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《世界胃肠病学杂志：英文版》
>Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation
Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation
AIM: To investigate the molecular mechanism of celladaptation and rapid replication of hepatitis A virus strainH2 in KBM17 cells,METHODS: Virus of strain H2 at passage 7 was consecutivelypassaged in KBM17 cells for 22 passages, every passagewas incubated for 14 days. Antigenic and infectious titers ofevery passage and one-step growth dynamics of passage22 were determined with ELISA. Genomes of passage 6,passage 12, passage 18 and passage 22 were sequencedand compared with H2K7.RESULTS: During continuous passage of vaccine strain H2at passage K7 in KMB17 cells, infectious and antigenic titersincreased with the increase of passages, infectious titers atday 14 reached 6.77LgCCID50ml-1 for passage 6 (P6), 7.0LgCCID50ml-1 for passage 12 (P12), 7.33 LgCCID50ml-1 forpassage 18 (P18) and 7.83 LgCCID50ml-1 for passage 22(P22), respectively. The one-step growth dynamics showedthat replicating peak of P22 appeared at day 14 withinfectious titers of 7.83 LgCCID50ml-1 and antigenic titer of1:1024. After passage 22 a new cell-adapted variant (P22)of H2K7 with rapid and shortened replication cycle from 28days to 14 days was obtained. Sequencing and comparisonsof genomes of P6, P12, P18 and P22 showed that mutationalnumbers in genomes of different passages increased withadaptive passages, and mutations scattered over thegenome. In comparison with that of K7, P6 had only 6nucleotides (nt) mutations, P12 had 7 mutational changes,in addition to 6 same mutations with P6, there appeared anew mutation in 5′NTR at nucleotide position 591 resultingin a nucleotide exchange from A to G. P18 had 10 ntmutations, among the 10 mutations, 7 mutational changeswere same as with P12, three new mutational changesappeared in the genome, one in 5′NTR, one in 3C codingregion, one in 3D coding region, at P22 there appeared 18nucleotide changes in the genome, on the basis of P18,there occured additional 8 nucleotide mutations, two in5′NTR, three in 2C, one in 3A, one in 3C and one in 3D. Theresults suggested that although H2K7 was already anattenuated strain, the mutations of genome is not sufficientto completely adapt the KMB17, further mutations causedrapid replication adaptation.CONCLUSION: 18-nt changes scattering over the genomeare cooperatively responsible for further adaptationcharacterized by rapid and shortened replication cycle from28 days to 14 days in KMB17 cells. The mutations in 2Ccoding region play more important role in increase ofinfectious titer than other mutations, the mutations in 2Bcoding region show less important role than it usually doesin cell adaptation, nucleotide changes in 5′ NTR seem to benot relevant to cell adaptation during initial stages (beforeP6), but do in late stages.
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