The enterovirus echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the α v β 3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3-and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this echovirus infection is controlled by the availability of α v β 3 integrin.

HBB [2-(α-hydroxybenzyl)-benzimidazole] and guanidine are potent inhibitors of picornavirus replication. Among other evidence, limited cross-resistance and a synergistic effect of both inhibitors suggest similar but not identical mechanisms of antiviral action. Echovirus-9 variants resistant to each of these drugs were characterized and sequenced. Complete resistance to HBB or guanidine was shown to be due to single but different point mutations in the non-structural protein 2C. Protein 2C was expressed as GST fusion and His-tagged proteins for the wild-type and various mutants. Although three mutations were located in or near conserved NTP binding motifs, NTPase activity was not altered in the presence of HBB or guanidine.

The recently analyzed sequences of the nonpathogenic prototype strain Hill and the mouse-virulent strain Barty of the human echovirus 9 differ particularly in an insertion coding for an RGD motif at the C-terminus of the capsid protein VP1 in the genome of strain Barty. To investigate molecular determinants of virulence, we generated a panel of recombinant viruses derived from cDNA clones of strains Hill and Barty. In this communication, we show that the mouse-pathogenic character of strain Barty correlates with a 310-aa segment including the RGD motif. By mutating the RGD to an RGE tripeptide, the infectivity of the resulting echovirus 9 clones for GMK cells is lost. Furthermore, we could show that synthetic peptides containing the RGD sequence influence binding of mouse-virulent echovirus 9 strains to GMK cells, whereas binding of apathogenic strains is not affected. These results suggest that the RGD motif is a significant factor affecting pathogenicity of echovirus 9 strains.

As part of a study of the molecular basis of pathogenicity of echovirus 9, the complete nucleotide sequence of the mouse-virulent echovirus 9 strain Barty was determined. Excluding the poly(A) tail, the complete RNA genome is composed of 7451 bases. The postulated open reading frame extends from nucleotide (nt) 741 to 7349 and predicts a polyprotein of 2203 amino acids (aa). As compared with the sequence of the echovirus 9 prototype strain Hill, which is apathogenic for newborn mice, 1492 nt are exchanged, leading to 9% divergence of the deduced amino acid sequence. The foremost difference between both strains is located at the C-terminus of the capsid protein VP1. In the case of strain Barty, an additional 10 aa fragment, including an RGD motif, is inserted.

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