In contrast to the production of virus and cell lysis seen in baby hamster kidney cells 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 (LysAGlu), 142 (LeuAPhe) and 180 (IleAAla). 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.

A recombinant encephalomyocarditis virus (rEMCV2887A-egfp) expressing the enhanced green fluorescent protein (EGFP) was produced. The EGFP gene was inserted in frame within the leader protein coding sequence of a full-length cDNA clone of EMCV. RNA transcripts derived from the recombinant full-length cDNA were synthesized in vitro and transfected into BHK-21 cells. The recombinant transcript RNA remained infectious despite the insertion of EGFP as shown by cytopathic effects on BHK-21 cells and by propagation of the rescued virus. The replication kinetics in BHK-21 cells and the pathogenicity in mice of rEMCV2887A-egfp did not differ significantly from that of the parental virus. The recombinant virus was shown to produce fluorescence in infected cells after at least five passages in BHK-21 cells. However, a decrease of EGFP expression was observed following serial passages, and this was associated with the accumulation of deletion mutations within the EGFP gene. Nevertheless, using EGFP autofluorescence, infected cells were easily detected in the brain of mice infected with the first-passage recombinant virus. These data demonstrate that rEMCV2887A-egfp could be a useful tool to study virus dissemination and pathogenicity when used at low passages.

Encephalomyocarditis virus (EMCV) can infect a wide range of vertebrate species including swine and non-human primates, but few data are available for humans. We therefore wanted to gain further insight into the mechanisms involved in EMCV infection of human cells. For this purpose, we analyzed the permissiveness of primary human cardiomyocytes towards two strains of EMCV; a pig myocardial strain (B279/95) and a rat strain (1086C). In this study, we show that both strains productively infect primary human cardiomyocytes and induce complete cytolysis. Binding and infection inhibition experiments indicated that attachment and infection are independent of sialic acid and heparan sulfate for B279/95 and dependent for 1086C. Sequence comparison between the two strains and three-dimensional analysis of the capsid revealed that six of the seven variable residues are surfaceexposed, suggesting a role for these amino acids in binding. Moreover, analysis of variants isolated from the 1086C strain revealed the importance of lysine 231 of VP1 in the attachment of EMCV to cell-surface sialic acid residues. Together, these results show a potential for EMCV strains to use at least two different binding possibilities to initiate infection and provide new insights into the mechanisms involved in primary human cell recognition by EMCV.

Samples of serum from 76 gilts, 1440 sows, 1473 piglets and 3093 finishing pigs from 96 farrow-to-finish herds were tested for antibodies to encephalomyocarditis virus (EMCV) in microtitre serum neutralisation tests employing two strains of virus, one associated with myocarditis and the other with reproductive failure. The total seroprevalence of EMCV infection was 2.48 per cent. There was no significant difference between the seroprevalence of the reproductive failure strain (1.6 per cent) and the myocardial strain (1.85 per cent). The seroprevalence was higher in the gilts (6.57 per cent) and sows (5.13 per cent) than in the piglets (1 per cent) and finishing pigs (1.84 per cent), and the highest titres were observed in the sows (1:540) and finishing pigs (1:640). In the gilts, the difference in seroprevalence between the reproductive failure strain (3.95 per cent) and the myocardial strain (5.33 per cent) was wider than in the other groups.