Optimization Of CRF01＿AE Strains Genetic Distance Threshold For Inferring HIV-1 Molecular Network Based On Next-generation Sequencing

Objective:Acquired immune deficiency syndrome(AIDS),caused by human immunodeficiency virus(HIV)infection,is a major infectious disease that seriously threatens human health.Controlling its spread is one of the main tasks for epidemic prevention and control.Based on viral genetic similarity,HIV molecular networks can reconstruct HIV transmission history,monitor network expansion in real time,and assess an individual’s HIV transmission risk for precise intervention and control.One of the most important parameters for constructing HIV molecular network is the threshold of genetic distance(GD),which can infer the potential transmission relationship based on the similarity degree of the virus carried by different infected persons,and can distinguish the rapidly expanding transmission clusters and assist precise intervention..Due to the different evolution rates of different HIV subtypes,the existing molecular transmission network parameters of subtype B may not be applicable to non-B subtypes.Compared with Sanger sequencing technology,the distribution of quasispecies in the infected patients can be presented more accurately by using next-generation sequencing technology.Therefore,this study aims to apply next generation sequencing technology to optimize the GD threshold range of the common HIV molecular transmission network of CRF01_AE subtype in China,so as to provide experimental basis for scientific construction of HIV molecular transmission network and precise intervention.Methods:Sanger sequencing data of CRF01_AE pol sequences(HXB2:2,253-3,300 base pairs,bp)in base point and Next-generation sequencing data of partial CRF01_AE pol sequences(HXB2:2868-3320 bp)in every points were obtained from 59 samples of 12 transmission pairs enrolled from a men having sex with men high-risk cohort from 2009 to 2014.The GD between any two sequences was computed using the Tamura-Nei 93 model of Mega7.0.14.2,019 CRF01—AE pol sequences(HXB2:2,253-3,300 bp)and information on recent HIV infection(RHI)were also collected from newly diagnosed individuals in Shenyang from 2016 to 2019.CRF01—AE molecular networks were constructed by using different GD.Results:1.A total of 12 transmission pairs,23 patients(R2D3 is both recipient and donor),were included in this study.There were 59 sampling points.Among them,17 points are in acute phase infection and 6 points are in chronic phase infection.There were 4(6.8%)sampling sites within 1 year before the event,22(37.3%)within 1 year after the event,21(35.6%)within 1-2 years,9(15.3%)within 2-3 years and 3(5.1%)within 3-4 years.2.Propagate the quality of next-generation sequencing results of 59 sampling points through Fast QC(version 0.11.7).The mean scores of Per base sequence quality was 83%(range:73%to 92%)and the mean scores of Per sequence quality scores was 84%(range:78%to 89%).The average number of quasispecies per sample was 4(range:1 to 23),and the average number of reads per sequence was 49969(range:118 to 411775 reads).3.Any viral sequences from donors and recipients of the same transmission pair within 1-4 years after the transmission event.The mean GD between columns were 0.008(95%CI:0.007-0.009),0.011(95%CI:0.010-0.011),0.013(95%CI:0.012-0.014)and 0.023(95%CI:0.022-0.023)substitutions/site(subs/site).4.When GD<0.013subs/site,the GD value reflecting the true transmission relationship accounted for more than 88%.When GD=0.023 subs/site,the proportion of GD value reflecting the real transmission relationship was 40.4%,which was not acceptable.5.To construct the molecular transmission network of CRF01_AE in Shenyang from 2016 to 2019 using different GD thresholds.With the increase of GD threshold(0.001-0.021 subs/site),the proportion of RHI in the network gradually increased(16.6%-92.3%),while the proportion of RHI connections gradually decreased(87.0%-48.2%).The two curves intersect at 0.008 subs/site.Conclusion:The GD threshold range(0.008-0.013 subs/site)was obtained for the construction of CRF01_AE molecular transmission network,which could be used to identify individuals with potential transmission relationships within 3 years.This study provided important data support for selecting appropriate GD threshold to construct the molecular transmission network of CRF01_AE subtype HIV-1 and provided reference for optimizing GD threshold methods for other non-B subtype HIV-1 strains.