Study On The Variation Characteristics Of Tibetan HBV Quasispecies And Its HLA Restriction

Backgrounds and objectives:HBV(hepatitis B virus)in patients in the form of quasispecies,has long been confirmed by scholars.The diversity of HBV virus variation is not only related to the lack of correction function of polymerase during virus replication,which is prone to random mutation,but also related to the host immune state and the intervention of antiviral therapy.Different populations and patients in different disease stages will have different characteristics of HBV quasispecies variation.HBV quasispecies variations are closely related to the diagnosis,treatment and prognosis of hepatitis B disease.At present,the variants most research focused on are within HBV BCP region,preC/C region,preS/S region and RT region.For example,double mutations of A1762T/G1764 A in BCP region and mutations of G1896 A in preC region will lead to abnormal transcription of C gene,which will cause HBeAg synthesis disorder,and both of them are related to the occurrence of liver cirrhosis and liver cancer.Tibet,located in the plateau in southwest of China,is geographically and culturally isolated from the outside world.Among the Tibetan population in Tibet,the rate of hepatitis B infection is over 12%,much higher than the average rate among Chinese(6.1%).At present,researchers focused on Tibetan hepatitis B virus have found that C/D recombinant genotype is the dominant strain of Tibetan HBV,while genotype B and C is the dominant strain of Han.There are few studies on Tibetan C/D genotype HBV quasispecies till now,and it is not clear whether it has unique quasispecies characteristics compared with other genotypes.HBV in the human body will be affected by the selection of host immune pressure,virus antigen was presented by HLAⅠclasses and classⅡmolecules,then it stimulated CD8+ T cell and CD4+ T cell to response.Variations in epitope peptide will affect the recognition capability of T cells and cause immune escape.Therefore,different HLA molecules can drive different HBV variation,which means restrictive to HBV variation.The present studies are more about HLA Ⅰ class molecules limited epitope peptide,such asHBcAg 17-28 aa in HBV core protein is HLA-A2 restricted antigen peptide.Currently,there is no relevant study on HLA restriction in Tibetan HBV.This research took Tibetans infected with HBV as the objects of study,and attempted to explore the characteristics of Tibetan HBV quasispecies characteristics and the restriction of HBV in Tibetan’s HLA genetic background,aiming to deepen the understanding of Tibetan HBV quasispecies variation and its HLA molecular restriction.Material and method:A genotype C HBV sample(pAAV-HBV plasmid)was used as the reference sequence for the third generations sequencing of HBV for HBV quasispecies analysis methodology.Firstly HBV full genome was amplified from pAAV-HBV plasmid by PCR,then adding the same amount of plasmid amplification products in three separate sequencing unit(cell).The sequences was analyzed by BioEdit 7.0,MEGA 7.0 and other bioinformatic tools,and the error rate of third generations sequencing,as well as the reasonable threshold of genetic distance and mutation rate for quasispecies analysis were studied.In 2016-2017,24 cases of HBsAg positive patients were collected during the epidemiological investigation in southern Tibet,and 24 cases of Han hepatitis B infected patients were matched and collected in the outpatient department of infection in southwest hospital as the control group in 2017.HBV DNA was extracted from 100 ul serum,and the whole HBV genome was amplified by PCR.Then,the full-length HBV was sequenced using the third-generation DNA sequencing platform PacBio RS II.At the same time,human gDNA was extracted from blood cell,and SBT(Sequence Based Typing),an HLA typing method,was used to classify HLA-A,B,C,DRB1,DPA1,DPB1,DQA1 and DQB1 with high resolution.PacBio SMRT Tools were used to split sequencing data and generate CCS(Circular consensus sequences)sequences.Sequence editing was operated by BioEdit 7.0;Sequence alignment was achieved with ClustalW 2.1,Muscle 3.8,T-coffee 11.0.The nucleic acid replacement model was tested by Modeltest 3.7.Phylogenetic analysis was conducted with MEGA 7.0,the construction of evolutionary tree was conducted with neighbor-joining,and the reliability analysis of evolutionary tree was conducted by Bootstrip Method with 500 times of Bootstrip repeat.FigTree v1.4.3 was used to analyze the evolutionary tree.The recombination analysis was carried out by SimPlot 3.5 developed by Stuart C.Ray.Characteristics of viral quasispecies were described in two ways: Shannon entropy(Shannon entropy,Sn)and the mean genetic distance(d),calculation formula of Sn is: Sn =-Σ(piInpi)/InN,which means pi is the frequency of occurrence of the ith population sequence while N is the total population.The genetic distance was calculated by MEGA 7.0.TN+G(Tamura-Nei model + Gamma distribution)was selected for nucleic acid replacement model.Meanwhile,the corrected Nei-Gojobori method(Jukes-Cantor)+ G model was used to calculate Synonymous mutation rate(dS)and Nonsynonymous mutation rate(dN).What’s more,predictions of the affinity between HLA Ⅰ,Ⅱ molecules and HBV peptides were conducted by the artificial neural network online tools,NetMHC 4.0 Server and NetMHC Ⅱ2.3 Server respectively.Results:1.The third-generation sequencing platform PacBio RS II is feasible for the full-length HBV sequencing,with an average read length of 3.4 KB.The total sequencing error rate is2.13%,with insertion error as the main,followed by deletion and mismatch,which are 1.45%,0.62% and 0.06%,respectively.The longer reads was,the lower error rate of sequencing was.After removing insertions by bioinformatic methods,the total error rate dropped to 0.69%.In HBV quasispecies analysis,the threshold of genetic distance was set as 0.01,and the threshold of mutation screening was set as 3.5%.2.Tibetan HBV is dominated by C/D recombination type,and there are two recombination types: C2/D4 and C2/D3,the former is recombination of 1-750 nt fragment of D4 type and C2 type,the latter is recombination of 1-1500 nt fragment of D3 type and C2 type.C2/D4 was the dominant strain.3.C/D recombinant HBV has higher complexity(P=0.006)and non-synonymous mutation rate(P=0.006)in preC/C region than the control.C/D recombinant HBV variants mainly concentrated in the Enh Ⅱ area(1636-1744 nt),BCP area(1742-1849nt)and preS area(2848-152nt),including C1653 T mutations(44.4%),T1753V/A1762T/G1764 A triple mutations(44.4%),deletions of preS(33.3%),etc.The variation of Han control samples was concentrated in preC,S and X genes,including G1896A(35.0%),sS143T(70.0%),and xV5L(75.0%).4.Among the HLA Ⅱ molecular genotypes,Tibetans have more DRB1*12:01:01G(55.6% vs 5.6%,P = 0.008),DQA1*05-DQB1*03:01(77.8% vs 16.7%,P = 0.004)than Han,while the HLAⅠmolecular HLA-B*40:01 in Tibetans were less than Han controls(0% vs50.0%,P = 0.012).It is suggested that the genetic background of HLA in Tibetan is different from that of Han.5.Variations of 36aa(1479nt)in HBV X gene were more common in HLA-DRB1*12:01:01G samples(66.7% vs 14.3%,P=0.024),and variations of 268aa(3108nt)in P gene were more common in HLA-DQA1*05-DQB1*0301 samples(50.0% vs 5.9%,P=0.015).The former site may be located in the DRB1*12:01:01G restrictive epitope,while the latter may be located in the DQA1*0501-DQB1*0301 restrictive epitope.It is suggested that under the different HLA genetic background of Tibetan and Han,different HBV quasispecies variations will be driven.Conclusion:We explored the specific method of the third generation sequencing technology for the full-length HBV quasispecies analysis,and established the genetic distance and mutation threshold of the HBV quasispecies.It was found that C2/D4 recombinant HBV was the dominant virus among Tibetans,and the characteristics of HBV quasispecies variation in Tibetan and Han populations were discussed on the whole HBV genome level.Combining the results of high-resolution classification for HLA Ⅰ,Ⅱ molecules,we had a preliminary understanding of the differences in Tibetan and Han HLA molecules,and their restrictive on the HBV variants.