We investigated the use of HPLC in analysis of picornavirus variants by comparing structural polypeptides of three stable mutants of encephalomyocarditis virus (EMCV). The variants are known to differ in their pathogenicity for mice: plaque variant 2 (PV2) is diabetogenic, PV7 is non-diabetogenic and PV21 induces a generalized lethal infection. We first used HPLC to separate the structural proteins at high purity levels. Detailed analysis of these structural proteins by HPLC-peptide mapping revealed differences in all four viral proteins of PV21 as compared with mutants PV2 and PV7. A single amino acid exchange was found in viral protein 1 between PV2 and PV7. Altered peaks were identified by calculating retention times of tryptic peptides using sequence data and a computer program. Since peak alterations could be attributed to the observed amino acid exchanges, the results correlate well with cDNA sequencing data. Thus HPLC proved to be a useful and fast tool for primary or additional characterization of picornavirus variants at the level of whole virus proteins.


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