| Hepatitis A is an acute infectious disease caused by hepatitis A virus(HAV)that remains an important public health issue throughout the world,spread largely by the fecal-oral route.There is one single serotype existed in HAV,which can be divided into six genotypes.The analysis of HAV sequences from sporadic or outbreak cases can provide effective and important information in transmission and tracking study.This study included two parts:Part 1:Next-Generation Sequencing and Whole-Genome Sequences Analysis of Hepatitis A VirusVP 1-2A junction region is most frequently used for genotyping in HAV molecular epidemiology,while strains from different geographic regions may share identical sequences.In this study,12 HAV serum samples were collected from four regions in China and their HAV sequences were divided into four groups,with each group share identical sequences in VP1-2A junction region.Next-generation sequencing were implemented to obtain quasispecies sequences of the isolates in VP 1-2 A junction region.Among all the 12 HAV serum samples,an average of 24352(21262-27285)HAV effective sequences were obtained,representing an average of 507(477-576)unique quasispecies.The dominant quasispcies in these 12 samples accounted for 41.26%-49.74%,while others accounted for no more than 1%.There are HAV serum samples collected from different place and time acted as controls in group G2 and G3,the HAV sequences average genetic distance within each sample or between samples of the unique quasispecies in each group showed no statistical significance(P>0.05).Phylogenetic trees were constructed using the top 20,top 100 and all the quasispecies sequences,no clear relationship was found between HAV strain from different serum samples.Strains in G4 were amplified by RT-PCR and sequenced.Then the sequences were further analyzed by genotyping,phylogenetic analysis and recombination analysis.The results showed that all the three HAV strains belonged to subgenotype IA,the nucleotide diversity in the whole-genome region was no more than 0.1%.It is reported that the nucleotide diversity of strains from the same outbreak could reach up to 0.31%,suggesting that the three strains(KH6,KH7,KH8)in this study may from the same outbreak.Phylogenetic analysis based on both whole genome and fragmented region showed that the three strains belonged to subgenotype IA,while there are some differences existed.No recombination event was found in HAV genome.With the complement of HAV sequences database,more HAV recombination events would be found in the future.The full-length VP1-2A regions of the three strains were cloned and sequenced,and 26 effective cloned sequences were obtained.The average genetic diversity within and between samples were 0.34%and 0.35%,respectively,which with no statistical significance(P>0.05),indicating that the genetic relationship among three strains was relatively close.The nucleotide and amino acid variation frequency of three strains in the VP 1-2A junction region were both higher than that in the partial VP1 region.The above results indicated that whole-genome sequencing can better reflect the sequence characteristics of the HAV strains,while in combination with field investigation and laboratory detection and analysis,could be able to do accurate tracking of HAV strains.Part 2:Genetic Characterization of HAV Strains in Hotan ChinaHAV is relatively stable in its antigenic structure and only one serotype existed,the antigenic neutralization sites were mainly located in structural protein VP3 and VP1 region.Hotan Xinjiang is located in the western part of China,its annual incidence of hepatitis A had been at a relatively high level throughout the country.Therefore,to determine the genetic characteristics of the HAV strains,especially the stability of the antigenic neutralization site regions,is very important for the control and prevention of HAV outbreak,and also useful for the widespread use of hepatitis A vaccines.In this study,we collected 43 sera from patients with acute phase hepatitis A in sporadic or outbreak cases during 2016 to 2017,and 2 samples of live attenuated hepatitis A vaccine.Viral RNA extraction,reverse transcription,and nested PCR amplification procedures were performed to obtain sequences of the structural protein VP3-VP1-2A region.The sequences were further subjected to phylogenetic relationships,nucleotide and amino acid homology,antigenic neutralization sites and selection pressure analysis.The results showed that all the 43 epidemic strains belonged to subgenotype IA.Their nucleotide and amino acid identities in structural protein VP3-VP1-2A region were 97.8%-100%and 99.3%-100%,respectively.Two vaccine strains belonged to subgenotype IB,and the nucleotide and amino acid identities between epidemic strains and two vaccine strains were 89.3%-90.2%and 99.1%-99.7%,respectively.All the epidemic strains and vaccine strains had no amino acid changes at the published antigenic neutralization sites.One amino acid substitution of Val(Val)?Ile(Ile)was detected at position 72 in the VP3 region,which is closed to antigenic neutralization site VP3-71 and worth further study.All epidemic strains were negatively selected in the coding region of structural protein VP3-VP1.The studies above described the genetic characteristics in VP3-VP1-2A region of the HAV strains in Hotan area,all the strains belonged to IA subgenotype,but there were many different strains existed.The amino acid sequences were conservative and found no substitutions in published antigenic neutralization site.In conclusion,VP 1-2A junction region of HAV is frequently used for genotyping and phylogenetic analysis.Accurate tracking can be accomplished using HAV whole-genome sequencing in combination with field investigations.The sequences of HAV epidemic strains from Hotan Xinjiang belonged to subgenotype IA and there were several different strains existed,no amino substitutions were found in antigenic nertralization sites,which is important for HAV molecular epidemiology investigation,as well as for the control and prevention of hepatitis A in this area. |
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