Optimization And Application Of HIV Molecular Transmission Network Based On Evolution Of CRF01＿AE

Objective:By the end of 2019,it is estimated that there are 960,000 HIV(Human Immunodeficiency Virus)infected people/living with AIDS(Acquired Immunodeficiency Sydrome)in China.The government invests billions of dollars in AIDS prevention and control every year,of which 2 of 3 is used for free antiviral treatment,but there are still 1.51 million newly diagnosed HIV infections in 2019,and sexual transmission has become the main route of transmission,making it more difficult to prevent and control the HIV epidemic in China.AIDS prevention and control work still needs the guidance of new technologies and new methods.The rapid evolution of HIV has left a measurable footprint in HIV genome.Because of the high variability of HIV,the sequence of HIV virus carried by each infected person is different,so it can be used for epidemiological surveillance through phylogenetic analysis.In recent years,a simplified HIV-1 transmission network based on gene distance method has been increasingly used in analysis and surveillance.The transmission relationship can be inferred from virus similarity,from individuals in the molecular network pointed to undiagnosed individuals,and even uninfected individuals with high risk.In addition,molecular transmission networks can also prompt the occurrence of rapid transmission and guide public health to respond quickly and implement precise interventions to block new infections.The molecular cluster represents a group of individuals infected with HIV similar in gene sequence.It is a group of HIV infection with similar HIV strain,in which the node represents HIV infection or a fragment of HIV sequence,while the edge represents the potential transmission relationship.When the genetic distance between the two sequences is less than a threshold,the nodes of the two sequences are connected in the network.The HIV molecular transmission network is based on the similarity of this gene sequence.Therefore,the setting of the threshold is very important for the construction of the network,using different thresholds,the clustering situation is completely different.There is no unified standard for the application of threshold.Most studies refer to the studies on subtype B in Europe and the United States,and most studies use 0.015 as the transmission network threshold.It has been reported that the evolution rate of HIV-1 virus varies with subtypes,and the evolution rate of the same subtype is also different in people with different transmission rates.Lineage4 and 5 represent MSM(men who have sex with men)people in the main local epidemic CRF01＿AE subtypes.To determine the threshold of local transmission network,we need to understand the evolution of the main local epidemic strains of CRF01＿AE within individuals and the evolution of homologous viruses among individuals.Therefore,in this study,the CRF01＿AE subtype infection in Shenyang was taken as the research object,and the next generation sequencing method was used to analyze the evolution of the virus longitudinally with the individuals who were followed up since the acute phase.Combined with the evolution among 7 pairs of"transmission pairs"with direct transmission relationship,the appropriate threshold of CRF01＿AE was set,and the HIV molecular transmission network was constructed by using this threshold.The effectiveness of intervention measures such as the advance of antiretroviral treatment standards were evaluated retrospectively.It provides a theoretical basis and practical example for the development of HIV molecular transmission network and network-based intervention and other regional research in China.Methods:Subjects:Part 1:The subjects of this study were collected from the Red Ribbon Clinic of the first affiliated Hospital of China Medical University in Shenyang,Liaoning Province from 2002 to 2017.The diagnosis of acute HIV infection(AHI)is based on negative or incomplete Western blotting to confirm the experimental results and HIV-1 RNA test results are positive.A total of 22 cases of CRF01＿AE subtype single strain infection were included in the study.Part 2:The subjects of this study came from 60 self-reported"transmission pairs"in the Red Ribbon Clinic of the first Hospital of China Medical University from 2000to 2014.Part 3:The subjects of this study were 1669 untreated patients with HIV CRF01＿AE subtype diagnosed in the first affiliated Hospital of China Medical University from 2008 to 2016.All patients have signed informed consent and approved by the Ethics Committee of the first affiliated Hospital of China Medical University.Experimental methods:1.HIV-1 limiting antigen(Lag)avidity enzyme immunoassay(EIA)Recent HIV infection(RHI)was distinguished from chronic HIV infection(CHI)using the LAg-Avidity EIA kit(Maxim Biomedical,Rockville,MD,USA)according to the manufacturer’s instructions.The normalized optical density(OD)of each sample was calculated as OD of the sample divided by that of the calibrator.RHI was defined as OD≤2.0 in the screening test,and OD≤1.5 in the confirmatory test.2.Extraction of viral Nucleic Acid from Plasma and Nest PCR Amplification of Target fragment.2.1 The nested PCR for sanger sequence:Collected the whole blood of HIV infected patients and separated the plasma.Using QIAamp RNA Mini Kit kit,60μl HIV virus RNA,was quickly extracted from 140μl infected plasma by protocol according to the instructions.HIV-1 pol 1.0Kb partial gene fragments(HXB2:2147-3326)were amplified by nested PCR in two rounds.2.2 The nested PCR for deep sequencing sequence:Super Script(?)III First-Strand Synthesis Super Mix kit was used to reverse transcribe plasma RNA into cDNA,KOD high fidelity enzyme nested PCR amplification nest PCR method was used to amplify partial 0.45kb gene fragments of HIV-1 pol RT region in two rounds(HXB2:2868-3320).3.Deep sequencing.3.1 Purification and quantificationThe purification of PCR products is carried out according to the instructions of Beckman magnetic bead purification kit.PCR products were quantified by QUBIT fluorescence quantitative analyzer.The prepared library was quantified by KAPA q PCR kit.3.2 Library construction:According to the QUBIT quantitative results of PCR products,the library was constructed with input 150ng.According to the operation flow of Truseq Nano DNA HT Library Prep protocol,the steps are as follows:end modification and fragmentation,3’"A"tail modification,connection adapter,library amplification,library q PCR quantification,library standardization,then Miseq.4.Bioinformatics processing:Sanger sequence:the alignment of the sequence was applied to the manual correction of the HIV Align,of the online website database,and the reserved length of Bio Edit,was 1015bp(HXB2:2253～3268).NGS sequence:on the Windows operating system,use Oracle VM Virtual Box-5.2.22 software package to build a virtual environment,run QIIME2Core-2018.4(1525276946)to analyze the packets generated by deep sequencing,report the sequence and number of HIV-1 quasispecies in each sample,and remove<1%quasispecies.5.Phylogenetic analysis.5.1 Distinguishing subtypesThe maximum likelihood phylogenetic tree of sanger sequence was constructed using Fast Tree 3.0,the periphery was N subtype reference sequence,and the nucleotide substitution model was calculated by SH test(Shimodaira Hasegawa-like test,SH Test)embedded in the software of GTR+G+I,phylogenetic tree node support value.5.2 Determination of single strain infection.The maximum likelihood tree is constructed by the next generation sequencing sequence.if the infected sequence is all in the same branch and the support value of the cluster branch is greater than 0.8,multiple infections are excluded,and if the quasi-species at the baseline point is unique,it is considered to be infected with a single virus strain.5.3 Determine the direct transmission relationship.We formed the fas file of the sanger sequence of"transmission pair",several Shenyang CRF01＿AE sequences downloaded from HIV database and corresponding years of"transmission pair"sequence,and several corresponding year CRF01＿AE sequences obtained by our laboratory.Fast Tree was used to construct the maximum likelihood tree,GTR+G+I nucleotide substitution model,Shimodaira-Hascgawa(SH)test,and local support values were used to determine the reliability of the phylogenetic tree.Figtreev1.4 is used to edit the phylogenetic tree.6.Diversity calculation.Each sampling site generates a sequence set with original constituent ratio(accounting for more than 1%of quasispecies),and the gene diversity,in the group calculated by Within group mean distance of MEGA software is gene diversity.7.Divergence calculation.Each sampling site generates a sequence set with the original constituent ratio(accounting for more than 1%of the quasispecies).The gene distance between the baseline point and the late follow-up point sequence is calculated by between group mean distance in the MEGA software,which is the degree of divergence.8.Construction of HIV molecular transmission network.Use HIV-TRACE to build HIV-1 molecular transmission network(www.hivtrace.org).All sequences are aligned with a reference HIV-1 pol sequence,and the Tamura-nei 93 pairwise distances of each pair of sequences are calculated.Based on the optimal threshold,the molecular network is constructed,and the information of transmission clusters and nodes is extracted and brought into the follow-up analysis.9.Proportional detection rate(PDR),cluster growth predictor,and effective reproductive number(R_e)To describe the dynamics of a given cluster,three parameters—i.e.,PDR,cluster growth predictor,and Re—were calculated as follows.PDR for a given year(j)was calculated as the cumulative number of cases in the cluster sampled up to and including year j,divided by the cumulative number of cases up to and during the last sampling year(i),per observation time between years j and i.PDR≥2(i.e.,a 2-fold increase in size in 1 year)was considered as a significant change.Cluster growth predictor was calculated as previously described as the number of newly diagnosed individuals in a given year divided by the square root of cluster size at the end of that year.A declining curve indicated that a given cluster had a very low probability of causing an outbreak.R_e of each large cluster(≥10 cases)was estimated with the birth–death skyline serial model in BEAST v2.4.2.R_e represents the average number of secondary infections caused by a typical infected individual when only part of the population is susceptible.The value is often used to describe temporal changes of an epidemic in a population,with R_e>1 and R_e<1 indicating the growth or decline of the epidemic,respectively.10.Statistical analysisThe standard statistical test method was used for statistics and analysis.The classified data are tested by chi-square test or Fisher accurate test with SPSS v21.0(SPSS Company,Chicago,Illinois,USA).Graphpad v7.0 linear regression was used to fit the trend of viral divergence with time,t-test was used to compare the difference of gene distance between groups,Sperman rank correlation test of SPSS 21.0 was used to analyze the correlation among quasispecies number,gene diversity,divergence and estimated infection time,CD4+T cell count and VL.P<0.05,the difference was statistically significant.The chart is constructed with excel,SPSS,graphpad and R.Results:1.Study on the evolution of CRF01＿AE intra-individualsa).Screening of single strain infection in the cohort of acute infection:in the cohort of 200 cases since acute infection,we excluded those infected with an estimated infection time of more than 6 months from the baseline point,non-CRF01＿AE subtype infection and known multiple infection,and selected 44 cases of infection with at least two continuous sampling sites,and then we applied the quasispecies sequence obtained from the next generation sequencing.If the baseline quasispecies is1,it is suggested that it is infected with a single strain.Among the 44 subjects,we screened 22 cases with single virus infection.b).The number of quasispecies and diversity of CRF01＿AE single strain infection changed with time in the natural course of disease:among the patients with 15 or more sampling sites,there were three kinds of changes,first rising and then decreasing,rising and always being 1.c).Divergence analysis of virus quasispecies at different time after infection with single strain infection:the intra-individual evolution of all patients infected with single strain was no more than 0.015s.19 of the 22 patients with single strain infection did not exceed 0.01s/s.The average evolution rate of all infected patients was 0.0024±0.001s/s.After divergence in groups≤1,1-2,2-3 and>3 years was0.003±0.003s/s,0.004±0.002s/s,0.007±0.002s/s and 0.009±0.004 s/s,respectively.d).Analysis of the correlation between the number of quasispecies,diversity,divergence and infection time,viral load and CD4 in single virus infection:there was a positive correlation between the number of quasispecies and viral load at the follow-up point(r=0.252,P=0.095).Diversity was positively correlated with infection time and viral load(r=0.307 and 0.322,respectively,P<0.05),and divergence was also positively correlated with infection time and virus load(r=0.508 and 0.386,respectively,P<0.05).e).By comparing the evolution rate of CRF01＿AE subtype in China with that of subtype B in Europe and America,it was found that the evolution rate of subtype B in the United States and Sweden was significantly lower than that of subtype CRF01＿AE in China.The evolution rates of subtype B strains in the United States,Sweden and China were 0.0006±0.001 s/s/y,0.0012±0.001 s/s/y and 0.0026±0.002s/s/y,respectively.2.Study on the evolution of CRF01＿AE virus when it spreads among individuals.a).Screening and differentiate of"transmission pair":We collected 60 pairs of self-reported"transmission pairs"in the Red Ribbon Clinic of the first affiliated Hospital of China Medical University from 2000 to 2014.First of all,we excluded the"transmission pair"of non-CRF01＿AE subtypes and excluded the"transmission pair"of non-plasma samples.After that,the sanger sequence was used to exclude the"transmission pair"of unreal transmission relationship by phylogenetic analysis.Then the next generation of samples from all the"transmission pairs"continuous sampling points were sequenced,and the maximum likelihood tree was constructed to further determine the transmission relationship.Combined with the final confirmation two pairs of"transmission pairs"of the real transmission relationship were included in the follow-up study.A total of 19 transmission pairs of the real transmission relationship were confirmed,and the recipient was finally selected as the 7"transmission pairs"of the single strain.b).The number and polymorphism of quasispecies in donors and recipients changed with time:there were individual differences in the diversity of quasispecies between donors and recipients.Then we compared the changing trend of gene diversity between donor and recipient over time,and found that the diversity of all donors was greater than that of recipients,and the initial gene diversity of recipients was very small,which proved the bottleneck of transmission.c).The divergence changes of donor-recipient homologous virus:when the donor-recipient baseline sampling time was close,the virus coevolved in the donor-recipient body,and the donor-recipient estimated infection time was far apart.The evolution rate of the virus from the recipient to the donor baseline point is generally slow.d).Distribution of divergence between donors and recipients:the genetic distance between all"transmission pairs"was less than 0.3s/s.5 of the 7 pairs of"transmission pairs"were less than 0.015s/s.The genetic distance between donors and recipients did not change linearly with time,but decreased from 2 to 3 years after infection.The average genetic distances within 1 year,1-2 years,2-3 years and more than 3 years after infection were 0.008±0.006 s/s,0.012±0.007 s/s,0.009±0.003 s/s and 0.015±0.009 s/s.3.Construction of molecular transmission network of CRF01＿AE strain and analysis of its dynamic changes.a).Characteristics of the subjects:1669 cases of CRF01＿AE subtype infection were identified from 2221 newly diagnosed cases from 2008 to 2016.Compared with the other two major subtypes,CRF01＿AE subtype infection had more RHI,MSM and male,and more people with lower CD4+T cell count and higher viral load were detected clinically(P<0.05).b).Optimize the gene distance threshold of molecular transmission network:according to the threshold setting principle of maximum network resolution,we observe that the number curve of CRF01＿AE transmission clusters is in the highest platform period when the threshold is 0.004-0.007.In addition,we use one in vivo evolution data of the first part,the evolution data of"transmission pair"in the second part and some data of the third part,to draw the frequency distribution of genetic distance satisfies the normal distribution curve.The threshold of genetic distance peak in individual is 0.005s/s,and the threshold of"transmission pair"is 0.01s/s.Combined with these results,we choose a smaller threshold:0.005 is more specific,that is,the network connection is more likely to be a real transmission relationship.c).Characteristics of molecular transmission network of three main subtypes of HIV:1669 CRF01＿AE subtype sequences of HIV molecular transmission network were constructed by HIV-TRACE technology.A total of 138 transmission clusters(sizes between 2 and 107)were formed,64.5%(89/138)were MSM transmission clusters,and there were 9 CRF01＿AE large transmission clusters.d).Large transmission clusters development history and expansion rate:according to the period of the fastest expansion of large transmission clusters(defined as 45%of the size of the final transmission clusters reached by the number of infected persons in a certain period at the end of 2008),these large transmission clusters are divided into historical group(2008-2010),intermediate group(2011-2013)and recent active group(2014-2016).PDR,cluster growth predictor and R_e are calculated to evaluate the expansion rate of each large transmission cluster.Although the shapes of the three curves of each large transmission cluster are not the same,it is confirmed that all large transmission clusters showed a downward trend during the study period.e).Retrospective evaluation of the effect of HIV treatment and prevention intervention in Shenyang based on molecular transmission network the decline of large transmission clusters is consistent with the ART standard in advance:since the establishment of a long-term follow-up cohort in Shenyang in 2008,the ART treatment standard for people with HIV infection has been gradually advanced.Our data show that the proportion of people infected with ART who began treatment within 6 months after diagnosis increased from 24%in 2008-2010 to 54%in2011-2013 and 79%in 2014-2016.At the same time,the proportion of people infected with ART two years after diagnosis decreased from 29%in 2008-2010 to 15%in 2011-2013 and 1%in 2014-2016.Conclusion:1.The intra-individual evolution rate of CRF01＿AE strain was 0.002±0.001,and the degree of divergence increased gradually with the time of infection:the degree of divergence in groups≤1,1-2,2-3 and>3 years was 0.003±0.003s/s,0.004±0.002s/s,0.007±0.002s/s and 0.009±0.004s/s,respectively.The number of quasispecies,diversity and divergence were correlated with viral load,and the evolution rate of CRF01＿AE strains in China was faster than that of subtype B in Europe and America.2.The evolution of CRF01＿AE"transmission pair"homologous strain is complicated,and the genetic diversity of the donor is greater than that of the recipient.The genetic distance between the recipient and the donor within 1 year,1-2 years,2-3years and more than 3 years is 0.008±0.006s/s,0.012±0.007 s/s,0.009±0.003 s/s,0.015±0.009 s/s.3.Characteristics of the subjects:1669 cases of CRF01＿AE subtype infection were identified from 2221 newly diagnosed cases from 2008 to 2016.Compared with the other two major subtypes,CRF01＿AE subtype infection had more RHI,MSM and male,and more people with lower CD4+T cell count and higher viral load were detected clinically.According to the first and second parts,0.005 was selected as the optimal threshold for CRF01＿AE strains,and the new threshold was applied to retrospectively analyze the CRF01＿AE molecular transmission network.Three kinetic parameters were used to prove that the expansion of large transmission clusters was effectively controlled,which was consistent with the continuous advance of antiretroviral treatment standards,which confirmed the effectiveness of these strategies.