Study On The Emergence And Epidemic Characteristics Of Novel HIV-1 Recombinant Strains In Shenzhen,China,2011-2018

Human immunodeficiency virus（HIV）is divided into HIV-1 and HIV-2.HIV-1 is divided into M,N,O,and P groups.Among them,HIV-1 group M has caused widespread epidemic and transmission in the world.Due to the high genetic polymorphism of HIV-1,it has been found that HIV-1 M group includes at least 10 pure subtypes（A-D,F-H,and J-L）,and its subtypes are prone to recombination.118 circulating recombinant forms（CRFs）have been reported,and a large number of unique recombinant forms（URFs）have also been discovered.HIV-1 recombination is the main reason for its genetic diversity,and HIV-1 different genotypes may cause different disease progression in infected persons.Since the discovery of HIV,HIV-1 global infections caused by HIV-1 recombinant strains have risen rapidly from 9.3%（1990-1999）to 22.8%（2010-2015）.East Asia,including China,has the highest recombination among HIV-1 subtypes in the world,up to 80.5%.A study among men who have sex with men（MSM）in China shows that high levels of URFs（9 out of 10 cases）are spreading rapidly,suggesting the current prevalence of CRFs may be underestimated.Many URFs have actually formed CRFs and caused prevalence and transmission.Shenzhen is an important entry-exit port city in China.Due to a large number of people flowing and rapid economic development,HIV-1 has a complex subtype distribution and epidemic trend in Shenzhen.In previous study in Shenzhen,the research group found that more than 10 genotypes were identified in MSM population,which also provided more ideal conditions for the production of URFs in this area.Based on the HIV international Database and abundant HIV-1 gene data produced by the laboratory in Shenzhen in the early stage,this study analyzed the prevalence of HIV-1 recombinant strains in China,identified the genome structure of URFs,and clarified the mechanism and characteristics of the recombination.Objective:1.To clarify the actual prevalence of novel HIV-1 recombinant strains in China.2.To clarify the production and genome characteristics of novel HIV-1 recombinant strains in Shenzhen.3.To analyze the occurrence of dual infection and minor drug resistance among people infected with HIV-1 URFs strain in Shenzhen.Methods:1.Based on the HIV-1 pol（2253nt-3252nt）gene fragment sequence from China from the HIV International Gene Database（as of 2018）,identify potential circulating recombinant strains（pCRFs）and novel CRFs through subtype analysis,phylogenetic analysis,and cluster analysis,infer the genome characteristics of pCRFs and CRFs and the recombination time of new CRFs.2.Take newly diagnosed HIV-infected persons in Shenzhen as the research object,combined with background information,analyze sequences of HIV-1 pol region gene fragments（about 1.3kb）,and explore the HIV-1 URFs infection proportion changes in Shenzhen from 2011 to 2018,identify novel CRFs;randomly select some HIV-1 URFs plasma samples（N=126 cases）,extract viral nucleic acid（RNA）,and use Nested Polymerase Chain Reaction（Nested PCR）to amplify HIV-1 5’ and 3’half-molecules,respectively,splicing the Nearly Full-length genomes（NFLGs）,analyzing its genome characteristics,and discovering new CRFs.3.Using viral nucleic acid as template and Barcode labeled primers,the partial gene fragments of HIV-1 gag（p24:1186-1879nt）,pol（A:2147nt-2605nt;B:2525nt-2976nt;C:2808nt-3250nt）,env（gp41:7758nt-8795nt）were amplified by nested PCR,further purified and quantified.Based on the NGS to analyzed the occurrence of HIV-1 URFs dual infection and minor drug resistance in Shenzhen from 2011 to 2018,and clarified the recombination mechanism.Results:1.HIV-1 has a high degree of genetic diversity in China,and there are multiple potential CRFs and new CRFs.As of 2018,there are more than 30 HIV-1 genotypes that China has submitted to the international HIV gene database,with CRFs and URFs as high as 75.72%.Some HIV-1 CRFs prevalent in West Africa and Cuba,such as CRF06_cpx and CRF18_cpx,have also appeared in China,and some new recombinant strains such as CRF55₀1B（323,2.12%）developed rapidly in a short period of time.Gene phylogenetic analysis revealed 17 pCRFs whose genomes were mainly derived from CRF01_AE,B,and C subtypes.Nearly half（41.18%,7/17）of pCRFs were transmitted in MSM.The sequence composition analysis of pCRFs shows that pCRF4 and pCRF7 contain more sequences（16 and 9）,suggesting that they may have been prevalent and spread in China.The novel CRF（CRF115₀1C）was recombined by CRF01_AE and C and was divided into 11 segments.CRF115₀1C occurred around 2009.1-2009.7.2.Shenzhen has a high level of HIV-1 inter-subtype recombination,and the genome composition is mainly based on CRF01_AE and CRF07_BC as the parent strains.Shenzhen is dominated by HIV recombinant strains（94.29%）,which was higher than that national（75.72%）.It is worth emphasizing that CRF55₀1B,the new recombinant strain that was prevalent in Shenzhen in the past,has become the main epidemic strain in Shenzhen,second only to CRF07_BC and CRFOI AE.The main recombination sources of URFs（2.16%）were CRFOI-AE,B,and C,which mainly occurred in unmarried young males aged 30-39,and also mainly occurred in MSM.NFLGs amplification and sequencing obtained 19 HIV-1 URFs NFLGs sequences,all of which are based on CRFO1_AE,B,and C subtypes.Compared with reference sequences showed that both B and C subtype fragments originated from CRF07_BC,and the discovery of CRF109₀107 also indicated that a large number of second-generation recombinant strains with CRF01_AE and CRF07 BC as the parent strains have appeared in Shenzhen.3.Shenzhen has a relatively high rate of dual infection and second-generation sequencing is easier to detect minor drug-resistant strains.NGS analysis showed that the prevalence of dual infection among people infected with HIV-1 URFs in Shenzhen was 38.10%,and the results of multi-gene segment analysis showed that the main genotypes of HIV-1 dual infection were CRF01_AE and CRF01_BC.Based on the detection limit of 1%of minor drug-resistant strains,the incidence of minor drug resistance was 26.19%.First-generation sequencing and second-generation sequencing to detect protease inhibitors（PIs）,non-nucleoside reverse transcriptase inhibitors（NNRTIs）and nucleoside reverse transcriptase inhibitors（NRTIs）minor resistance rates were 0.00,11.90%;0.79%,3.97%（P=0.219）;0.79%,13.49%（P=0.000）.Especially in the second generation of NRTIs,the detection rate of minor resistance was significantly higher than that of the first generation（P=0.000）.Conclusions:1.Through the analysis of the HIV Database,it was determined that China has a large number of potential circulating recombinant forms（pCRFs）and novel CRFs,confirming that the actual prevalence of HIV-1 CRFs in China is underestimated.2.Among newly diagnosed HIV-1 infected persons in Shenzhen from 2011 to 2018,a large number of novel HIV-1 recombinant strains（URFs）were found.The genome of these strains is composed of CRF01_AE and CRF07_BC,and novel CRFs are prevalent.3.Next-generation sequencing can be applied to the study of quasispecies of HIV-infected people in different populations,can detect more HIV minor drug resistance sites,and can be used for HIV-1 dual infection epidemiological analysis.