Study On HIV-1 Molecular Transmission Network In Gansu Province And Construction Of HIV-1 Pseudovirus

Objectives:Because of the HIV-1 gene high mutation,the sequence of HIV-1 in every case is unique.Based on the differences in these HIV-1 sequences,transmission network of HIV-1 molecular can be constructed.One node can show the HIV gene sequence and relationships.A smaller distance between two nodes indicates a greater association on the two in HIV infection.So,we constructed a molecular transmission and analyzed the potential transmission relationships of AIDS in Gansu Province.Due to the particularity of HIV/AIDS transmission routes,the HIV or AIDS patients might not tell us how they infect HIV.Thus,HIV molecular genotypes and networks are constructed to reflect HIV/AIDS transmission truly.This study investigated the characterization of HIV-1 genotypes in Gansu Province.And we tested the more samples and then constructed HIV-1 molecular transmission network to analyze the potential transmission relationships between HIV-1 patients in Gansu Province.This study can provide gene information for HIV/AIDS prevention and control.Based on the results,we also constructed HIV-1 pseudoviruses of CRF01-AE,CRF07-BC,CRF55-01B and CRF65-cpx,for HIV-1 neutralizing antibodies,phenotypic resistance and related research in the future.Methods:1.Samples:Plasma samples from HIV positive patients with viral load over 1000copies per microliter were collected from July 2018 to December 2018 in Gansu Provincial CDC.All participants signed informed consent approved by the Gansu Provincial CDC.2.Viral RNA was extracted and the gag,pol,env genes of HIV-1 were amplified by nested RT-PCR.Nested RT-PCR products were verified by 1%agarose gel electrophoresis.Successfully amplified samples were sent to the Beijing Genomics Institute for sequencing.Then,the genotypes of HIV-1 were determined using HIV-BLAST tool of the HIV sequence database of Los Alamos National Laboratory in the United States.The Kimura's 2-parameter method was used to calculate the gene distances and the Neighbor-joining method was used to construct a phylogenetic tree by using MEGA 4.1.TN93 nucleotide substitution model was used to calculate the gene distances in HYPHY2.2.4 software.After determining the optimal gene distance threshold,the gene distance less than the threshold was selected,and HIV-1 molecular transmission network was constructed using Cytoscape 3.2 software for visual analysis.3.Viral RNA was extracted from HIV-1 positive plasma samples whose genotypes were CRF01-AE,CRF07-BC,CRF55-01B and CRF65-cpx.The full length of env gene was amplified by nested RT-PCR and were cloned directly into vector plasmid pEGFP-N1 to construct pEGFP-N1-env.Pseudoviruses were generated after co-transfection of pEGFP-N1-env and the backbone plasmid pNL4-3.Luc.R^-E^-?1:4?.The fluorescence expression of EGFP was observed by a fluorescence microscope 36 hours post co-transfection.The culture medium were collected and centrifuged at 2500 rpm for 10min after another 12-hour culture for pseudoviruses collection.Protein gp120 in cell lysis was detected by Western Blot.Luciferase were detected by Luciferase method to check the infectivity and titer of HIV-1 pseudoviruses.Results:1.Combining the sequence analysis of gag,pol,env,we successfully genotyped74 samples,while 8 samples showed different genotypes in gag,pol and env,making us undetermined the genotypes,which need to be confirmed by full-length gene sequence analysis.CRF07_BC was predominant in Gansu Province,which proportion is 56.8%?42/74?,followed by CRF01_AE?31.1%,23/74?,subtype B/B'?4.1%,3/74?,CRF55₀1B?4.1%,3/74?,CRF08_BC?2.7%,2/74?,CRF65_cpx?1.4%,1/74?.The characteristics of the phylogenetic tree based on gag,pol or env region were similar and the sequences of different samples were scattered on both sides of the reference sequence in the same cluster.In the HIV-1 molecular transmission network,the number of clusters was eight,number of both cluster and node is smaller,showing that the HIV-1 molecular transmission network was incomplex.Most samples possessed degree=1or 2.Among all the samples,GS-141 had the highest degree with a higher transmission risk.2.According to the result of double enzyme digestion,PCR and sequencing,pEGFP-N1-env for different genotypes were successfully constructed.The fluorescence expression of EGFP can be observed using a fluorescence microscope and expression of protein gp120 was verified using Western blot.HIV-1 pseudoviruses were successfully constructed and had infectivity.Conclusion:1.The distribution of HIV-1 genotype in Gansu Province is consistent with that in Northwest China,with CRF07_BC ranking the first,followed by CRF01_AE.And the epidemic trend of HIV-1 genotype in Gansu Province is consistent with that in China,with proportion of CRF01_AE gradually increasing.According to HIV-1 molecular transmission network,the transmission is uncomplicated.Due to the existence of people with high transmission risk,further surveillance to reduce HIV-1 transmission is still needed.2.HIV-1 pseudoviruses were constructed successfully.