Molecular Evolution Of Hepatitis A Virus (H₂W Strain) In A Human Diploid Cell Line

Methods of molecular evolution may be useful for virologists to visualize molecular events of viruses evolved in a known/given environment. We aimed at employing hepatitis A virus (HAV) as a model virus to investigate the hotspots, direction, and velocity of evolution of HAV in the course of consecutive cell culture passage in human KMB₁₇ diploid cells. In this study, we serially propagated wild type H₂W in KMB₁₇ cells, applied cold adaptation in the middle of consecutive passage, and sequenced the full-length genomes of wild type H₂W and its six chosen progenies by directly sequencing RT-PCR products amplified from viral genomic RNA. Alignment comparison of sequences from H₂W with its six progenies and phylogenetic analysis of the whole VP1 region from H₂W, progenies of H₂W, and other cell culture adapted HAV was then carried out to obtain information concerning molecular evolution of HAV in the process of consecutive passage in KMB₁₇ cells. Phylogenetic analysis of H₂W and its cell adapted derivatives with other known HAV was also carried out to evaluate the direction of molecular evolution. Theresults showed that most hotspot mutations seemed to have finished at the vicinity of passage 5, which indicated that the velocity of mutation we observed was quite different from what described by other authors. About 648 mutations accumulated in the first 5 passages, among those 40 caused mutations in deduced amino acid. The hotspot mutations concerning adaptation of HAV to cells, e.g., 9bp deletions at ntl28 to ntl36,11 insert mutations at ntl76 to ntl77, point mutation at nt3889 causing Ala to Val mutation in amino acid, seemed to have finished before passage 5. When the time course of mutation concerning virulence determinant was investigated, the result showed that a crucial mutation at nt4222 causing Phe to Ser mutation in amino acid occurred between passage 10 and passage 14. There were as few as only 3 mutations accumulated through passage 5 to passage 30, which suggested that the cell adapted HAV genome was quite stable. The output of phylogenetic analysis of the whole VP1 region suggested that progenies of H₂W evolved closely to other cell culture adapted HAV, i.e. MBB, L-A-1, other than its progenitor H₂W. The technical information accumulated in this paper may help us to solve some crucial problems in the process of cultivating seed virus for live viral vaccine production, and provide technical assistance on monitoring the genetic stability of live viral vaccine, etc.Part I Molecular evolution of wild type hepatitis A virus in the process of consecutive cell passage in a human diploid cell lineObjectiveTo study the hotspots, time course and direction of molecular evolution of hepatitis A virus in a given environmentMethodsWild type H₂W was adapted to grow in human diploid cells KMB₁₇ for 30 passages. Six cell-adapted derivatives were chosen out for whole genome sequence analysis. Alignment of sequences from H₂W and its six derivatives were done by software package to evaluate the hotspots and time course of HAV in the long time of consecutive cell culture in KMB₁₇ cells. Phylogeny study of sequences obtained above was performed to compare to other known HAV sequences to obtain the direction of molecular evolution of HAV in the process of consecutive cell culture in human diploid cells.Results1. Nearly whole genomic sequences were obtained for H₂W and itsprogenies with GenBank accession numbers, H₂w: EF406357; H₂K₅: EF406358; H₂K₁₀: EF406359; H₂K₁₅: EF406360; H₂K₂₀: EF406361; H₂K₂₅: EF406362; H₂K₃₀: EF4063632. The Wild type H₂W switched its sub genotype for IA to IB in the process of cell adaptation in KMB₁₇ cells, which was based on diversity comparison of a 168bp fragment located in VP1/2A region.3. There were as many as 648 mutations in nucleotide accumulated in the first 5 passages. Among these mutations, 40 caused mutations in amino acid. There occurred several hotspot mutations concerning cell adaptation of HAV, e.g., 9 bp deletion at nt 128 to nt 136, 11bp insert mutation at nt176 to nt177, point mutation C3889U causing Ala to Val mutation in amino acid. Another hotspot mutation, U4222C occurred between passage 10 and passage 15, which caused Phe to Ser mutation in amino acid.4. The time course of mutations was summarized as following: There occurred 648 mutations in the first 5 passages. Only one mutation occurred throughout the genome from passage 5 to passage 10, and two mutations occurred from passage 10 to passage 15. No new mutations accumulated in the rest part of consecutive cell passage.5. Phylogeny analysis demonstrated that cell culture derivatives evolved closely to other cell culture adapted strains, e.g., L-A-1, MBB, otherthan its wild type progenitor H₂W.Conclusions1. The wildtype H-2w almost evolved its genes concerning adaptation to grow in cells before passage 5, and evolved its virulence determinants between passage 10 and passage 15.2. The cell culture adapted genomes of H₂W were found to quite stable, although many mutations occurred at the very beginning of cell adaptation.3. Molecular evolution may be useful in determining virulence gene of HAV and studying the condition for the occurrence of mutations within virulence determinants, as well as investigation of genetic stability of cell culture adapted HAV genome.4. HAV acted as an effective model for visualization of hotspots, time course and direction of evolution of virus in given conditions, because it can be cultivated in many types of cells and full length genomic sequence can be easily determined.Part II Cultivation of seed virus (strain H₂K) for live hepatitis A vaccine ObjectiveTo cultivate a seed virus for live hepatitis A vaccine via cell culture adaptation of a wild type HAV in human diploid fibroblasts.MethodsWildtype H₂W was adapted to grow in KMB₁₇ cells for 30 passages, among which cold adaptation was employed to attenuate HAV in the middle of consecutive passage. Molecular evolution was also adopted to determine the virulent gene of HAV, and to study the exotic conditions for attenuating HAV, the time course of mutations within virulence determinants, as well as the stability of cell culture adapted viral genomes. Animal test of a certain progeny virus to evaluate virulence attenuation and immunogenicity was then carried out according to the time course of mutations of virulence determinants.Results1. The derivative of H₂W grew more efficiently to a high stable titer after passage 5.2. Sequence comparison of virulent progeny and attenuated progeny demonstrated that nt4222 was the main virulence determinant. 4222 U/C mutation occurred between passage 10 and passage 14, and the mutation were quite stable throughout the rest consecutive passage in KMB₁₇ cells.3. Cold adaptation may not evolve the virulence gene, maybe cell passage acted as the main reason for attenuation, where cell substrate played a crucial role.4. The wildtype H₂W took about 5 passages to get adapted to KMB₁₇ cells. No more than 4 mutations were then accumulated thereafter in the derivatives of H₂W through 25 passages in KMB₁₇ cells.5. Monkeys inoculated with H₂K₂5 all developed anti HAV antibodies to a high titer. No abnormity was observed during ALT test and liver autopsy. H₂K₂₅ sounded to be an optimal seed candidate for production of live hepatitis A vaccine.Conclusions1. Wild type HAV got attenuated through longterm cell culture adaptation in human diploid cells KMB₁₇, among which a certain progeny was proven to be attenuated and of good immunogenecity by animal test.2. The genomes of cell culture adapted derivatives were quite stablethrough long time passage in KMB₁₇ cells, as well as the mutated virulence determinants.3. The 25th derivative of H₂W may be an appropriate seed candidate for live viral vaccine, which grew to a high stable titer in KMB17 cells and kept a genome with attenuated virulence and good immunogenicity as well as high stability.