Research On Molecular Epidemiology Of HIV-1 Infection Among IDUs In Yunnan

Acquired immune deficiency syndrome (AIDS) is a kind of immune deficiency disease which is caused by a chronic infection with HIV. Since the first case was identified in 1981, AIDS has become a serious global epidemic disease that has bad effect on the health of humankind. One of the significant characteristics of HIV is its high genetic variability, which makes HIV-1 generate a lot of subtypes and recombinant types during its propagation process, and presents the time and regional distribution characteristics. At present, intravenous drug users (IDU) in Yunnan Province of China are still the people who have the highest AIDS infection rate among the crowd. The epidemic has already begun to diffuse from the high-risk groups (IDU, as the center of diffusion) to general population. In view of the special status of Yunnan Province in Chinese HIV-1 epidemiology, it's necessary to research the actual HIV-1 molecular epidemiological characteristics of Yunnan Province. The research will not only help to know HIV-1 disseminative laws, to predict the future trend of the epidemic, but also have great significance on HIV-1 infection prevention and control as well as the manufacture of vaccine.Using an easy-and-fast RT-Nested-PCR method to amplify three regions (respectively named:gag(P17-P24), gag-Prot, RT; length:1160bp,885bp, 1010bp) of forty-two HIV-1 samples from KaiYuan Prefecture and Dali Prefecture in Yunnan Province. Ultimately, we got 31 (73.8%)pieces gag(P17-P24),41 pieces (97.6%) Pr and 30 pieces (71.4%) RT effective gene sequences from the 42 samples respectively. By segmentalizing or jointing effective gene pieces, we obtained 8 regions--P27, P24, Pr, RT, P17-P24, P17-Pr, Pr-RT and P17-RT. Phylogenetic trees analysis shows that both the methods of using P17,P24 gene separately and of using P17-P24 jointed regions have their own limitation for determining the subtype, especially there is a lack of effective ability to distinguish the target gene fragments which have high homologous similarity. Genotyping assay based on Pr,RT,gag(P17-Pr),gag-pol(Pr-RT) gene regions can effectively and steadily identify general subtypes and two kinds of B'/C recombinant strains mainly prevalent in China. But the method cannot identify the strains that have complex recombination structure precisely. Identification of genetic subtypes based on sequencing gene region P17-RT (2.6kb) is a good way to replace full-length sequencing, because the region has both completely different recombination sites of CRF07 and of CRF08-BC strains. Little workload in the lab will be enough to define all of subtypes and the recombinant types in China. Using this method, we found that the majority of IDUs were infected with CRF08-BC, (50.0% in Kaiyuan; 92.3% in Dali), whereas CRF07-BC was present in only low prevalence (3 of 42, 7.1%). Interestingly, there are six islates(14.3%), that branch out on the top of the phylogenic tree. These divergent viruses are probably derived from some novel B'/C recombinants, temporarily defined as URFs-BC.We had a focused analysis on 27 gag-pol (2.6kb) gene regions which are highly conservative to do some further study on the breakpoint distribution state of the HIV-1 recombinant strains among IDUs in Yunnan. By Simplot software analysis, we've got the result:CRF08_BCs and CRF07_BCs were comprised of subtype C throughout the majority of 2.6kb genome with two and three distinct B'subtype segments respectively. Moreover, these six URFs-BC appeared to be different recombinants, which showed structural similarity to both CRF07-BC and CRF08-BC in different regions of the gag-pol genome. The breakpoint distributions across the six HIV-12.6kb genome regions were scanned against a pair of parental strains of CRF07-BC and CRF08-BC. Three relative breakpoint regions were identified in sample DL310 at positions 2448-2472nt,3109-3207nt and 3250-3372nt(nucleotide numbering relative to HXB2). For the sample KY104, the first breakpoint is at approximately 1534-1619nt, while another breakpoint at position 1654-2134nt. Sample KY137 also presented two relative breakpoints at about 1612-1619nt and 1654-1968nt. Breakpoints of sample KY117 and KY118 are at 1612-1619nt, 2170-2188nt,2617-2943nt,3069-3255nt and 1425-1507nt,2067-2134nt,2617-2712nt, 3207-3255nt, respectively. Finally, sample KY139 has the breakpoints at positions 2067-2092nt,2397-2511nt and 2601-2616 nt.By further analysis of Explorotory trees and information sites, we have the finding that the Bootstrap value of most recombinants is more than 70% relatively, and the Chi-Square (Pearson) tests boast outstanding statistical significance. It can prove to be true that 6 samples belong to a new URFs-BC, which is likely to be generated by the recombinants between the CRF07-BC and CRF08-BC spreading in China.In summary, a simple and effective assay for genetic subtyping is established. Using this method, the systematic molecular epidemiology of HIV-1 infections among IDUs in Yunnan province have been investigated in our study, the observation will provide an important data for the therapy, prophylaxia and vaccine research of HIV-1.