title: [Tracing hepatitis C virus transmission by using high-throughput sequencing technique].
abstract: OBJECTIVE
  An approach for analysis of hepatitis C virus (HCV) quasispecies using Hiseq high-throughput sequencing (hereinafter referred to as Hiseq sequencing) technique was developed and then applied to investigate a possible case of HCV needle sharing transmission.
  
  
  METHODS
  One case of HCV antibody seroconversion (P1) was found in a methadone clinic on January 15, 2015. Four HCV antibody positive injecting drug users (IDUs), 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 lived in the same town with P1. The RNAs were extracted from the plasma specimens and then reverse-transcribed into cDNA. After HCV subtyping, Hiseq sequencing was performed to detect and sequence the HCV quasispecies (263 bp) in the specimens with the same subtype as P1. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated.
  
  
  RESULTS
  The HCV subtype of specimen P1 was 3b. All the other specimens with the same subtype were P2, C7, C12, C14, C15, C16, C19, C20 and C28. Hiseq sequencing was successfully performed in 9 out of these 10 specimens, and 249 753 to 1 086 333 (average 869 608) cleaned sequences representing 3 to 172 (average 48) unique HCV quasispecies were obtained. The medians (P50) of intrapersonal genetic diversities from the 9 specimens were 0.4% to 12.3%. The P50 (P25, P75) of genetic diversities between P1 and the other 8 specimens were 19.0% (18.4%, 19.8%), 10.4% (2.8%, 18.3%), 19.6% (17.8%, 21.4%),24.9% (23.8%, 26.1%), 19.8% (18.7%, 20.7%), 20.1% (18.9%, 21.2%), 20.6% (20.0%, 21.1%), 23.6% (22.4%, 24.8%). There were no significant difference between the genetic diversities of P1 and P2 and those of P1 and other 7 specimens (H=9.40, P=0.100). The genetic diversities between few HCV quasispecies from P1 and few ones from C7 were 0. Phylogenetic tree analysis indicated that there was no HCV transmission relationship between P1 and P2, but there was HCV transmission relationship between P1 and C7.
  
  
  CONCLUSIONS
  With the feature of high-throughput, easier operation and lower cost, Hiseq sequencing technique has high practical value in tracing HCV transmission at the quasispecies level.
authors.affiliation: National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: J N
authors.initials: JN
authors.lastname: Zhang
authors.affiliation: Department of AIDS/STD Control and Prevention, Dehong Center for Disease Control and Prevention, Dehong, Yunnan 678400, China.
authors.forename: Y K
authors.initials: YK
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Jiang
authors.forename: Y C
authors.initials: YC
authors.lastname: Long
authors.forename: J B
authors.initials: JB
authors.lastname: Wang
authors.forename: K D
authors.initials: KD
authors.lastname: Feng
authors.forename: R H
authors.initials: RH
authors.lastname: Tang
authors.forename: Z M
authors.initials: ZM
authors.lastname: Zhang
authors.forename: S
authors.initials: S
authors.lastname: Duan
authors.forename: Q
authors.initials: Q
authors.lastname: Zhao
authors.forename: G Y
authors.initials: GY
authors.lastname: Zhang
authors.forename: M F
authors.initials: MF
authors.lastname: Qiu
mesh_terms: D000328:Adult; D000914:Antibodies, Viral / Q000032:analysis; D002681:China; D016358:Contact Tracing; D014644:Genetic Variation; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D006526:Hepatitis C / Q000097:blood / Q000473:pathology / Q000635:transmission* / Q000821:virology; D006801:Humans; D008691:Methadone / Q000627:therapeutic use*; D008969:Molecular Sequence Data; D016485:Needle Sharing; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D016320:Substance Abuse Treatment Centers; D015819:Substance Abuse, Intravenous / Q000150:complications / Q000534:rehabilitation / Q000821:virology*
chemical_list: D000914:Antibodies, Viral; D012367:RNA, Viral; D008691:Methadone