Five Near Full-Length Hepatitis C Virus Sequences Were Identified from HIV Coinfected Injection Drug Users of China

HIV and hepatitis C virus (HCV) share transmission routes, including contaminated blood transfusion, sexual intercourse, and needle-sharing in injection drug users (IDUs). ^1,2 HIV/HCV coinfection is associated with increased risk for HIV disease progression, AIDS-related mortality, and more rapid progression of HCV-related liver disease. ³ In China, HCV prevalence among IDUs have been estimated to be 67%. ⁴ Previous study have considered that there exist at least six major genotypes of HCV globally, which differ in 30%–35% of nucleotide sites among each genotype. ⁵ The high degree of genetic diversity creates big challenge for drug treatment and vaccine development. At present, the duration of treatment, cure rates, and the need for interferon and ribavirin with the new direct-acting antiviral (DAA) therapies remain dependent in part on the HCV genotype. ⁶ Therefore, HCV transmission with various genotypes in small geographic area of certain risk group brings considerable challenge to pandemic control.

In this study, we obtained near full-length genome (NFLG) of HCV derived from five HIV-1-coinfected IDUs patients in China. This study was approved by the Institutional Review Board of the National Center for AIDS/STD Control and Prevention. Blood plasma samples were collected from five HCV-positive consenting subjects (SCIDU31, SCIDU206, SCIDU126, XJN0021, and XJN0041), residing in Sichuan and Xinjiang provinces. NFLGs (∼9.0 kb) were determined from plasma HCV viral RNA with nested polymerase chain reaction that have been described previously. ⁷ Amplicons were purified using the QIAquick gel extraction kit (Qiagen) and then sequenced directly by an ABI 3730XL sequencer using BigDye Terminator (Applied Biosystems, Beijing, China). All sequenced data were cleaned and assembled into contiguous sequences using the Sequencher program version 5.1 (Gene Codes Corporation). The codon-aligned nucleotide sequence alignment was constructed using the HCVAlign online tool, available from the Los Alamos National Laboratory HCV Sequence Database (http://hcv.lanl.gov/content/sequence/VIRALIGN/viralign.html), and then minor manual adjustments according to codon-aligned nucleotide sequence alignment were performed using BioEdit v7.0.9. ⁸ Phylogenetic analyses were performed using MEGA v6.0. ⁹

The NFLGs from the five subjects were 9154, 9044, 9066, 8994, and 8967 bp in size for isolates SCIDU031, SCIDU126, SCIDU206, XJN0021, and XJN0041, respectively. All the five NFLGs were used to construct a neighbor-joining phylogenetic tree (Fig. 1); all the reference sequences used in Figure 1 were downloaded from Los Alamos National Laboratory HCV Sequence Database, and the result showed that the five isolates were clustered with four different genotypes (1b, 3a, 6a, and 6n) and revealed a large pairwise genetic distance between each other. The five sequences were translated to amino acid sequences using GENE Runner v3.05 and aligned to compare the amino acid positions with reference sequence (H77, accession number: NC_004102), and amino acid substitutions was analyzed by the Geno2Pheno [hcv] v0.92 program (http://hcv.geno2-pheno.org/index.php). ¹⁰ The amino acid substitutions compared to reference sequence in NS3 of HCV were listed in Table 1; and the amino acid substitutions (170I) in XJN0021 related to resistant to protease inhibitors (Grazoprevir) were also identified. ¹¹

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FIG. 1.

A NJ phylogenetic tree of the five isolates (SCIDU031, SCIDU126, SCIDU206, XJN0021, and XJN0041) with reference sequences was constructed using MEGA 6.0. The five isolates (SCIDU031, SCIDU126, SCIDU20, XJN0021, and XJN0041) were marked with solid circle. Bootstrap values above 80% are shown at the corresponding nodes. The scale bar represents 5% genetic distance. NJ, neighbor-joining.

Sichuan and Xinjiang provinces are two major areas in China severely affected by heroin, with HCV and HIV epidemic among IDUs. This is also the first report of near full-length HCV genome sequences from these regions. Different from that subtype CRF07_BC dominates the HIV-1 epidemic among IDUs in Sichuan and Xinjiang provinces, China. ¹² The four genotypes of HCV detected in this study may indicate the presence of complex transmission networks of HCV infections among the IDUs in these regions.