| This study is divided into 3 parts. Part 1 is about the sequence characteristic and genetic variation of predominant circulating strains of the Human Immunodeficiency Virus Type 1 in Shandong province, part 2 is about the construction of HMA system for HIV-1 subtyping, part 3 is about the effect and the distribution of HIV-1 anti-gene (CCR2-64I, SDF1-3'A) among people infected with HIV-1 and their consanguineous contacters.First Part We use the measures of molecular biology, bioinformatics and epidemiology, to study the sequence characteristic and genetic variation of predominant circulating strains of the Human Immunodeficiency Virus Type 1 in Shandong province in 2002-2003, and analyse their effects on HIV-1 biological characteristic and HIV-1 spreading in this province, this is the first time to do this kind of research roundly in Shandong province. The results are as followings:1. There are 7 HIV-1 subtypes in Shandong province, which are B' (71/93, 77.2%), CRF01AE (9/93, 9.8%), CRF07-B'C. CRF0B-B'C(3/93,3.3% respectively ), B, C and CRF02_AG (2/93, 2.2% respectively). Subtype B' distributes in 10 cities, covering 4 kinds of groups of high risk population, especially paid blood donors, subtype B'/C distributes in 5 cities, mainly involving injecting drug users (IDUs), CRF01_AE and other subtypes distribute in several developed cities, mainly involving sex workers and whorehouse visitors.2. Gene divergence and amino acid sequence analysis of section env, gag, tat shows each subtypes' origins, B' strains are closely related to B.CN.RL42 and B'/C strains to 97CN54A and 97CNGX6F, CRF01-AE strains to 01_ae.th.90.cm2, 01_ae.jp.93.93j, while A/G and C strains are closely related respectively to standarded strains from Cameroon and Ethiopia, B strains to American B. Gene divergence of env of B' strains shows that subtype B' has come into Shandong for 7-10 years. From gene divergence and amino acid sequence, we also know that A/E strains' variation is the largest, B' strains take the second place and B7C is the least.3. Amino acid sequence analysis of section env shows V3 is more conservative than C2, V4. The top four peptides of V3 loop shows variously, besides GPGQ and GPGR, there were 6 kinds of them in all, there is a drifting evidence from GPGR to GPGQ in B' strains. Electron affinity of 11th and 25th Amino acid shows that the NSI strains are changing to SI strains. Gag and Tat proteins take on intrinsic invariableness at their constant section, but 2 strains at Gag's and 14 strains at Tat's important section develop variation which may lead to characteristic changes.4. Gag protein shows 4 signature Amino acid sites, Env shows 7 and Tat shows 4 of B' strains. This displays that B' strains have developed a special group in Shandong province. B' strains at Env section have 9 N-glycosites, among which 7 are lost at the various extent, at Gag section, 1 of 2 is lost.All above shows that there are many kinds of subtype and CRFs, and their genomes generate obvious variation, there is a quickening trend of HIV-1 transmitting in Shandong province, The results also provide important reference for study and production of vaccine and diagnostic agents, and can be a gist to make measures of HIV-1 prevention and control for Shandong province.Second Part We select 3-4 strains of subtype B', BVC and A/E , whose env sequence are most closely related to their consensus sequence as standarded strains, construct clones with their vl-v5 segment of section env. We construct 11 standarded plasmids in all for HMA subtyping system, we also explore the best ways of PCR, heteroduplex hybridization and polyacrylamide gel electrophoresis(PAGE), et al, for this HMA system. We use the sequencing subtyping ways to evaluate this system, show that subtyping rate of 3 types get to 94%, 90% and 90% respectively.Third Part We detect the anti-gene (CCR2-64I> SDF1-3 'A) by PCR and RFLP, and analyze the relationship between anti-gene and the disease progression using statistics method. The allelic frequencies of CCR2-64K SDF1-3'A were 23.64%x 23.94% respectively, and their polymorphism conformed to the Hardy-Weinberg balance distribution. The incubation periods for AIDS patients with SDF1-3'A1 ? CCR2-64I and wild type were 8.33±1.16> 8.94±1.17,... |
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