Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 07 Dec 2018 20:17:25 Insertion and recombination events at hypervariable region 1 over 9.6 years of hepatitis C virus chronic infection Brendan A. Palmer, 1 Isabelle Moreau, 1 John Levis, 1 Ciara Harty, 1 Orla Crosbie, 2 Elizabeth Kenny-Walsh 2 and Liam J. Fanning 1 Correspondence Liam J. Fanning l.fanning@ucc.ie Received 18 June 2012 Accepted 12 September 2012 1 Molecular Virology Diagnostic and Research Laboratory, Department of Medicine, Clinical Sciences Building, University College Cork, Cork, Ireland 2 Department of Gastroenterology, Cork University Hospital, Cork, Ireland Hepatitis C virus (HCV) exists as a quasispecies within an infected individual. We have previously reported an in-frame 3 bp insertion event at the N-terminal region of the E2 glycoprotein from a genotype 4a HCV isolate giving rise to an atypical 28 aa hypervariable region (HVR) 1. To further explore quasispecies evolution at the HVR1, serum samples collected over 9.6 years from the same chronically infected, treatment naı ¨ve individual were subjected to retrospective clonal analysis. Uniquely, we observed that isolates containing this atypical HVR1 not only persisted for 7.6 years, but dominated the quasispecies swarm. Just as striking was the collapse of this population of variants towards the end of the sampling period in synchrony with variants containing a classical HVR1 from the same lineage. The replication space was subsequently occupied by a second minor lineage, which itself was only intermittently detectable at earlier sampling points. In conjunction with the observed genetic shift, the coexistence of two distinct HVR1 populations facilitated the detection of putative intra-subtype recombinants, which included the identification of the likely ancestral parental donors. Juxtaposed to the considerable plasticity of the HVR1, we also document a degree of mutational inflexibility as each of the HVR1 subpopulations within our dataset exhibited overall genetic conservation and convergence. Finally, we raise the issue of genetic analysis in the context of mixed lineage infections. INTRODUCTION Hepatitis C virus (HCV) is a hepacivirus that exists as a family of seven major genotypes and numerous subtypes (Murphy et al., 2007; Simmonds et al., 2005). The positive- sense, ssRNA genome encodes a single polyprotein that is processed by cellular and viral proteases giving rise to 10 proteins (Penin et al., 2004). Progression from acute to chronic hepatitis C infection represents 50–80 % of cases, with outcomes ranging from gradual hepatic fibrosis to hepatocellular carcinoma over several decades (Timm & Roggendorf, 2007). Genotypes are not equipotent with respect to the development of chronic infection (Lehmann et al., 2004; Simmonds, 2004). Persistence is enhanced through changes in antigenic epitopes arising as a con- sequence of low fidelity of the virally encoded, RNA- dependent RNA polymerase and selective pressures exerted by the host immune system (Schmidt-Martin et al., 2012; Timm & Roggendorf, 2007). The virally encoded RNA-dependent RNA polymerase has an estimated error rate of the order of 10 23 to 10 25 mutations per nucleotide per genomic replication (Duffy et al., 2008). These mutations are not distributed evenly over the viral genome and three hypervariable regions within the E2 envelope glycoprotein have been character- ized (Farci et al., 1996; Troesch et al., 2006). The HVR1 is typically a stretch of 27 aa at the N-terminal extremity of E2 and demonstrates the highest degree of heterogeneity. In spite of this flexibility, overall conservation of the physio-chemical properties of replacement amino acids is evident (Hino et al., 2002; Penin et al., 2001). Following HCV infection both strain-specific and cross-reactive neutralizing antibody responses develop. This antibody response in part targets the HVR1 (Farci et al., 1996; Kato et al., 1993; Logvinoff et al., 2004). It is known that the HVR1 undergoes repeated change over time in an attempt to mask recognition of the HCV glycoprotein, resulting in immune escape (von Hahn et al., 2007). There are several reports of atypical HVR1 sequences containing insertions at the 59-terminal end of this domain implying that such events constitute part of the virus escape repertoire (Abe et al., 1992; Gerotto et al., 2001; Ho ¨hne et al., The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this study are presented in Table 1. Supplementary figures are available with the online version of this paper. Journal of General Virology (2012), 93, 2614–2624 DOI 10.1099/vir.0.045344-0 2614 045344 G 2012 SGM Printed in Great Britain