| Hepatitis A Virus (HAV) known to cause acute hepatitis has significant implications on public health, and it is a "C" class infectious disease in China. There is only one serotype identified so far for HAV but six related genotypes. Hepatitis A is mainly transmitted via the fecal-oral route either by person to person contact or by contaminated food or water. Through analyzing the full length sequences of HAV in sporadic or an outbreak cases, it can provide the theoretical and technical support for the understanding of molecular evolution of HAV.In this study, five samples were collected from five provinces of China during2006-2009. The RNA was extracted from the serum, then amplified by RT-PCR and sequenced. The sequences were further analyzed to evaluate the aspects of homology, phylogenetic analysis, neutralizing epitope sites, selection pressure, recombination event, evolutionary history and population dynamics, etc.The homology analysis of nucleotide acid and amino acid sequences showed that the five sequences had the highest homology with IA subgenotype reference strains; Phylogenetic analysis based on complete genome and different segments exhibited that the five sequences belongs to IA subgenotype branch, but slightly differences in some segments; No amino acid mutations were found at the published neutralizing epitope sites, several unique amino acid substitutions were identified closed to the immunodominant sites; The nonsynonymous and synonymous substitution rates were compared to estimate the natural selection pressure, the calculated mean nonsynonymous/synonymous distance ratio was less than1. indicating there is a clear negative selection in these isolates, and the negative selection sites mainly concentrated on the nonstructural protein coding area; No intertypic recombination was detected, however, recombination between subtypes IA and IB was detected, intratypic recombination signal were also detected in the study using Simplot software, besides, it is believed that more recombination events will be detected in the future with a growing number of HAV full length genome sequence; Molecular evolution analysis using BEAST showed the estimated mean substitution rate of HAV full genomes in Asia was3.64×10-4substitution/site/year, the time for the most common ancestor (tMRCA) could date back to1055years ago, and estimated mean substitution rate of genotype I in the world was3.19×10-4substitution/site/year, the time for the most common ancestor was about266years ago.Through analyzing the full length genomes of HAV in China, the results indicated that most strains circulated in China were IA subgenotype. The amino acid was highly conserved, especially at neutralizing epitope sites. The isolated strains in this study were undergoing negative selection pressure, and experienced potential recombination in some regions. The research also exhibited the evolution trends of HAV in China and in Asia. Our study laid a foundation for the molecular epidemiological research of HAV.The viral quasispecies and population dynamics have a close relationship with drug resistance and cross-species transmission etc., but HAV quasispecies distribution studies are mostly at VP3, VP1regions. To further understand the genetic diversity in hepatitis A patients in China and in the full length genome, we cloned four clinical samples and analyzed the quasispecies distribution.Firstly, we cloned the VP3and VP1-2A genes in three clinical samples from2005-2007to evaluate the quasispecies spectrum, nucleotide mutation frequency, amino acid mutation frequency and Shannon entropy. Then another one sample was selected to evaluate the quasispecies distribution in whole genome sequence. The phylogenetic analysis based on VP1-2A regions showed that the all three sequences belong to IA subgenotype branch. In each sample a mean of20molecular clones were analyzed, the clones exhibited an intra-isolate nucleotide sequences identity of more than98.7%and amino acid sequences identity of more than96.1%. The nucleotide mutation frequency ranged from7.22×10-4to2.33×10-3substitution per nucleotide, and the amino acid mutation ranged from1.55×10-3to5.04×10-3per amino acid in three isolates. The nucleotide Shannon entropies ranged from0.38-0.98and the amino acid Shannon entropies ranged from0.26-0.77. The amino acid substitutions at the published neutralizing epitope sites were observed at two clones, and some clones lost the segments, which contains the published neutralizing epitope sites. Then we analyzed the complete genome of HAV with eight segments, the nucleotide mutation frequency ranged from7.26×10-4to2.30×10-3substitution per nucleotide, the amino acid mutation frequency ranged from1.38×10-4to4.27×10-3substitution per amino acid, the nucleotide and amino acid Shannon entropies both ranged from0.65-1.00. No amino acid mutations were observed at the neutralizing epitope sites in the clones.The results indicated that HAV quasispecies distribution inside the viral populations was presented in China. Many clones differ by only one nucleotide or amino acid, but at different sites within the genome, and some clones contained several deletions or insertions. The amino acid mutations were identified at neutralizing epitope sites in the clones, and more researches will be required to investigate whether this change would affect the neutralizing antibodies reaction. The full length genome analysis indicated that there was a wide difference of nucleotide mutation frequency in different segments, and high mutation frequency regions mainly at the nonstructural protein coding area. The research first conducted the quasispecies distribution analysis in full length genome, which provides the basis for the further HAV quasispecies study in the future.In conclusion, this study provides the information about the genotype distribution. selection pressure, neutralizing epitope sites mutations, recombination events and molecular evolution in China, and also analyzed the evolution status of different genotypes in Asia and genotype I in the world. The results will supply reference for the molecular epidemiology studies of HAV in the future. Furthermore, this study explored the quasispecies distribution of HAV in VP3-VP1-2A regions and whole genome sequence, which give us a primary understanding about quasispecies complexity in different areas. |
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