Study On The Differences Of HIV-1 Quasispecies In AIDS Patients Through Different Modes Of Transmissions

Backgrouds and Objectives Quasispecies that were firstly proposed by Eigen are used to describe genetic heterogeneity of the same organism. In the research field of virology, quasispecies are used to describe the heterogeneity of the same virus in one infected individual. It has been confirmed that quasispecies characteristic is the general biological of all RNA viruses. HIV-1 is one kind of retrovirus with highly active variations. Because transcriptase of HIV-1 are lacking in proof-reading function, variations such as recombinations, deletions, repetitions and inversions are very common and become more and more common as time goes on. Conformation sensitive gel electrophoresis (CSGE) developed from the method of heteroduplex analysis and now is a good tool for analyzing gene polymorphism. In this study, CSGE was modified. First, urea was added into the polyacrylamide gel by the proportion of 15% in order to make the straps thinner and clearer. Second, the concentration of formamide was raised from 15% to 20% and ethylene glycol from 10% to 15% so as to raise the sensitivity of CSGE. Third, driver was selected by pre-electrophoresis in order to make the distance between straps farther and easier to identify. Regarding the negative influence of non-specific PCR products on CSGE, PCR was strictly optimized so as to decrease non-specific PCR products maximatily. Sex and vertical transmissions are two important modes of HIV-1 transmission in which HIV-1 is transmitted from one host to another. Though theoretically HIV-1 in the new host should be exactly the same as that in the original host, it has been rebutted by more and more experiments. Our study was made on the differences of HIV-1 quasispecies in the individuals infected through vertical and sexual transmission by CSGE in order to understand more about the changes of HIV-1 in the transmission process and in the new environment of new hosts. Our results might disclose the regularity of HIV-1 quasispecies evolution and provide some experimental evidence for clarification of the mechanism of vertical and sexual transmission of HIV-1. Materials and Methods 1. Objects Plasma from HIV-1 infected individuals through vertical and sexual transmission. 2. Main reagents QIAamp Viral RNA mini kit(Qiagen, U.S.A); QIAGEN OneStep RT-PCR Kit(Qiagen, Germany); TA-Cloning kit(Invitrogen, USA), etc. 3. Methods To extract HIV-1 RNA from the plasma→RT-PCR for external DNA fragments(1 712 bp)→The second round PCR for internal DNA fragments(762 bp)→To purify internal DNA fragments→To insert the target DNA fragments into T vectors→To transform vectors into competent Escherichia coli→To identify positive clones by PCR→To perform CSGE→To select clones of major and minor quasispecies for sequencing→To analyze the sequencing results with the sequencing analysis softwares. Main Results 1. HIV-1 RNA was extracted from serum of the HIV-1 infected individuals of vertical and sexual transmission and reverse-transcribed to cDNA. We performed nested-PCR with cDNA as template. Finally we acquired env C2-V5 fragment of 2 pairs of vertical transmission and 1 pair of sexual transmission individuals. 2. There were about 300 colonies forming on the white-blue screening plate and 80% colonies were white colonies that were positive clones. 3. After mixed with driver, PCR products of positive clones were performed CSGE. The CSGE results indicated that there were significant differences between the HIV-1 quasispecies from individuals through either vertical or sexual transmission respectively. 4. Having analyzed the sequencing results of major and minor quasispecies of individuals of vertical and sexual transmission, there were significant differences between the nucleotide and amino acid sequences of major and minor quasispecies of individuals through either vertical or sexual transmission. 5. Analyzing the sites and styles of V3 loop mutations in the sequences of nucleotides and amino acids indicated that there were significant differences of nucleotide sequencesbetween the individuals of two pairs of vertical transmission and one pair of sexual transmission and of amino acid sequences between the individuals of two pairs of vertical transmission and one sexual transmission group. A deletion occurred at the start site of V3 loop nucleotide of the mother of one pair, so V3 loop amino acids could not be translated normally. Discussion 1. I acquired high quality images of electrophoresis by using the improved CSGE protocol that meaned this method was feasible for experimental studying of quasispecies, especially for the fragments from 400 bp to 1 000 bp(normal HDA couldn't analyze the fragments over 400 bp); 2. The subtypes of HIV-1 didn't change in the process of vertical transmission but there were significant differences between the quasispecies of HIV-1 in the individuals of vertical transmission which provided the experimental evidences for the theory of selected vertical transmission; 3. The subtypes of HIV-1 didn't change in the process of sexual transmission but there were significant differences between the quasispecies of HIV-1 in the individuals of sexual transmission which provided the experimental evidences for the theory of selected sexual transmission; 4. A deletion occurred at the start site of V3 loop's nucleotides in one mother of vertical transmission group, so I concluded that V3 loop of gp120 couldn't be translated normally or the products of translation couldn't work functionally.