A single-base deletion in the middle of the wild-type hepatitis A virus (HAV) HM-175 cDNA clone was discovered. This deletion resulted in a frame-shift mutation which probably accounted for the lack of infectivity of this full length cDNA clone. This deletion was corrected by oligonucleotide directed mutagenesis yielding a genome that was infectious when the 5' non-coding region of the cell culture adapted mutant was substituted for the corresponding wild type sequence. Analysis of the growth of chimeric HAV viruses differing only in the 5' non-coding region established that mutations in this region can impart a host range effect; viruses containing the 5' wild-type sequence were restricted in their replication in the simian CV-1 cell line compared to viruses containing the 5' sequence of the cell-culture adapted mutant. Thus, both the 5' non-coding region and the 2B/2C region of the viral genome contain important determinants of host range and replicative capacity in vitro. Greater than 95% of the sequence of a simian isolate of HAV was determined by direct sequencing of the polymerase chain reaction product obtained using specific primers and a homogenate of the liver of an infected African green monkey. The sequence differs substantially from that of any known human isolate of HAV. Preliminary studies demonstrated that this virus grows very efficiently in vitro in primary African green monkey kidney cells or in a continuous cell line of fetal rhesus kidney cells. The polymerase chain reaction was used to determine the sequence of the 5' non-coding and 2B/2C regions of the master seed pools of passage 21 and passage 32 lots of HAV candidate vaccine virus. A continuous simian cell line stably transformed with a plasmid containing the P2 region from the Sabin 1 strain of poliovirus was shown to support growth of HAV. In addition, the infectivity of the cell-adapted HAV mutant was increased 10 to 100-fold when assayed in the poliovirus P2 transformed cell line compared to the non-transfected parental cell line.
| Graff, Judith; Emerson, Suzanne U (2003) Importance of amino acid 216 in nonstructural protein 2B for replication of hepatitis A virus in cell culture and in vivo. J Med Virol 71:7-17 |
| Kabrane-Lazizi, Y; Emerson, S U; Herzog, C et al. (2001) Detection of antibodies to HAV 3C proteinase in experimentally infected chimpanzees and in naturally infected children. Vaccine 19:2878-83 |
