Characteristics Of HIV-1 Genotypes In Gansu Province In Recent Years And Meta-analysis Of HIV-1 Molecular Epidemics In Injected Drug Users In China

Objectives:Human immunodeficiency virus（HIV）is the pathogen of acquired immune deficiency syndrome（AIDS）,which can be classified as the HIV-1 and HIV-2 subtypes.Among them,HIV-1,which has resulted in prevalence of AIDS in the world,can be further divided into different subtypes and circulating recombination forms（CRF）.This study aimed to investigate the characteristics and genotype of HIV-1 based on the clinical samples in Gansu Province in recent years,so as to analyze the epidemiological data to provide reference for the accurate diagnosis,prevention strategies and treatment for HIV in Gansu Province.Methods:A total of 114 HIV-1 positive cases with the virus load of≥1000 that were retained by the Gansu Provincial Center for Disease Control and Prevention（CDC）from January 2016 to May 2018 were selected in this study for HIV-1 genotype study using Nested RT-PCR.In addition,the positive PCR products were verified by agarose gel and then sent to the Beijing Genomics Institute（BG1）for sequencing.Afterwards,the sequencing results were passed through the HIV-BLAST tool（Los Alamos National Laboratory,USA）.Moreover,the genotypes of gag,pol and env were initially identified based on homology,and the preliminary identification results were further confirmed through phylogenetic tree analysis.Besides,the Clustal X 1.83 software was employed for sequence alignment and homology analysis.A phylogenetic tree was constructed by means of Neighbor-joining using the Mega 4.1 software.On the other hand,the different standard reference sequences were downloaded from the HIV databases at the Los Alamos website（https://www.hiv.lanl.gov/content/index）.The gene distance between sequences was calculated according to the Kimura dual-parameter method in the distance program of Mega 4.1.A total of 19 samples were collected to analyze the HIV-1 near-full-length-gene（NFLG）sequence using the SimPlot software,including 6 samples of CRF55₀1B and CRF65_cpx selected in the previous study,10 positive samples in gag,pol and env genes,and 3 both positive in gag and env genes.Additionally,27 plasma samples with confirmed positive HIV-1 and the viral load of>1000 that were retained in the Gansu Provincial CDC were selected to investigate the characteristics of the HIV/AIDS resistant genotypes in Gansu Province.Results:A total of 114 HIV-1 positive plasma samples were detected by Nested RT-PCR,and the expression of 39 HIV-1 gag genes,24 pol genes,and 57 env genes could be detected.Meanwhile,expression of at least one of the gag,pol,env structural genes could be detected in 68 samples.Additionally,10 samples were detected in all the three structural genes,while 46 were not detected in any of the three structural genes.Our results suggested that the recent epidemic genotypes of HIV-1 in Gansu Province were dominated by CRF01_AE,CRF07 BC,CRF08 BC,subtype B,subtype C,CRF55₀1B and CRF65_cpx.Among them,CRF07 BC and CRF01_AE were predominant,which accounted for 41.18%and 27.94%,respectively.At the same time,the demographic characteristics of library information of the analyzed samples were as follows,men had taken up a higher proportion（85.29%,58/68）among the HIV positive people in Gansu province.Meanwhile,young adults aged 20-50 years were the most susceptible population,holding 82.35%（56/68）.Moreover,the distribution of HIV-1 genotypes in Gansu Province had displayed significant regional characteristics.To be specific,CRF01_AE was dominated in Linxia city,taking up 37.0%（10/27）,while CRF07_BC was predominant in Dingxi city,which held 58.8%（10/17）.When analyzing the HIV-1 near-full-length gene sequence（NFLG）in Gansu Province,the gene sequences of 6 samples were successfully amplified,among which,CRF55₀1B had not been reported in Gansu Province previously.Besides,NFLG was compared with each reference virus of HIV-1 sequence,which suggested the presence of similar recombinant patterns and recombinant breakpoints,as confirmed by the corresponding genotype.To analyze the resistant gene sequence of the pol gene reverse transcriptase（RT）encoding region,27 HIV-positive samples were selected,and the gene sequences were successfully amplified,in which 13 samples were BC-recombinant,10 were CRF01_AE and 4 were uncertain regarding their genotypes.Afterwards,the gene sequences were submitted to the HIV-resistant database（Stanford University）for drug-resistant mutation analysis.Among them,8 samples with drug-resistant mutations were discovered from the HIV-1 infected cases.Conclusion:CRF07 BC and CRF01_AE still take the predominant places in Gansu Province recently.Findings in this study can supplement the HIV-1 genotype information,and the epidemic CRF55₀1B and CRF65_cpx genotypes are found in Gansu Province.The distribution and epidemicity of the HIV-1 genotype have displayed regional characteristics.Additionally,there are HIV-1 drug-resistant genotypes spreading in Gansu Province.Objectives:Injected drug users（IDUs）have been blamed for early increases in the number of HIV-positive cases in China.Early in the epidemic,HIV was mainly prevalent among high-risk groups of IDUs in China.Over time,the primary HIV transmission mode has gradually shifted to the sexual route.The HIV genotypes transmitted through IDUs in China may control the primary HIV genotype in the epidemic in China.The HIV genotypes in IDUs were comprehensively analyzed to trace them to the source and establish their relationships to the AIDS epidemic in China.Therefore,a systematic review and Meta-analysis was conducted of HIV genotypes and their sources in China.Methods:The PubMed,Web of Science,Cochrane Library,China National Knowledge Infrastructure（CNKI）,China Biological Medical Database（CBM）,Wanfang and Weipu databases were searched from the date of database inception to May 2018.The following text terms and medical subject heading（MeSH）terms were used:（"human immunodeficiency virus" OR "HIV" OR HIV-1" OR "AIDS" OR"acquired immunodeficiency syndrome"）AND（"subtype" OR "genotype" OR"molecular epidemiology"）AND（"China" OR "Chinese" OR "location=province,e.g.,Yunnan"）AND（"intravenous drug users" OR "IDU"）.The included studies were managed in Endnote X7,the data were organized with Microsoft Excel 2007,and the data analysis was performed with SPSS 18.0 and MATLAB R2016b software.RevMan 5.3 was used to search the databases and screen the studies for inclusion.The Meta-analysis was performed with R 3.3.1 software.Heterogeneity was determined using Cochran’s Q test and the I2 statistic.A random effects model was used.Funnel plots and Egger’s tests were used to determine the presence of publication bias.Subgroup analyses were performed according to the study period,study area,and gene location.The Chi-square（χ2）test was used to analyze the differences in the proportions of genotypes between subgroups.A sensitivity analysis was performed to test the robustness of the results.Results:In total,7,149 publications were identified from seven electronic databases:1,307 from PubMed,1,002 from Web of Science,0 from Cochrane Library,2,496 from CNKI,1,195 from Wanfang,744 from CBM,and 405 from Weipu.According to the inclusion and exclusion criteria,882 duplicate publications were removed.In total,6,267 articles were screened,3,987 of which were excluded,and the remaining 2,280 papers were evaluated by reading the full text.Finally,forty-five articles with 2,765 cases that met the inclusion and exclusion criteria were identified and included in this Meta-analysis;these articles were published from 2005 to 2017.The overall genotype proportions were as follows:CRF07 BC 45.18%（95%CI:33.55%-57.08%）,CRF01_AE 16.00%（95%CI:9.39%-23.82%）,CRF08_BC 13.43%（95%CI:7.32%-20.84%）,subtype B/B’ 3.58%（95%CI:1.52%-6.24%）and subtype C 0.90%（95%CI:0.04%-2.43%）.Between 1991 and 2015,the most prevalent HIV-1 genotype in Chinese IDUs was CRF07_BC.The subgroup analysis based on time period showed that the overall prevalence of CRF07_BC and CRF08 BC fluctuated over time.According to the results of the subgroup analysis based on geographic regions,in the eastern region,CRF07_BC（63.26%,95%CI 50.87-74.89）and CRFO1_AE（21.70%,95%CI 11.92-33.24）were the predominant circulating viruses.In the central region,CRF07 BC（54.17%,95%CI 20.10-86.32）and subtype B/B’（23.44%,95%CI 0.02-64.29）were the major epidemic viruses.In the southeastern region,the distribution of HIV-1 genotypes was the same as in the eastern region,with CRF07_BC（45.30%,95%CI 23.77-67.75）as the most prevalent virus and CRF01_AE（39.84%,95%CI 19.64-61.96）ranked second.In the southwestern region,CRF08 BC（47.39%,95%CI 32.17-62.84）was the most common circulating virus.In order starting from the highest proportion,CRF07_BC（13.52%,95%CI 8.96-18.74）,CRF01_AE（6.89%,95%CI 2.15-13.49）and subtype C（2.56%,95%CI 0.00-8.41）were the three most frequently observed circulating viruses in southwestern China.Conclusion:The main epidemic HIV-1 genotypes in IDUs in China were CRF07 BC,CRFO1_AE and CRF08 BC;this finding remained consistent in the subgroup analyses based on geographic region and time period and is similar to the overall pattern observed in the current HIV-1 epidemic,which is dominated by HIV transmitted via the sexual route.