Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation

Abstract

Aim: To investigate the molecular mechanism of cell adaptation and rapid replication of hepatitis A virus strain H2 in KBM17 cells.

Methods: Virus of strain H2 at passage 7 was consecutively passaged in KBM17 cells for 22 passages, every passage was incubated for 14 days. Antigenic and infectious titers of every passage and one-step growth dynamics of passage 22 were determined with ELISA. Genomes of passage 6, passage 12, passage 18 and passage 22 were sequenced and compared with H2K7.

Results: During continuous passage of vaccine strain H2 at passage K7 in KMB17 cells, infectious and antigenic titers increased with the increase of passages, infectious titers at day 14 reached 6.77LgCCID(50)ml(-1) for passage 6 (P6), 7.0 LgCCID(50)ml(-1) for passage 12 (P12), 7.33 LgCCID(50)ml(-1) for passage 18 (P18) and 7.83 LgCCID(50)ml(-1) for passage 22 (P22), respectively. The one-step growth dynamics showed that replicating peak of P22 appeared at day 14 with infectious titers of 7.83 LgCCID(50)ml(-1) and antigenic titer of 1:1024. After passage 22 a new cell-adapted variant (P22) of H2K7 with rapid and shortened replication cycle from 28 days to 14 days was obtained. Sequencing and comparisons of genomes of P6, P12, P18 and P22 showed that mutational numbers in genomes of different passages increased with adaptive passages, and mutations scattered over the genome. In comparison with that of K7, P6 had only 6 nucleotides (nt) mutations, P12 had 7 mutational changes, in addition to 6 same mutations with P6, there appeared a new mutation in 5'NTR at nucleotide position 591 resulting in a nucleotide exchange from A to G. P18 had 10 nt mutations, among the 10 mutations, 7 mutational changes were same as with P12, three new mutational changes appeared in the genome, one in 5'NTR, one in 3C coding region, one in 3D coding region, at P22 there appeared 18 nucleotide changes in the genome, on the basis of P18,there occurred additional 8 nucleotide mutations, two in 5'NTR, three in 2C, one in 3A, one in 3C and one in 3D. The results suggested that although H2K7 was already an attenuated strain, the mutations of genome is not sufficient to completely adapt the KMB17, further mutations caused rapid replication adaptation.

Conclusion: 18-nt changes scattering over the genome are cooperatively responsible for further adaptation characterized by rapid and shortened replication cycle from 28 days to 14 days in KMB17 cells. The mutations in 2C coding region play more important role in increase of infectious titer than other mutations, the mutations in 2B coding region show less important role than it usually does in cell adaptation, nucleotide changes in 5' NTR seem to be not relevant to cell adaptation during initial stages (before P6), but do in late stages.

Figure 1

Sequencing strategy of HAV genome. A-I represent 9 amplified fragments of HAV