Mutation Analysis And Spatio Temporal Dynamics Analysis Of The Whole Genome Of Hepatitis B Virus From Tibetans In Qinghai-Tibet Plateau

Objective:To analyze the genetic variation of hepatitis B virus(HBV)CD recombinants;to investigate the incidence and causes of surface antigen(HBsAg)and anti-surface antigen antibody(HBsAb)double-positive serum in HBV-infected Tibetans;to analyze the spatio-temporal dynamics of HBV/CD recombinant and evolutionary genetic features(including genome sequence origin,nucleotide substitution rate and population dynamics).Methods:Blood samples were collected from community population in the Tibet Autonomous Region and Hainan State,Qinghai Province using a multi-stage random sampling method.A total of 852 HBsAg positive serum samples were collected from seven cities in Tibet,and 411 cases of HBsAg-positive serum samples were collected in Qinghai Province.HBsAg positive serum samples were selected by geographical distribution of Tibetan population,then HBV DNA was extracted and primers were designed,and the full HBV and BCP regions were amplified by nested PCR,then they were sequenced and aligned,respectively.Recombinant analysis was performed on the obtained full sequences.Between different recombinants,the baseline information,genetic variations,and serotypes were compared;phylogenetic analysis was performed on different fragments in recombinant genomes.To compare the Amino Acid(aa)mutation rates,deletions,and genome-wide nucleotide mutation distributions of the two groups in the S region and PreS region,HBsAg and HBsAb double-positive serum samples from the Tibetan population were selected as the observation group,and HBsAg-positive/HBsAb-negative serum samples which were homogeneous in age,gender,viral DNA level,and HBeAg status were selected as the control group using a 1:4 ratio,The database contains full-length sequences of C2 subgenotypes,D genotypes and CD recombinants which were retrieved from NCBI,together with 185 sequences obtained in this study.Phylogeography analysis of D genotype fragments in CD recombinants and D1-5 subgenotypes;C genotype fragments in CD recombinants and C2 subgenotypes was performed using the BEAST software package,respectively.At the same time,CD1 and CD2 were analyzed under coalescent theory and historical changes in population size were inferred using Bayesian Skyline Plot,respectively.Results:One hundred and eighty-five full-length sequences were genotyped,most were CD recombinants(181/185,97.84%),there were 132 CD1 with nt1 0-799 as genotype D and the rest as genotype C;49 CD2 with nt10-1499 as genotype D and the rest as genotype C;four HBV/C2 subgenotypes were also obtained.The HBeAg-positive group had higher viral DNA level than the HBeAg-negative group in 185 sequenced samples(P=0.001);the HBeAg-negative group was also older than the HBeAg-positive group(P=0.006).The genotype C fragment of both CD1 and CD2 was closest to the subgenotype C2;the genotype D fragment of the CD recombinant(nt10-799)was closest to the subgenotype D4,while the CD2 nt800-1499 fragment was closer to subgenotype D1-D3 in the phylogenetic tree.The CD2 recombinant was mainly distributed in the Shanan and Rikaze regions which were on the border of India(P=0).The CD recombinant differed from the genotype D at amino acid position aa76,aa120 and aa129 in the S region of genome.The major serotype of HBV/CD recombinant was ayw2(175/181,96.7%).Compared to CD1,CD2 has more S207N mutations.Compare to results in previous study,HBV/CD recombinants have a lower incidence of the T1762/A1764 double mutation than genotype C,but close to genotype D.HBV/CD strains have a lower frequency of mutations in G1896A(11.1%vs.25.6%)and A2189C(11.1%vs.50.0%)than the C2 genotype.While mutation G1613A has a similar level between CD recombinant and subgenotype C2(8.9%vs.10.3%).The double-positive rate of 1263 HBsAg-positive samples from the Qinghai-Tibet Plateau was 2.14%(27/1263).The mutation rates in the full-length,N-terminal,MHR(aa100-169),"a" determinant(aa124-147),first Ioop(aa124-137),second loop(aa139-147)and C-terminal were significantly higher in the double-positive group than in the control group.The PreS deletion was significantly higher in the double-positive group than in the control group(P<0.001).There was no significant difference between two groups(P=0.19;P=0.89)in the aa mutation rates of PreS1 and PreS2.The distribution of C2002T,A2159G,A2189C,C2198A in the PreC/C region was significantly different between the two groups.Nucleotide point mutations in the PreS/S region C3189A and T825C,P region C930A were significantly different between the double-positive group and the control group Specially,PreS region C129T(S155L)was identified only in the control group;the PreS region C3189A(D103E)and S region T825C(V224A)mutations were identified only in the double positive group.D fragment(nt10-799)of HBV D genotype sequence and CD recombinants sequence could date back to southern Asia about 271 years ago(95%HPD 138-477)with an average evolutionary rate of about 1.26×10-4(95%HPD 5.92 × 10-5-1.93 × 10-4)/site/year.D4 subgenotype is the closest branch of CD1 and CD2,entering the Qinghai-Tibet Plateau around 1850(95%HPD 1743-1934)and dividing into corresponding segments within CD1 and CD2 around 1911(95%HPD 1845-1971).It seems CD1 was generated earlier than CD2,suggesting the formation of CD1(with D region nt10-799)at first and then CD2(with D region nt10-1499).The global spread of the C2 subgenotype fragment originated in eastern Asia,firstly moved to northwestern China and northern Asia,and then from east Asia to the Americas by further distributions(around 1988 and 1994).Conclusions:HBV/CD recombinants were the predominant genotypes prevalent in the Qinghai-Tibet plateau,and patients in the HBeAg-negative group had higher levels of viral DNA and faster viral replication.Age was one of the main factors of HBeAg seroconversion in the Tibetan population infected with HBV/CD.HBV/CD1 recombinant is of D4 and C2 subgenotypes;HBV/CD2 may have multiple recombination processes among D1-3,D4 and C2 subgenotypes.Based on the geographical distribution of the genotypes,the main sources of recombination for the formation of CD recombinants are likely to be China(C2 subgenotype)and India(D4 subgenotype).Compared with genotype D,the serotypes of HBV/CD recombinants had no significant change.The HBV/CD recombinants were all isolated from high altitudes areas,which may be due to similar genetic background of the highlanders or to the similar folk customs due to long periods of high altitude living.The incidence of HBsAg and HBsAb double positivity among HBsAg positive population in the Qinghai-Tibet Plateau is close to the results of most previous studies about different genotypes.In the whole of the S region and in each region within it,the rate of aa mutations was significantly increased in the double-positive group.S gene hypermutation is not a sufficient and necessary condition for serum double-positivity and could not fully explain HBsAg/HBsAb coexistence.PreS region variation may be a factor of double-positivity.S region mutation T825C(aaV224A),PreS region C3189A(aaD103 E)were present only in the double-positive group.These aa mutations lead to changes in protein structure and may be an important condition for double-positivity phenomena.The most likely origin of the HBV D genotype is in South Asia,where the prototype D genotype gradually spread from India to Central Asia and then to Europe,the Mediterranean region and Africa in the mid-1800s.In the process,the D1,D3 and D2 subgenotypes were differentiated in the mid-19th century.At the same time,the D4 subgenotype spread from India to the northeast direction in the mid-19th century,gradually forming the D4 subgenotype and recombinanted with the C2 subtype around the Qinghai-Tibet Plateau in about 1910,which coincided with the two times of invasion in Tibet by British forces.The spread of the major HBV D subtypes in Europe dates back to the early 20th century,including the period of World War I and World War II.This process may be related to the rapid spread and evolution of HBV due to trauma,blood transfusions and vaccinations,as well as organized mass population migrations during wartime.While the worldwide spread of the C2 subgenotype is related to the large numbers of Asian populations emigration from East Asia to developed countries in Europe and America in the 1980s and 1990s.