Distribution Of Hepatitis B Virus Genotypes And Characteristics Of Its Quasispecies In Uighur Patients With Chronic Hepatitis B In Xinjiang

Hepatitis B virus(HBV),a major human pathogen,infects more than 350 million people across the world,ranging from chronic hepatitis through liver cirrhosis to eventual hepatocellular carcinoma.HBV infection is a big problem all over the world.Genotypes and quasispeices are the general biological characteristics for HBV hetero-geneity.HBV genotypes and quasispeices may influence the course of disease and therapy.There are eight genotypes of hepatitis B virus(HBV)designated A to H based on greater than 8%nucleotide variation over the entire genome.The eight genotypes of HBV show a distinct geographical distribution.Genotypes A and D have global distributions (especially in Asia,Africa,Europe and American),genotypes B and C are predominantly in East and South East Asia,the prevalent genotypes in China are genotypes B and C,genotype E is predominant in West Africa,and the most divergent genotype F is found exclusively amongst indigenous peoples in Central and South America.The geographical location HBV H is restricted to Central and South America.Mutations of hepatitis B virus(HBV)viral genomes are normal biological events and result in the heterogeneity,based on less than 4%nucleotide variation over the entire genome,we named 'quasispecies'.The HBV DNA heterogeneity is ubiquitous all chronic HBV infected patients.Quasispecies is different with genotypes and serotypes,with important biological and clinical characteristics,playing an important role in the patho-genesis,drug-resistance,and clinical outcomes in HBV infection patients.Also,quasis-pecies may contribute to a revolutionary re-evaluation of the mutations of HBV DNA.Objective:1)To study hepatitis B virus(HBV)genotype distribution and its characteristic in Uighur patients with chronic hepatitis B in Xinjiang.2)To determine the complete nucleotide sequence of a HBV strain of genotype D from Uighur patient with chronic hepatitis B in Xinjaing.3)We further investigated quasispecies of HBV. Methods:1)We used type specific primers and PCR to detect HBV genotype of 127 patients with chronic hepatitis B among Uighur population from Xinjiang.Genotyping results were confirmed by PCR product sequencing.2)The complete nucleotide sequence of a HBV drived from a Uighur patient with chronic hepatitis B was amplified by PCR and cloned to conduct sequence analysis.Homology of the resulted nucleotide sequence with those of published HBV strains was assessed by using BIOEDIT and NCBI-BLAST, and complete nucleotide sequence analysis of the phylogenetic tree of 34 genotype D HBV strains conducted by the assistance of BIOEDIT and PHYLIP.3)We choose five Uighur patients with chronic HBV infectin,including chronic hepatitis B and liver cirrhosis,and one of those is acute exacerbation on chronic hepatitis B.We determine quasispecies of HBV reverse transcriptase(HBV RT)region by using PCR- Clone-sequence analysis and CSGE(conformation-sensitive gel electrophoresis).HBV RT region was amplified and cloned by nested PCR and TA cloning.33 clones from a single sample was measured by sequence analysis and CSGE to determine the different clonetypes.Results:1)Among 127 patients,the proportions of genotype D,B,C and B/D,C/D, B/C/D were 39.4%(50/127),22.0%(28/127),16.5%(21/127)and 9.5%(12/127),8.7% (11/127),3.9%(5/127),respectively.Genotype distribution showed no significant differences between male and female patients(x~2=8.058,P=0.623),in patients positive or negative with HBeAg(x~2=6.033,p=0.303),in patients with difference ages(x~2=3.137, P=0.679).2)To determine the complete nucleotide sequence of a HBV strain of genotype D from Uighur patient with chronic hepatitis B in Xinjaing.We found,the complete nucleotide sequence of Xinjiang HBV strain is 3174bp,and deletion of 9 bp be in nt1760 to nt1768(ATT AAA GGT).This Xinjiang HBV strain belongs to ayw2 subtype and D genotype,and no combination with other genotypes.The evolutionary relations of the complete nucleotide sequence of Xinjiang HBV strain is the closest to those of the two Turkey HBV strains out of the 34 genotype D strains published in GenBank.Its complete nucleotide homology is 92%～98%with the published sequence of genotype D HBV strains,91%with the published sequence of genotype D/C combination HBV strains(accession number AY862865)from China.3)We found,There does exist quasispecies phenomenon in HBV RT region in Uighur CHB patients in Xinjiang.The contribution of the quasispecies of this region changed during the clinical course of this CHB patient.The complexity and heterogeneity of quasispecies of this region were low at the time points of exacerbation of chronic hepatitis and high at the time points of recovery of chronic hepatitis,and were higher with lamivudine treatment than no treatment.The complexity and heterogeneity of quasispecies of RT region may be unity without under drug pressure,rtA222T mutation has been detected in RT gene in the predominant clones of HBV strain that can not be detected usually.Conclusions:1)genotype D are predominant in Uighur patients with chronic hepatitis B in Xinjiang.Genotype distribution among the same spectrum of disease with chronic HBV infection patients showed no significant correlations with HBeAg,sex and ages.2) we finished the complete nucleotide sequence of a HBV strain of genotype D from Uighur patient with chronic hepatitis B in Xinjaing.The complete nucleotide sequence of Xinjiang HBV strain is 3174bp.This Xinjiang HBV strain belongs to ayw2 subtype and D genotype,and no combination with other genotypes.3)We further investigated quasispecies of HBV.We found,There does exist quasispecies phenomenon in HBV RT region in Uighur CHB patients in Xinjiang.The contribution of the quasispecies of this region changed during the clinical course of this CHB patient.The complexity and heterogeneity of quasispecies of this region were low at the time points of exacerbation of chronic hepatitis and high at the time points of recovery of chronic hepatitis,and were higher with lamivudine treatment than no treatment.The complexity and heterogeneity of quasispecies of RT region may be unity without under drug pressure,rtA222T mutation has been detected in RT gene in the predominant clones of HBV strain that can not be detected usually.It is possible that the shift of quasispecies of HBV RT region is related with antiviral treatment and clinical courses.