Analysis Of HIV-1Transmission Relationship And The Transmitted Virus Sequence Characteristics

Background and objectiveThe randomized clinical trial (HPTN052) has proved that early initiation of ART (antiretroviral therapy) in HIV-infected partner can reduce the risk of HIV transmission among the HIV serodiscordant couples (SD)。In China, One national retrospective observational cohort study in serodiscordant couples has also make the critical conclusion. The ART can be used as prevention. HPTN052exclude the unlinked couples by molecular analysis, the prevention efficiency changed from90%to96%. However, the investigation in China has lacked this kinds of research. So identify the transmission relationship among the sera-concordant couples(SC) in China by molecular analysis, know about the extra-family HIV transmission phenomenon, has the important significance to support the results of the investigation in China. Guangxi province has been choose as the first study site, cause its high rate of HIV incidence and large numbers of both the serum-discordant couples and the sera-concordant couples. At the same time, based on the transmitted couples’samples, we continued some other researches including the identification of unique recombinant form, the identification ways of transmission direction and the analysis of transmitted virus sequence characteristics,which can provide the data and guidance for the China HIV-1transmission research and vaccine antigen design.Methods:1. The plasma samples were collected from the Guangxi province. There are47couples’ plasma from both sides of the partners and60samples from only one side of the partners. Among the47couples’ samples,20couples came from the the serodiscordant couple cohort and27couples from the seroconcordant couples. All the sample date choose as close as to the seroconversion date and the seroconcordant samples were from the first identified sample. 2. Extraction of the plasma RNA, amplification of the1.2kb pol region sequences (encodes all99amino acids in HIV protease and the first304amino acids in HIV reverse transcriptase) and env C2V5region, then identified the transmission relationship by the combination of the Phylogenetic trees and the epidemiology data. Some couples with indeterminate relationship or inconsistent within two gene results can be further determined by SGA analysis.3. The unique and transmitted recombinant form identified by the near full genome amplification and breakpoint analysis.4. All the couples which has the transmission relationship try to get the C2V5sequences by SGA. The transmission direction of the seroconcordant couples has been deduced by the simplified model based on the genetic distance within and between the partners.5. Analysis of the quasispecies data represented by the SGA sequences deduce the transmission related element.Results1. Analysis of HIV transmission within the couplesThere are135pol sequences and118env C2V5sequences obtained from all the samples. The transmission relationships of the42couples were identified by the combination of the Phylogenetic trees, the epidemiology data and SGA analysis. The results followed:Final linkage status was determined for37of42couples(37/42,88%), unlinked status was determined for3of42couples(3/42,7%),2of42(5%) was indeterminate. Selected four couples’samples were analyzed using SGA and identified2couples belong to the linked group while the other two still indeterminate. The couples’sequences clustering with the bootstrap value of80%or more were selected and the pairwise distance were calculated. The pairwise distance of pol is0.009and the env is0.05. The dominant subtype of the transmitted virus among37couples was CRF01_AE(29/37,78.3%), the second subtype is CRF08_BC(13.5%,5/37), CRF07_BC(2/37,5.4%), URF(1/37,2.7%). All the samples were genetyped by pol region:CRF01AE (108/135,80%), CRF08_BC (19/135,14%), CRF07_BC(6/135,4.5%), URF(2/135,1.5%).2. Identification of the unique recombinant form transmitted within one couple by near full genome amplification.Unique recombination forms(URF) has important significance in HIV molecular epidemic research. Two subtype HIV infected one person named superinfection and can create new recombinant virus which has the potency to spread. There is a unique recombinant form transmitted within the couple in this survey. Through the breakpoint analysis, the virus comprised of CRF01_AE as backbone and CRF08BC derived envelop protein. The transmitted virus has the trend of less PNGS number than the donor unrelated quasispecies. Amino acids in V3loop region has obvious selectivity.3. The approach to determine the transmission direction.There are total933sequences obtained from the28linked pairs, average16per subject. The approach to determine the transmission direction is based on the bottleneck effect and the virus evolve dynamics. Evaluated by the published data about30pairs, the accuracy of the approach is93.3%; detect the samples from this research which transmission direction has known, the accuracy is83.3%; using this way to detect16seroconcordant couples,13couples transmission direction are consistent with epidemiology data(81.2%) and3couples are not (18.8%).4. The characteristics of the transmitted virus.There are28linked pairs got enough sequences to analyze the number of transmitted virus,68%(19) couples were transmitted by single virus which is consistent with the severe bottleneck effect in heterosexual transmission way. Although one or two samples has the decline trend of PNGS number, there is no significant variance in PNGS number between the donor and recipient. The CCR5co-receptor tropism is dominant among the subtype CRF08BC recipients; the tropism of CRF01_AE is composed of59%CCR5and41%others(CXCR4and R5/X4). The high frequency of amino acid changes/exchanges between the donors and recipients happens at10sites located by HXB2. Some of them at conserved site maintain the spatial interaction on the three dimensional graph.Conclusion:This research is first time to investigate the transmission relationship of Guangxi couples by the genetic molecular analysis combined the epidemiology data and SGA. The results showed linked viruses in88%(37)couples, the first dominant subtype of the transmitted virus is CRF01AE and the second is CRF08BC. This data suggest that most of the seronegative partner is infected within family and the early ART and related involvement should be initiated to prevent transmission among the sera-discordant couples. Based on the model, we predict the seroconcordant couples transmission direction and most of the results (81.2%,13/16) is consistent with the epidemiology data. We first time identify a URF (CRF01_AE and CRF08_BC) by near full genome amplification. Monitor this kind of URFs to prevent new recombinant transmission events. The high proportion of non-R5tropism virus in recipient infected by CRF01_AE suggest fast HIV progression is associated with the transmitted virus tropism. The rule of amino acid changes between the donor and recipient is related to the virus escape and the conformation maintain.