SUMMARYThe nucleotide sequence of cDNA from a high-passage, cell culture-adapted variant of hepatitis A virus strain HM175 was compared with the previously determined sequences of wild-type virus and two other cell culture-adapted variants. A total of 42 nucleotide changes were detected when the sequence was compared with wild-type virus. Five of these changes were common to all cell culture-adapted strains and a further two changes were shared by the strains that had experienced the greatest number of cell culture passages. The mutations were distributed throughout the genome coding for amino acid substitutions in regions 2B, 2C and 3D with silent changes in 1C and the 5' non-coding region. The possible relevance of these mutations to cell culture adaptation and attenuation is discussed.Hepatitis A virus (HAV) is a positive-stranded RNA virus which has been classified as an enterovirus within the Picornaviridae . Early attempts to isolate and passage HAV in cell culture were hampered by the absence of a cytopathic effect and the lack of a sensitive means of detecting viral antigen. Following the development of a sensitive immunofluorescence assay (Mathieson et al., 1977) for the detection of viral antigen, HAV was isolated in cell culture after several passages in non-human primates (Provost & Hilleman, 1979). Subsequently, primary isolation was achieved in a variety of cell cultures (Balayan et al., 1979; Frosner et al., 1979;Daemer et al., 1981). Serial passaging of HAV in these systems resulted in an increased yield of virus and a reduction in the time before maximum virus production (Provost & Hilleman, 1979; Frosner et al., 1979). Prolonged passage of HAV in cell culture has been shown to be accompanied by a decrease in virulence for experimentally infected animals and man (Provost et al., 1986;Purcell et al., 1984;Karron et al., 1988), but the molecular basis of these alterations in viral phenotype is not known.In an effort to define the genetic changes associated with adaptation to cell culture and attenuation of virulence, two groups have compared the complete nucleotide sequences of different cell culture-adapted variants of the HM 175 strain of HAV (Cohen et al., 1987 a;Jansen et al., 1988) with wild-type virus (Cohen et al., 1987c). Previously, we have described a partial nucleotide sequence comparison of HAV HM175 at passage level 59 (Ross et al., 1986;B. N. Anderson et al., 1988). The present paper compares the complete nucleotide sequence of this variant with both wild-type and the two other cell culture-adapted variants.Restriction enzyme fragments of the HM 175 cDNA clones depicted in Fig. 1 were subcloned into mp8 and mp9 derivatives of bacteriophage M13 and sequenced by the chain termination method (Sanger et al., 1977). All nucleotide changes from the wild-type HM175 sequence were confirmed by sequencing in both orientations in the M13 polylinker.The passage histories of the molecularly cloned variants ofHAV HM 175 are illustrated in Fig. 2. HM175m3 was derived from the initial human faecal...