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Molecular Cloning of cDNA from Hepatitis A Virus Strain HM-175 after Multiple Passages in vivo and in vitro

Virus Laboratory, Fairfield Infectious Diseases Hospital, Fairfield, Victoria 3078, Australia

Hepatitis A virus (HAV) strain HM-175 was passaged six times in marmosets, 59 times in cell culture and purified from infected cell culture supernatant fluid. The viral RNA was extracted, copied into cDNA and the cDNA:RNA hybrids were cloned into the PstI site of plasmid pBR322. The cDNA clones were authenticated by hybridization to RNA extracted from HAV-infected cells and clones representing the 3' end of the genome were identified using a previously authenticated cDNA clone. The clones represented all but 29 bases of the HAV genome. They were compared to HAV strain HM-175 cDNA cloned from viral RNA after three passages in marmosets on the basis of restriction endonuclease mapping and DNA sequencing. No differences were found in either the presence or absence of restriction endonuclease sites using 33 different restriction enzymes. Sequencing of cDNA representing bases 29 to 1002 of the HAV genome revealed eight base changes all of which were within the 5' non-coding region.

Keywords: HAV, cDNA cloning, multiple passage

Received 16 December 1985; accepted 17 April 1986.