Analysis Of HIV Transmission Network Among Men Who Have Sex With Men By Using Next Generation Sequencing

Objective:To analyze the relationship and HIV transmission network among men who have sex with men(MSM)by using next generation sequencing data,and provide laboratory evidences for searching new HIV infections and key HIV transmitters.Methods:The HIV infections founded in an MSM sexual partner detection project in a city of Yunnan province from 2016 to 2017 were enrolled as the research subjects.HIV nucleic acids were extracted from venous blood specimens.The env,gag,and pol gene fragments(lengths:625bp,1028bp and 1036bp)were amplified for sequencing and subtyping,while fragments(length:376bp,384bp and 387bp)were amplified for next generation sequencing.Before next generation sequencing,PCR products were sequenced for confirmation and gene libraries were built.After HIV quasispecies analysis,we analyzed the genetic distance and phylogenetic trees and transmission networks.And then explored the key HIV transmitters.Results:Among 38 source-infections,13 failed to mobilize their sexual partners for HIV testing,but other 25 mobilized 68 persons for HIV testing,in which 8 were HIV positive.The blood specimens from 45 out of the above 46 HIV infections were tested.Sexual partners presented the transmission pairs numbered 1 and 2,2 and 12/13,3 and 8,6 and 9,4 and 19,34 and 40.The new reported infections were accounted for 48.89%(22/45)while the previous reported ones were accounted for 51.11%(23/45).24 out of 45 infections received antiretroviral therapy before blood sampling,of whom 25%(6/24)had been treated for?3 months,29.17%(7/24)had been treated for 3 months to 1 year and 45.83%(11/24)had been treated for?1 year.(2)93.33%(42/45)PCR amplicons from all specimens were successfully sequenced while 3 were failed.The HIV genotypes were confirmed as CRF01_AE and CRF07_BC,accounting for 95.24%(40/42)of the total.The pairs of 3 and 8 were excluded from further study because of being confirmed as different genotypes.(3)At env and gag and pol gene regions,an average of 347585 and 400702 and 201117 effective sequences(range 23681?1669877 and 125523?1027642 and 18092?441024)were obtained from each specimen.An average of 533 and 652 and 495 unique quasispecies(range 23?2658 and 354?1035 and 57?895)were obtained.The next generation sequencing data supported the epidemiological information of HIV transmission between infections 34 and 40.(4)The HIV transmission networks of specimens with subtype CRF01_AE indicated 4,13 and 8 key transmitters by analyzing the data of env,gag and pol gene regions,respectively.The key transmitters identified by three gene regions data were numbered 4 and 17,while number 16,20,25,26,36 and 45 were identified by two gene regions data,and number 1,10,15,18,24,27 and 43 were identified by only one gene region data.These key transmitters revealed more risk of HIV transmission.The HIV transmission networks of specimens with subtype CRF07 BC indicated 1,1and 5 key transmitters,with numbered as 27,11,5,28,37,39 and 44,respectively.(5)The HIV transmission networks of specimens with subtype CRF07_BC indicated a more accurate threshold in gag and pol gene regions at 6.5%and 4.5%,respectively.Conclusion:Next generation sequencing data can reveal the relationship and HIV transmission network at the quasispecies level.The results suggested the HIV transmission relationship was much more complex than the clue of MSM HIV infections provided.This is helpful for optimizing the implementation protocol of current MSM sexual partner detection project,and identifying key HIV transmitters and searching more HIV infections.