Efficient infection of buffalo rat liver-resistant cells by encephalomyocarditis virus requires binding to cell surface sialic acids.

In contrast to the production of virus and cell lysis seen in baby hamster kidney cells (BHK-21) infected with the strain 1086C of encephalomyocarditis virus (EMCV), in buffalo rat liver cells (BRL) neither virus replication nor cytopathic effects were observed. After 29 passages in BRL cells, each alternating with boosts of the recovered virus in BHK-21 cells, the virus acquired the ability to replicate effectively in BRL cells, attaining virus titres comparable to those in BHK-21 cells and producing complete cell destruction. The binding of virus on BRL cells was increased after adaptation and was similar to that observed on BHK-21 cells. Treatment of BRL cells with sialidase resulted in an 87 % reduction in virus binding and inhibition of infection. Sequence analyses revealed three mutations in the VP1 amino acid sequence of the adapted virus at positions 49 (Lys-->Glu), 142 (Leu-->Phe) and 180 (Ile-->Ala). The residue 49 is exposed at the surface of the capsid and is known to be part of a neutralization epitope. These results suggest that the adaptation of EMCV to BRL cells may have occurred through a mutation in a neutralizing site that confers to the virus a capacity to interact with cell surface sialic acid residues. Taken together, these data suggest a link between virus neutralization site, receptor binding and cell permissivity to infection.

Prevalence of anti-hepatitis E virus antibodies in French blood donors.

Accumulating evidence suggests that hepatitis E virus (HEV) infection is an emerging disease in regions where HEV is nonendemic. In France, the prevalence of anti-HEV antibodies in the general population has never been studied. Using blood donors' samples, we have found a prevalence of 3.20%, which is similar to that of other industrialized countries.

Long-term monitoring of classical swine fever in wild boar (Sus scrofa sp.) using serological data.

In the European Community, epizootics of classical swine fever (CSF) in the wild boar (Sus scrofa) are compulsorily monitored because transmission may occur between wild boars and domestic pigs, causing heavy economic losses to the pork industry. The estimation of incidence in populations of wild boars is generally based on viroprevalence. However, viral isolation becomes rare when the incidence is low because the virus cannot be detected for more than a few weeks following infection. On the contrary, seroprevalence is detectable at low incidence levels, because antibodies can be detected for the lifetime of the infected animal. We thus attempted to analyse the long-term evolution of CSF incidence using serological data. The data came from France, where CSF had been monitored from 1992 to 2002, and where the virus has not been detected since 1997. We assumed that the overall seroprevalence would estimate the proportion of immune wild boars, that seroprevalence in juveniles would approximate incidence and that seroprevalence in different age classes would show the evolution of incidence in a given cohort. Spatial and temporal trends of incidence and seroprevalence were explored using logistic modelling and the spatial trend was analysed using polynomial regression. In 1992, incidence peaked in the northern area. After 1993, incidence decreased but remained the highest in the northern area. After 2000, no seropositive juvenile was observed, suggesting the extinction of the epizootic. Our results support the reliability of serological monitoring since it allowed a longer detection of viral transmission and provided more information on the spatio-temporal evolution of incidence than did viral isolation. We advocate that the highest persistence of infection in northeastern France is not independent from infection persistence in Reinland-Pfalz (Germany). Such persistence may be due to favourable local conditions and/or the social organisation of wild boars.

Isolation of foot-and-mouth disease virus specific bovine antibody fragments from phage display libraries.

Foot-and-mouth disease virus (FMDV) is an important veterinary pathogen which can cause widespread epidemics. Due to the high antigenic variability of FMDV, it is important to undertake mutation analysis under immunological pressure. To study the bovine antibody response at a molecular level, phage display technology was used to produce bovine anti-FMDV Fabs. CH1-VH chains with FMDV specific binding could be isolated after selection from a library made from vaccinated cattle. Though their involvement in the bovine immune response remains to be ascertained, it is planned to express the five different selected VH domains in bacterial or insect systems as sequence homologies with integrin beta6 chain could shed light on the basis of FMDV type receptor specificities.

Diversity of enterovirus sequences detected in oysters by RT-heminested PCR.

Oysters harvested in western France, from five sites associated with outbreaks of food-borne norovirus gastroenteritis between February 2000 and March 2001, were assayed for enterovirus RNA by reverse transcriptase-heminested polymerase chain reaction (RT-heminested PCR). Forty percent (21/52) of shellfish samples (pool of seven oysters) were contaminated by enteroviruses. Infectious coxsackieviruses serotype A21 were isolated from three of these positive samples. Amplicons corresponding to 65 base sequences in the 5' untranslated region of the enteroviral genome were analyzed by direct sequencing. Interpretable results were obtained from 18 amplicons, but mixtures of sequences confused the results from 3 samples. Sequences isolated from samples from the different sites were different but similarities were observed between sequences detected in shellfish from two sites at different dates. Sequences were also compared to sequences of human, bovine and porcine enteroviruses. Both human and animal origins of enterovirus contamination of shellfish seemed likely.

Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture.

Fruits and vegetables may act as a vehicle of human enteric virus if they are irrigated with sewage-contaminated water or prepared by infected food handlers. An elution-concentration method was modified to efficiently detect, by reverse transcriptase-polymerase chain reaction (RT-PCR) or by cell culture, contamination by poliovirus, hepatitis A virus (HAV), and Norwalk-like virus (NLV) of various fresh and frozen berries and fresh vegetables. The protocol included washing the fruit or vegetable surface with 100 mM Tris-HCl, 50 mM glycine, and 3% beef extract, pH 9.5 buffer, which favors viral elution from acid-releasing berries, supplemented with 50 mM MgCl2 to reduce the decrease in viral infectivity during the process. The viral concentration method was based on polyethylene glycol precipitation. Copurified RT-PCR inhibitors and cytotoxic compounds were removed from viral concentrates by chloroform-butanol extraction. Viruses from 100 g of vegetal products could be recovered in volumes of 3 to 5 ml. Viral RNAs were isolated by a spin column method before molecular detection or concentrates were filtered (0.22-microm porosity) and inoculated on cell culture for infectious virus detection. About 15% of infectious poliovirus and 20% of infectious HAV were recovered from frozen raspberry surfaces. The percentage of viral RNA recovery was estimated by RT-PCR to be about 13% for NLV, 17% for HAV, and 45 to 100% for poliovirus. By this method, poliovirus and HAV RNA were detected on products inoculated with a titer of about 5 x 10(1) 50% tissue culture infectious dose per 100 g. NLV RNA was detected at an initial inoculum of 1.2 x 10(3) RT-PCR amplifiable units. This method would be useful for the viral analysis of fruits or vegetables during an epidemiological investigation of foodborne diseases.

Nucleotide sequence and construction of an infectious cDNA clone of an EMCV strain isolated from aborted swine fetus.

A full-length cDNA clone of an Encephalomyocarditis virus (EMCV) strain (2887A) isolated from aborted swine fetus was constructed and sequenced. Sequence comparison showed more than 99% nucleotide and amino acid sequence identity with two other EMCV strains, EMCV-PV21 and -R. However, the 2887A genomic sequence showed only about 84% nucleotide identity and 96% amino acid identity with EMCV-B, -D and -PV2 variants. RNA synthesized by in vitro transcription of this cDNA clone was infectious upon transfection of BHK21 cells, as shown by cytopathic effects and identification by neutralization test, and by propagation of the virus released into the culture media. The transcript RNA led to the production of infectious particles despite the presence of two nongenomic nucleotide residues at the 5' end, the short poly(C) tract (C(10)TCTC(3)TC(10)), the short poly(A) tail (7A), and the presence of six nongenomic nucleotides at the 3' end. The rescued virus was also found to be highly pathogenic for mice by intra-peritoneal inoculation producing a fatal disease indistinguishable from that of wild-type virus. An important finding concerning the molecular basis of infectivity was that the in vitro synthesized EMCV RNA transcript is infectious, although it contains a very short poly(A). The availability of the infectious cDNA clone of the reproductive failure strain of EMCV should prove to be useful for studying the molecular basis of the pathogenicity of EMCV in pig.