| Background:Acquired immunodeficiency syndrome(AIDS)is an infectious disease caused by human immunodeficiency virus(HIV)infection.Since the first case was repoted in the United States in 1981,HIV has experienced fast evolution and developed extraordinary global genetic diversity.Currently,the main strains circulating in the world are from HIV-1 M group,which has evolved into nine subtypes.Meanwhile,the frequent occurrence of recombination between different subtypes also contributed to the generation of 103 circulating recombinant forms(CRFs)and numerous unique recombinant forms(URFs).The emerging CRFs and URFs are the best circumstantial evidence for HIV-1 dual infection that defined as infection of the same individual with two or more genetically distinct strains,including co-infection(double infection occurring before antibodies are detectable in the blood)and super-infection(second infection takes place after seroconversion).According to the number of infected strains,HIV-1 dual infection can be divided into double infection and triple infection and so on.The occurrence of HIV-1 dual infection not only provided the condition for recombination,but also increased the chance of infection with drug-resistant strain and always associated with faster disease progression.Therefore,the study on HIV-1 dual infection is of great significance to the prevention of HIV-1.Men who have sex with men(MSM)have a high risk of exposure to HIV-1infection due to their sepecial sexual behaviors.The high prevalence of HIV-1 infection and the sexual characteristic(multiple sexual partners)increased the possibility of HIV-1 dual infection occurrence among MSM.Currently,MSM has been the main population with HIV-1 infection,accounting for more than 80%of newly reported cases in Beijing.Objectives:In this study,the MSM with HIV-1 infection in Beijing were enrolled to investigate the incidence of HIV-1 dual infection,analyze the relevant factors that would have impact on disease progression and thus establishing the HIV-1 dual infection detection method.The main objectives are to elucidate the occurrence of HIV-1 dual infection and the epidemic characteristic of HIV-1 recombinant strains,and thus laying foundation for the detection and prevention of HIV-1 among MSM.Methods:Firstly,146 MSM subjects with acute/early HIV-1 infection were recruited from Beijing Youan hospital between September 2010 and July 2012.HIV-1full-length gag and partial pol,gp41 genes amplification and direct bulk sequencing were performed to determine the viral genotypes and then elucidating epidemic characteristics of recombinant strains.Secondly,70 individuals from the Beijing PRIMO cohort were included in this study.Of these subjects,97.69 person-years were followed up and total of 281 plasma samples were obtained with 2-5 samples per person.HIV-1 gp41 and p24 bulk squencing sequences are used to screen for HIV-1 dual infection.The suspect cases with dual infection were further confirmed by phylogenetic analyses using within-patient single genome sequences.To evaluate the effect of dual infection on disease progression,the rate of CD4~+T-cell decline were compared between different groups.Finally,total of 96 different barcodes were respectively attached to the 281 samples through gp41 and p24 primers before sequencing using the PacBio Sequel System.A pipline of bioinformatic analyses was performed to identify the HIV-1 dual infection,and then the results were compared to that conducted by SGA sequences analyses.Results:(1)Totally,the information of HIV-1 genotype in 135 infected subjects was obtained.CRF01?AE was the most common genotype(51.9%,70/135),followed by CRF07?BC(23.0%,31/135),subtype B(14.8%,20/135),URFs(7.4%,10/135),CRF65?cpx(2.2%,3/135),and CRF55?01B(0.7%,1/135);(2)Among 10 subjects with URF infection,a novel CRF was identified and named CRF80?0107.The information of this novel CRF was recoeded in HIV database;(3)A total of 249 gp41 SGA-derived sequences was obtained from 10 subjects with URF infection.Nine of the 10 individuals harboring URFs were infected by onward transmission of URFs,while the remaining one individual was coinfected.(4)Among the 70 individuals from the Beijing PRIMO cohort,we identified 12dual infections including 9 double infections(2 co-infections and 7 super-infections),and 3 triple infections(1 co-infection and 2 super-infections).The overall incidence of dual infection was 12.28 per 100 person-years,and the incidence of double infection was up to 9.21 per 100 person-years.Notably,this is first report identifying the incidence of triple infection,which was 3.07 per 100 person-years.The incidence of superinfection was 8.19 per 100 person-years,and for co-infection,the prevalence reached 4.29%;(5)In the survival analysis,triple infection showed significant faster CD4~+T-cell decline(p<0.05);(6)The Pacbio sequencing method identified 12 dual infections(3 co-infections and 9 super-infections)using 193382 circular consensus sequences(CCS).Compared to the results conducted by SGA sequences analyses,three triple infections(YA199,YA310 and YA339)were identified as dual infections and one co-infection(YA286)was identified as super-infection by Pacbio sequencing method.Conclusion:(1)HIV-1 epidemic is complicated among MSM in Beijing,China.The identification of a novel CRF and the confirmation of URF onward transmission indicate that new recombinant strains have been prevalent;(2)The incidence of triple infection was high among MSM in Beijng.This study also described the first reported incidence of triple infection and demonstrated that triple infection is associated with faster CD4+T-cell decline.All these results indicated that the HIV-1 epidemic is complicated among MSM in Beijng,and the more tailored interventions are urgently needed to be implemented among this population subgroup;(3)A detection pipline of HIV-1 dual infection by Pacbio Sequel sequencing was established.However,the current next-generation sequencing(NGS)method based on the amplicons needs to be further optimized to effectively detect minor quasispecies. |
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