Tracing Hepatitis C Virus Transmission By Using High-throughput Sequencing Technique

BackgroundOutbreak of hepatitis C virus (HCV) occasionally occurs in China in recent years.To investigate the source of the outbreaks, track the root of transmission, and formulate effective prevention and control measures, we need reliable traceable technology for HCV infection.Laboratory methods on tracing transmission of HCV reported abroad mainly included direct sequencing of nest PCR products, sequencing of cloned PCR products and end-point limiting-dilution PCR, our country also had reported direct sequencing of nest PCR products.Direct sequencing of nest PCR products is simple, but only one dominated quasispecy can be detected; the latter two methods can obtain more major and minor quasispecies, but the disadvantages of being sophisticated and time-consuming limit their applications.The Next-generation sequencing technology(NGS) is built on the concept of ’sequencing by synthesis’(SBS) to determine the sequence of DNA by identifying new end of the sequence, it can produce millions of DNA sequence reads in a single run.At present, NGS technology platforms mainly include Illumina/Solexa Genome Analyzer (Illumina company, USA),Applied Biosystems SOLID system (ABI company, USA) and Roche 454 FLX pyrotechnical platform (454 Life Sciences company, USA).Roche 454 FLX pyrotechnical platforms had been used on quasispecies variability of the natural history of HCV infection and HCV patients who had antiviral therapy, HCV transmission bottlenecks in primary acute HCV infection and HCV transmission in outbreaks.,There is little practical value for tracing transmission of HCV infection because of the high cost of sequencing.Our research group had used Miseq high-throughput sequencing on tracing transmission of HCV infection suspected by the hemodialysis treatment successfully.Approach for analysis of HCV quasispecies using Hiseq high-throughput sequencing technique what is newly and more high-throughput was developed and then asked to investigate a possible case of HCV needle sharing transmission in this study, and provide technical reserves for future HCV outbreak investigation.Objective1.To develop and optimize an approach for analyzing HCV quasispecies basing on the Hiseq platform;2.To learn a wide range of quasispecies diversity during HCV infection in intravenous drug users;3.To use this method to investigate a possible case of HCV needle sharing transmission.Subjects and Methods1.Subjects1.1 Tracing investigation of a case of recent HCV infectionA case of HCV antibody seroconversion (P1) was discovered in a methadone clinic on January 15,2015. Four HCV antibody positive injecting drug users(1DUs), P2 to P5, suspected to be involved in needle sharing transmission with P1 during the period (after March 24,2014) that P1 may be infected with HCV were investigated,and another 28 HCV antibody positive IDUs were selected as controls (C1 to C28). These controls came from the same methadone clinic or resided in the same town with P1. Thirty-three whole blood specimen was collected. Plasma was centrifugation.1.2 Tracing HCV transmissions in IDUs from 2 methadone clinicsCollect plasma specimen of IDUs who has HCV antibody positive from January to September of 2015 in methadone clinics H and L (numbered H1～H13, L1～L32).2.Methods2.1 RNAs was extracted from plasma, and then Reverse-transcribed the RNA into cDNA.2.2 Direct sequencing of nest PCR products:NS5B gene region and Core/E1 gene region was respectively amplified by nest PCR, products were sequence directly.Subtypes were considered based on the sequence of HCV.2.3 Designed primers of HCV HVR1 gene region, and streamline the reaction conditions.Primer should cover multiple gene subtype as much as possible.Designed several sets of primers, if necessary.2.4 For HCV HVR1 gene region, each cDNA specimen was amplified by nest PCR; PCR products were purified and structural DNA library, then Hiseq sequence.2.5 After Hiseq sequencing data was preliminary processed, statistical frequency of each unique sequence to understand the quasispecies distribution of HCV; Each specimen can get multiple quasispecies sequence to do phylogenetic analysis after excluding repeats.Results1.Tracing investigation of a case of recent HCV infection1.1 The gene subtype of HCV:HCV nucleic acid of 11 specimens (P3 and 10 controls) are negative (specimen P3 viral load was negative) in P1～P5 and 28 control specimen,the nucleic acid of remaining specimens are positive,and achieve HCV subtypes.The HCV gene subtype of specimen P1 and P2 was 3b, while P4 and P5 were 3a and 6n respectively, excluding the possibility of HCV transmission in PI.The HCV gene subtype of 8 specimens(C7, C12, C14, C15, C16, C19, C20, C28) was same as P1 in 18 specimens of nucleic acid positive.1.2 The distribution of HCV quasispecies:HVR1 gene region in 9 out of 10 specimens(except C20) with subtype 3b was amplified and Hiseq sequencing successfully.Hiseq sequencing results show that each specimen obtains about one million nucleic acid sequences(about 1M data), after the data were preliminary processed,249753-1086333(average 869608) cleaned sequences representing 3-172 unique HCV quasispecies were obtained from these specimens.Statistical frequency quasispecies sequences of each specimen,from high to low in descending order,the frequency of top 2 quasispecies in total sequences more than 38%,of which 4 specimen (C7,C15,C19, C28) exceeds 85%,even 3 specimen (C7,C19,C28) more than 97%.1.3 The range of genetic diversity:Intraspouse genetic diversity was from 0.5% to 13.2% in 9 plasma specimens, in which P1, C7, C16 were higher than 6%.Interspecies genetic diversity was from 10.8% to 25.0% in 9 plasma specimens, interspecies genomic diversity of P1 and P2 was 19.0%, there was no significant statistical between the genetic diversity of P1 and control specimens (P> 0.05).1.4 Phylogenetic analyses:Twenty-five HCV representative sequences from the Los Alamos National Laboratory HCV database were download and build a phylogenetic tree with quasispecies sequences of all specimens.The phylogenetic tree showed that all quasispecies sequences of specimen P1 and partial quasispecies sequences of specimen C7 gathered in a cluster. Another part quasispecies sequences of specimen C7 separately gathered in a cluster.Other specimens(P2, C12, C14, C15, C16, C19, C28) were in separate clusters, which were separated from each other by HCV representative sequences respectively.Separate analysis gathered part quasispecies sequences of P1 and C7, the average genes distance between them is 6.4%(0 to 13.3%), far less than the average genes distance between P1 and other specimens(P<0.05).Phylogenetic analysis and genes distance noted that a relatively close evolutionary relationship of PI and C7.In view of probable existence of HCV multiple infection, the minimum gene distance between specimens is more valuable than the average gene distance in the investigation of HCV transmission.2. Tracing HCV transmission in IDUs from 2 methadone clinics2.1 The gene subtype of HCV:HCV nucleic acid of 13 specimens was negative in methadone clinics H and L, the nucleic acid of the remaining specimens is positive, and achieves HCV subtypes.HCV nucleic acid of 2 specimens (H8, H12) was negative in methadone clinic H.The HCV gene subtype of 1 (H11) was 3a/3b,7 specimens(H1,H3,H4,H7,H9,H10,H13)were 3b,2 (H5, H6) were 6a,and the remaining 1 (H2) was 6u in 11 nucleic acid positive specimens in methadone clinic H.HCV nucleic acid of 11 specimens (L3,L5,L6,L7,L11,L15,L17,L20,L25,L29,L32) were negative in methadone clinic L.The HCV gene subtype of 5 specimens (L8,L18,L21,L28,L31) were la,l (L14) was 3a/3b,6 specimens(L1,L10,L12,L13, L16,L24) were 3b,1 (L19) was 6m/6n,4(L4,L22,L23,L30) were 6n,the remaining 4 (L2,L9,L26,L27)was 6a,1b,6u,3a respectively in 21 nucleic acid positive specimens in methadone clinic L.Only 1 specimen obtain the HCV gene subtype of lb,excluding the possibility of HCV transmission between this infected person and others.2.2 The distribution of HCV quasispecies:Analysis of the subtypes, the same genetic subtypes of not less than 2 specimens(total of 31) was amplified by PCR HVR1 region.HVRl gene region of 27 specimens from H and L was amplified and sequence with Hiseq technique successfully.Hiseq sequencing results show that each specimen obtain about one million nucleic acid sequences7, after the data were preliminary processed,366,980-2,732,511 (average 1,432,153) cleaned sequences Representing 2-207(average 45) unique HCV quasispecies were obtained from these specimens.Statistical frequency quasispecies sequences of each specimen,from high to low in descending order,the frequency of top 2 quasispecies in total sequences more than 27%,of which 12 specimens(H1,H3,H4,H6,H9,H11,H13,L1,L8,L16,L18, L31) exceeds 82%, even 8 specimens(H3, H4, H6, H9, L1, L16, L18, L31) more than 96%.2.3 The range of genetic diversity:Intraspouse genetic diversity was from 0.4% to 26.0%in the H and L methadone clinic, in which H9, L14, L19, L23, L30 were more than 5%.The minimum gene distance between most specimens is larger than 5% in H and L methadone clinic.The minimum gene distance between partial specimens is 0.0%, the minimum gene distance between specimens H6 and H9 was 0.4%,the minimum gene distance between specimens H11 and L23,L30 were 0.4%,the minimum gene distance between specimens L8 and L21 was 1.9%,the minimum gene distance between specimens L21 and L28 was 4.8%.2.4 Phylogenetic analysis:Phylogenetic tree showed that quasispecies sequences of specimens L28 and L31 each gathered in a cluster,all quasispecies sequences of specimens L8,L18,L21 and partial quasispecies sequences of L23,L30 gathered in a cluster,the minimum gene distance between specimens L8 and L21 was 1.9%,L8 and L23,L18 and L23,L18 and L30,L21 and L23,L21 and L30 were 0.A1I quasispecies sequences of H9 and partial quasispecies sequences of H6 gathered in a cluster, the minimum gene distance between specimens H6 and H9 was 0.4%.Another phylogenetic tree showed that quasispecies sequences of specimens H3,L10,L12,L14 each gathered in a cluster,quasispecies sequences of specimens L23 and L24 gathered in a cluster,quasispecies sequences of specimens H1,H4,H9,H1O,H11,H13,L1 and partial quasispecies sequences of L23,L30 gathered in a cluster respectively.The minimum gene distance between specimens Hll and L23 and L30 was 0.4%, the minimum gene distance between specimens L23 and L24was 0, and the minimum gene distance between specimens L23, L30 and the remaining specimens was 0.Phylogenetic analysis and genes distance indicated that a relatively close evolutionary relationship of partial specimens in the same and different methadone clinic.Conclusionsl.This study established HCV quasispecies analysis method based on Hiseq sequencing technology.2.There is HCV quasispecies diversity in HCV antibody positive injecting drug users.3.Investigation one patient recently infected with HCV,excluded the potential spread relationship between him and the 3 needle sharing HCV antibody positive injecting drug users,and find a new communication clues.4.By analyzing HCV antibody positive injecting drug users in 2 methadone clinics, the results suggest the relationship existence between the same methadone clinic, and 2 methadone clinics.5.In view of the possible existence of HCV multiple infection, the minimum gene distance between specimens is more valuable than the average gene distance in the investigation of HCV transmission.6.Hiseq sequencing is high-throughput, the operation is relatively simple, relatively low cost and high practical value, there is high practical value in tracing post exposure HCV infection.