The Molecular Epidemiological Study On HIV Prevalent In Shenzhen And Construction Of Infectious Clone Of Dominant Strainin In China

Chapter 1 Molecular epidemiological study on HIV prevalent in Shenzhen cityThe first HIV-positive patient in Shenzhen was found in 1992.After that,the number of HIV / AIDS cases reported in Shenzhen has increased year by year,with an average annual growth rate of 24.7%.The dominant transmission route also changed from IDU in the early time to sexual transmission,which suggesting that HIV has spread out of high-risk populations into general population.The emergence and rapid spread of HIV in Shenzhen City has caused an important public health problem.Considering the frequent flow of people in Shenzhen,the complicated characteristics of population structure,the large number of population not living with spouse,it is urgent to fulfill epidemiological survey on HIV prevalent in Shenzhen city.OBJECTIVE: To fulfill epidemiological survey on HIV prevalent in Shenzhen area.METHODS: HIV positive cases newly repored in 2015 were randomly selected.The corresponding demographic information was investigated and peripherial blood samples were collected.Genomic DNA was extracted by using the nucleic acid extraction kit.HIV gag,pol gene fragments were amplified by using nested / reverse transcription PCR method.The amplified fragments were purified and sequenced.All of the sequenced segments were assembled by using Contig Express software.The gene fragments were submitted to Los Alamos HIV Databases to analyze subtypes and construct subtypes of ML trees.The full length of URF virus was amplified and sequenced.Our sequences were aligned with HIV sequences from the whole nation before constructing the phylogenetic tree to explore the origin of Shenzhen sequences.Pol gene sequences were submitted to the Stanford University drug resistance database to detect drug resistance sites.Enzyme-Linked Immuno Sorbent Assay was used to detect HCV antibody.The epidemic of HIV prevalence in Shenzhen in 2015 was compared with that in 2013 to explore the tendency of HIV prevalent in Shenzhen.Phylogenetic tree based analysis was used to detect peoples who were infected by homosexual activity but reported through heterosexual contact.RESULT: The 250 newly reported HIV-postive individuals were enrolled.Among them,88.4% individuals were male;the HIV positive participants were mainly composed of young and middle-aged people.Among them,individuals of 15²9 years old composed of 49.2% of total people;96.8% individuals were infected through sexually contact,among of them,52% individuals were MSMs.The dominant subtypes were CRF01_AE,CRE07_BC and CRF55₀1B,accounting for 31.96%,37.90% and 10.50% of total infections respectively.The proportion of URFs was 7.76%.Primary resistance was detected in 0.046% individuals.HCV nuclic acid was detected in 7 individuals?2.8%?.In the phylogentic trees,HIV CRF01_AE strains isolated in HESs and MSMs grouped into separated clusters,while CRF07_BC strains distributed equally.The comparation of characteristics of those participants collected in 2015 and 2013 showed that the proportion of 15 to 29-years-old cases in 2015 was significantly higher than that in 2013?P <0.05?.The distributions of the proportion of HIV subtypes in those two yeare were similar..The ratioes of drug resistance were 0.9% and 2.74% in 2013 and 2015 respectively.The total numbers of HCV co-infections were 23?9.2%?and 7?2.8%?,respectively,which were mainly found in IDUs and HES.No significant difference between 2013 and 2015 was identified.Approximately 11% males who were infected by CRF01_AE strains and reported to beinfected through heterosexual contact were inferred to be infected through homosexual contact by using phylogenetic tree analysis.CONCLUSION: HIV prevalent in Shenzhen was mainly identified in the males who were infected throgh sexually transmission.Furthermore,the age of HIV infected people has become younger and younger.Diverse distribution of HIV subtypes were found in Shenzhen City.High ratio of URFs was found.High ratio of men who reported to be infected by HIV through heterosexual contact were actually infected through homosexual contact.Chapter2 Construction of Infectious Clone of Dominant HIV strain?CRF01_AE?in Shenzhen citySince the first CRF01_AE infected cases identified in 1994,CRF01_AE strains has spread quickly in the country and now become one of the dominant strain in China?except the southwestern region?.In some provinces,more than 80% of new HIV infections were caused by CRF01_AE strains.Due to the rapid increase of HIV infections through sexually contact,the proportion of CRF01_AE subtypes will also increase.In Shenzhen,CRF01_AE is also one of the dominant subtypes.Hence,it is urgent to study on the epidemiology and pathogenesis of CRF01_AE.The technology of infectious clon is not only a powerful tool to determine the biological effects of virus gene,but also an effective means to study the pathogenesis of HIV virus and the human immune response.Therefore,it is significant to construction the CRF01_AE infectious clone to study the pathogenicity mechanism of CRF01_AE.OBJECTIVE: To construction the infectious clone of CRF01_AE and further study the pathogenesis of that.METHODS: First construction the semi-molecular clones used by semi-nested PCR and T-vector.Second,the semi-molecular clones was digested with Aar I and Xhol I,respectively.And the two gene fragments was linked by T4 ligase to contruction the full-length infectious clone.Third,the full-length clones were transfected into HEK293 T cells to obtain the derivatized virus of CRF01_AE infectious clones.Then the derived virus infected the MT2 cell,and determined the value of p24 and TCID50 using the culture supernatant of MT2 cell.Forth,the replication kinetics curve of the derived virus on MT2 was plotted on the basis of the amount of p24 as the ordinate and the time as the abscissa to comparison of its ability to replicate with wild-type viruses.The last using U87 cells which express different receptors to determine the tropism of derived viruses.RESULT: The full length clones of CRF01_AE were successfully constructed after PCR,digestion,ligation and transformation.The length of the clone was 13630 bp.The sequence of the virus was 9702 bp,with 15 independent open reading frames.The full-length clone plasmid was transfected into 293 T cells.The p24 of cell culture supernatant was 0.375 ug / ml.Then transfected virus was infected with MT2 cells again.The obvious cytopathic effect was observed.The p24 was 19.1ug / ml,and the value of TCID50 was 15588.The replication kinetic curve of the derived virus was similar with that of the wild strain virus.The tropism of the derivatized virus is double-tropism virus which was determined by U87 cell.CONCLUSION:We have successfully constructed a CRF01_AE infectious clone that was completely derived from wild-borne viruses and has similar replication capabilities.And the clone provid a powerful tool for studying the pathogenesis of the virus and the study of the vaccine.