Characterization of the Ljungan virus

Jens-Ola Ekström, Susanne Johansson, Kjell Edman and A. Michael Lindberg

Department of Chemistry and Biomedical Sciences, University of Kalmar, S-391 82 Kalmar, Sweden.

Ljungan virus (LV), a novel virus isolated from rodents (the bank vole Clathrionomys glareolus and the montane vole Microtus montanus), has previously been proposed as a human pathogen. In the search for an etiological agent causing deaths from myocarditis among Swedish elite orienteers in the early 1990s, several strains of LV were isolated from bank voles. The presence of LV in humans is however only circumstantial. Nucleotide sequence analyses showed that these vole derived strains are closely related and are novel members of the Picornaviridae. LV carries several unique characteristics, e.g. lack of maturation cleavage of VP0 and the presence of two successive 2A and different protein motifs. The predicted cleavage sites and molecular mass of the structural proteins VP0 and VP3 were confirmed by SDS-PAGE analysis of disrupted virions and further verified by use of immuno-precipitation. In addition, the estimated molecular mass of VP1 indicated that the first encoded 2A protein sequence motif is a part of the carboxy-terminal end of VP1.

The parental strains of LV replicate, at best, displaying a slow cytopathic effect (CPE) and low titres of viruses in cell cultures. An efficient system for propagation of LV in cell cultures will facilitate further studies of the biology of LV. The prototype strain of LV, 87-012, was therefore adapted to grow at high titer in GMK cells resulting in rapid and complete cellular lysis. However, the observed CPE type is different from the type caused by enteroviruses. The genome of the cell culture adapted 87-012 was sequenced and showed 14 amino acid changes compared to the parental virus. Furthermore, one nucleotide change was observed in the 5’ untranslated region, a region previously known to influence the tissue tropism of several picornaviruses. The observed seven amino changes in the capsid coding region does not appear to cluster according to a structure model of the LV virion.

Physico-chemical characterization of LV virions is in progress. Thermal inactivation of LV virion indicates the absence of “A” particle formation. A particles are frequently observed as an intermediate in the uncoating process (release of viral RNA) during the entry of picornaviruses to the cell cytoplasm. LV virions are completely inactivated by heat treatment at 70° C for 20 minutes. No inactivation is observed at 20° C and 37° C, while a slight viability reduction is observed at 56° C.

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