Study On Hepatitis B Virus Genotype In Napo County Guangxi

Background:Chronic hepatitis B virus(HBV)infection is a major global public health problem.More than two billion people,one-third of the world's population alive today,have been infected with HBV at some time in their lives and,of these,around 257 million remain infected.The lack of a proof-reading activity of the viral polymerase leads to a high rate of mutation during the replication of the HBV genome.New genotypes may be form with these mutations accumulate.At present,HBV can be divided into 10 genotypes.Each genotype can be divided into multiple subtypes.With the further study of HBV,more and more genotypes and subtypes will be found.Meanwhile,more and more recombinants between different genotypes or subgenotypes are reported.Guangxi is a region with high prevalence of hepatitis B virus in China.The genotype of HBV in this region is relatively complex.Among them,genotypes B and C are more common,and so do genotype I(recombinant)in some areas.Our team found in 2011 that 6 samples from 863 HBsAg carriers in Napo country,Guangxi,could not be genotyped or subgenotyped or have different genotypes in different amplication by PCR from a single sample,based on phylogenetic analysis of the S gene sequence.Objective:To determine the genotypes of undetermined genotype of HBV isolates above using cloning,sequencing and phylogenetic analysis of the complete genomes.Methods:The serum samples and the basic information of samples collected in 2011 was collected.For confirmation,we collected serum samples from the same individuals in 2018.Informed consent in writing was obtained from the individual.The serological markers of hepatitis B virus were detected using enzyme-linked immunosorbent assay(ELISA).The hepatitis B virus load was measured by real time PCR.DNA was extracted from serum by Pronase digestion followed by phenol/chloroform extraction.The full length HBV genome was amplified using nested PCR.Amplicons were cloned and sequenced.Sequences obtained were edited using Sequencher 5.1 software.The sequences were aligned to 48 HBV sequences of all known genotypes retrieved from GenBank using Clustal W and visually confirmed with the sequence editor Mega 6.0,Bioedit7.2.6,RDP4.Results:(1)General information:Eleven serum samples in total were collected from6 asymptomatic HBsAg carriers in 2011 and 2018.Full length HBV genome from eight of them were obtained.However,three of them were failed in PCR amplification.(2)Subject NP137:The carriers had two serum samples:NP137Q and NP137,collected in 2011 and 2018,respectively.Viral loads of the latter was567.71IU/mL.The serological results of both samples were the same:HBsAg positive,HBsAb negative,HBeAg negative,HBeAb positive and HBcAb positive.Fifteen isolates from each sample were picked and obtained full length genome from all of isolates.Phylogenetic analysis showed that the carrier was infected by genotype B(3 isolates),D(8 isolates)and 4 unassigned isolates in 2011 while all isolates from the sample collected in 2018 belong to genotype D.Twenty-three of the 30 isolates form a cluster branching out from other subgenotype D sequences,supported by a 100%bootstrap value.Almost all of the estimated intragroup nucleotide divergence over the complete genome sequences,by pairwise analysis between our isolates and the other HBV D1–D10subgenotypes,exceed 4%.Therefore,we propose this be of a new subgenotype,provisionally designated HBV subgenotype D11.Thirty-seven amino acids were found to be unique to subgenotype D11,compared to the other subgenotypes(D1-D10).Fifteen of them were found in all our isolates.The serotypes of all of 23subgenotype D11 isolates were ayw2.The PreC mutation from G to A at nucleotide 1896,which generates the codon 28 termination signal,was found in15 of the 23 isolates.However,we found that the base at the relevant position of these 8 isolates was T rather than C.The A1762T/G1764A basal core promoter double mutations,C1766T and T1768A were not found in any of the isolates.However,18 of the 24 isolates have a T1753C mutation.Genotypes in the sample collected in 2011 include 3 isolates of subgenotype B2 and 4 isolates of B/D recombinant.All recombinants have subgenotypes B2 and subgenotype D11 as the major and minor parents.The serotype of B2 subtype and the recombinants are adw2,and that of NP137Q-20 isolate is adw.(3)Subject NP911:The carriers had two serum samples:NP911Q and NP911,collected in 2011 and 2018,respectively.The serological results of sample NP911Q were HBsAg positive,HBsAb positive,HBeAg negative,HBeAb positive and HBcAb positive.The serological results of sample NP911 were positive for HBsAg,negative for HBsAb,negative for HBeAg,positive for HBeAb and positive for HBcAb.The viral load was 3.23?10~7IU/mL.Ten isolates from sample NP911were picked and obtained full length genome from all of isolates.Sample NP911Q was failed in PCR amplification.Therefore,the full lenth genome was not obtained.Phylogenetic analysis of sample NP911 showed that the carrier was infected by subgenotype I1.All of them have deletion mutation,G1896A mutation,A1762T/G1764A double mutations.(4)Subjects NP808,NP872 and NP634:The first two of them had samples collected in 2011(NP808Q and NP872Q)and 2018(NP808 and NP872),respectively.However,the latter had samples collected in 2011(NP634Q)only.They were all infected with subgenotype C1.Their serotype were adrq~+.G1896A mutation and A1762T/G1764A BCP double mutation were not found.(5)Subject NP422:There were two serum samples collected in 2011 and2018,NP422Q and NP422,respectively.However,they were failed in amplification of the whole genome and the whole genome sequence was not obtained.Conclusions:(1)Some isolates of subject NP137 belong to a new subgenotype named subgenotype D11.(2)Some isolates of subject NP137 belong to a new recombinant named B/D recombinant.(3)Genotyping using sequences from direct sequencing may not be suitable for mixed infection and cloning sequencing should be applied.(4)Due to the replication of virus vary with time,the genotypes in individuals infected by a mixture of genotypes may be same at different time point.(5)Cloning,sequencing and phylogenetic analysis of the complete genomes should be applied in individuals infected with recombinants or a mixture of genotypes.