscle diseases, and fibromyalgia or chronic fatigue syndrome (fibromyalgia/chronic fatigue syndrome). These results suggest that skeletal muscle may host enteroviral persistent infection. To test this hypothesis, we investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay the presence of enterovirus in skeletal muscle of patients with chronic inflammatory muscle diseases or fibromyalgia/chronic fatigue syndrome, and also of healthy subjects. Three of 15 (20%) patients with chronic inflammatory muscle diseases, 4 of 30 (13%) patients with fibromyalgia/chronic fatigue syndrome, and none of 29 healthy subjects was found positive. The presence of VP-1 enteroviral capsid protein was assessed by an immunostaining technique using the 5-D8/1 monoclonal antibody; no biopsy muscle from any patient or healthy subject was found positive. The presence of viral RNA in some muscle biopsies from patients exhibiting muscle disease, together with the absence of VP-1 protein, is in favor of a persistent infection involving defective viral replication.

Although the transmission of coxsackievirus B3 occurs mainly via the oral route, little is known about the primary replication and persistence of this agent in the intestine. To address this question, BALB/c mice were inoculated by gavage with coxsackievirus B3, Nancy strain. The mice were killed from 1 hr to 90 days after infection. The viral markers were detected in the small intestine using RT-PCR, cell culture and detection of VP1 protein. Coxsackievirus B3 was detected positive by the three methods from hr 2 to day 45 after infection. By using monoclonal antibodies directed towards VP1, CD40 and CD26, the virus was shown to be present in the lymphocytes of the mucosa as soon as 2 hr after infection; in contrast, no virus was detected in the epithelial cells lining the intestinal lumen. Further experiments were performed to evaluate the capacity of coxsackievirus B3 to establish a persistent infection in two intestinal cell lines. In contrast to HT29 cells, the CaCo-2 cells were shown to develop a persistent infection for up to 20 passages, as demonstrated by the detection of viral RNA and VP1 protein. This study provides further evidence that, after infection by the oral route, the viral particles are concentrated in the lymphocytes of the mucosal layer. In addition, the results suggest that coxsackievirus B3 is capable of establishing a persistent infection in the small intestine that may act as a reservoir of viral particles for the delayed spread of the virus to other target organs.

In order to shorten the time required for the detection of enteroviruses in stool specimens, an 18-h immunoperoxidase test combining low-speed centrifugation and the use of a group specific anti-VP1 monoclonal antibody (5-D8/1, Dako) was developed. This rapid culture assay (RCA) was compared blindly to a conventional culture assay (CCA) on a panel of 180 children's stool specimens received for routine diagnosis of enterovirus infection. The same cell lines (human embryonic fibroblasts and KB continuous cell line) were used in both tests. Discrepancies in results were analysed by a PCR technique with primers located in a conserved part of the 5' non-coding region of the enterovirus genome. Fourteen specimens were positive and 158 were negative with both tests. Four samples were positive with the RCA yet negative with the CCA and 3 others showed the opposite pattern; an additional sample positive by RCA was uninterpretable by CCA due to bacterial contamination. Subsequent PCR testing of these 8 samples showed no discrepancies; all were positive. Using CCA as the reference, the sensitivity and specificity of RCA were 77.8 and 98% respectively. Kinetic studies using enterovirus isolates demonstrated that RCA was much more sensitive than CCA during the first three days of culture. These results further suggested that RCA sensitivity could be improved by a factor of at least 10 times by prolonging the incubation period by 24 hr. With this change, the RCA assay described below is suggested as a rapid alternative to CCA for the routine diagnosis of enterovirus infection in stool specimens. When an identification at the serotype level is required, samples found positive using RCA could then be subjected to CCA.

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