Study Of Inter-(sub)genotype Recombinants In Human Full-length Hepatitis B Viruses

Human Hepatitis B virus is representative species of the familyHepadnaviridae of the genes Orthohepadnavirus，known as the smallesthuman DNA viruses. Recombination is one of the main causes of HBVvariation, which may affect the pathogenicity and transmissibility even theenlargement of virus host, and change the treatment and prognosis inpatients. This dissertation reports the results with extensive analyses of thecomplet HBV genomes currently available in GenBank to indentify allpossible recombinants: Establish a standardized way to describerecombinant by "Genotype of Backbone genome (uppercase)/Genotypeof Insert genome (lowercase)"(such as B/c, D/a and C/a); Summary detailedinformation of recombinants, including hot or Cold Spots of recombinationbreakpoint、the characteristics about recombinants and the recombinationtendency of different genotypes; Propose first about "Circulatingrecombinant form and Sporadic recombinant form"to classify recombinantbased on the epidemic characteristics; Analysis of subgenotypecomprehensive about two parents genome; Try to analyze the evolutionaryhistory of all strains come from hybird B/c. The main results of this paper are as follows:Download all complet HBV genomes from GenBank by October2010,and deal with the data by standardization. Finally,3560complet HBVgenomes were obtained of length between3082bp to3384bp. Building localdatabase Seq-HBV by Access2007.Building a novel strategy of double subgentype representativescombined with Phylogenetic Tree to classify the3560complet HBVgenomes. The results show that:3450genomes belonging to (sub)genotypeA1-A7, B1-B8, C1-C10, D1-D8, E, F1-F4, G, H, I1-I2and J;110genomesbelonging to genotype A, B, C and D with uncertain subgenotype. Thelargest group of Genotype was from genotype C occupy the37.1%;Genotype B is next, occupy22.9%; followed by genotype D with14.6%,genotype A with13%, genotype F with2.3%, genotype I with1%, genotypeH and G with0.8%；The least is genotype J, only one genome. C1and C2arethe main subgenotype of genotype C, accounted for81.2%and12.5%respectively；Subgenotype B1and B2of genotype B accounted for76.5%and4.8%respectively; Subgenotype D1,D2and D3of genotype D ccountedfor44.2%,22%and17.7respectively; Subgenotype A1and A2of genotypeA, ccounted for51.7%and32%respectively.(Sub)Genotype show distinctgeographic distribution, on the other hand, a variety of genotypes are popularon one region.The78double representative genomes can be as reference genomes of (sub)genotypes base on an extensive analysis of accumulated HBV genomesequence date. Currently we propose the seven criterias of minimalrequirenents for defining genotype and subgenotype.Established a novel strategy of "Stepping fragment-genotyping" to findthe HBV Mosaic genomes from a large number of genome data. Therecombination breakpoints of the potential recombinants were fartherprecisely determined by Simplot. Two phylogenetic trees based on completgenome and mosaic fragment were inferred to farther determine thegenotype of each mosaic fragment using a70%bootstrap value cut off.Finally,918genomes were revealed as inter-genotype recombinantswith17hybird and61inter-genotype recombinants forms. Two hybird (B/iand D/c),22recombinants forms and648recombinants of which beingrevealed for the first time. Five cases of recombinants on one genotype wererevealed for the first time also.Composition and geographical distribution of recombinants.918recombinants belonging to different genotypes: Genotype A (30cases),Genotype B (743cases), Genotype C (74cases), Genotype D (63cases),Genotype E (3cases), Genotype F (1case) and Genotype G (5cases).New forms of22kinds with B/c (6kinds), c/B (7kinds), c/d and d/a ofthe two kinds, B/I and c/a etc. with one kind. We assign the subgenotypes oftwo parents genomes with each recombinants base on extensive analysis,trying to find the evolution clues of the genotype and recombinants. The five cases of recombinants in one genotype are come from Asia:including C1/c2(2kinds), D5/d1(2kinds) and D5/d3(1kinds). Ingeneral, the genotypes of two parents for one recombinant are poplur in local:strains of B/c are popular in southeast Asia, strains of A/d are popular inIndian, strains of c/d are popular in west China and Mongolia, tsrains of G/aare popular in Europe.Recombination Forms. This is the first attempt to classify therecombinants with Circulating recombinant form(CRF) and Sporadicrecombinant form(SRF) according to epidemic characters of strains. If thestrains of recombinants have the same form and come from differentcountries or regions, indicating that the recombinants spread in apopulation,we classified them as strains of Circulating recombinant form; Ifone or a few genomes sequences from the same specimens, it is a chance,with no evidence of further spread among people, we classified it as strainsof Sporadic recombinant form(SRF).Tendency of recombination. The genotype A, B, C, D, E and G are veryactive to recombine. Genetype B and C, A and D, C and D, A and E, E and Dand C, G and G are recombine each other frquently, but only one hybridwhich can inherit steadily, such as B/c, A/c, C/d, A/e and D/e, C/g and G/a,etc.; there aren’t recombination events of genotype B and D, B and E or Cand E. Genotype A and C easier to insert the genome as formes includingD/a, E/a, B/a, C/a, D/a,G/a and B/c, D/c, G/c, F/c; Recombination between Genotype G and other genotypes are occurs frequently; It is never involve inrecombination events of Gneotype H.Hot or Cold Spots of Recombination Breakpoint. The most popularregions of recombination appeared to be that from3’ of S gene to3’ of Pgene contain whole X gene, followd by the region of S gene and C gene.Different genotypes with a variety of ways to participate in recombinationand had differences recombination breakpoint. Region of nt2500-3000ongenome which rarely happen recombination events except C/b form.According to available information indicates that: It may not produce thestrains with high genetic stability when the recombination events appearedon the genome region with hot spots recombination breakpoint.This dissertation analyses all possible recombinants of the completHBV genomes currently available in GenBank. The results will be of benefitto explore the genetic and evolution mechanism of (sub)genotypes or hybird.Such Study are of great value in Basic Research and clinical trials.