Journal of General Virology (1999), 80, 717–725. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Variation of hepatitis C virus following serial transmission : multiple mechanisms of diversification of the hypervariable region and evidence for convergent genome evolution Carmela Casino, 1 Jane McAllister, 1 Fiona Davidson, 2 Joan Power, 3 Emer Lawlor, 3 Peng Lee Yap, 2 Peter Simmonds 1 and Donald B. Smith 1 1 Department of Medical Microbiology, University of Edinburgh, Medical School, Teviot Place, Edinburgh EH8 9AG, UK 2 Edinburgh and South East Scotland Blood Transfusion Service, Lauriston Place, Edinburgh EH3 9HB, UK 3 Irish Blood Transfusion Board, St Finbarr’s Hospital, Cork and Pelican House, Dublin, Ireland We have studied the evolution of hepatitis C virus (HCV) from a common source following serial transmission from contaminated batches of anti-D immunoglobulin. Six secondary recipients were each infected with virus from identifiable primary recipients of HCV-contaminated anti-D immunoglobulin. Phylogenetic analysis of virus E1/E2 gene sequences [including the hypervariable region (HVR)] and part of NS5B confirmed their common origin, but failed to reproduce the known epidemiological relationships between pairs of viruses, probably because of the frequent occurrence of convergent substitutions at both synonymous and nonsynonymous sites. There was no evidence that the rate at which the HCV genome evolves is affected by transmission events. Three different mechanisms appear to have been involved in generating variation of the hypervariable region ; nucleotide substitution, insertion/deletion of nucleotide triplets at the E1/E2 boundary and insertion of a duplicated segment replacing almost the entire HVR. These observations have important implications for the phylogenetic analysis of HCV sequences from epidemiologically linked isolates. Introduction The ability of hepatitis C virus to establish a persistent infection following transmission to a new host is generally thought to be related to continued evolution of the virus envelope glycoproteins E1 and E2. Attention has focused particularly on a 25–30 amino acid hypervariable region (HVR) at the NH # terminus of the E2 protein that induces the production of specific antibodies that may be neutralizing in vivo (Farci et al., 1996) and may be capable of blocking virus attachment to cells in vitro (Zibert et al., 1995). Previous work has identified both invariant and variable positions within the HVR ; amino acid replacements at some variable sites are limited to a small number of residues with similar chemical Author for correspondence : Donald Smith. Fax ›44 131 650 6531. e-mail Donald.B.Smith!ed.ac.uk The GenBank accession numbers for the nucleotide sequences reported here are AF119717–AF119768, AF056764–AF056785 and AF056795–AF056812. properties, while at other sites almost every residue can occur (Sekiya et al., 1994 ; Sherman et al., 1996 ; McAllister et al., 1998). Detailed comparisons of the HVR sequences in different individuals infected from a common infectious source has provided evidence for the operation of selection against amino acid substitution at some positions, and positive selection for amino acid replacement at others (McAllister et al., 1998). However, most previous studies of the evolution of the HVR have been confined to longitudinal studies of acutely or persistently infected individuals, or in a few cases to individuals infected from an identifiable source (Weiner et al., 1993 ; Hohne et al., 1994 ; Esteban et al., 1996). To date, there is no information about the effect of multiple transmission events on virus evolution in humans, and it is possible that very different patterns of variation might be observed under these cir- cumstances. For example, continued selection of antigenically distinct variants of the HVR might drive the virus population away from a fitness optimum, and subsequent transmission to a new host might result in selection for less divergent variants, as observed previously for human immunodeficiency virus (Zhang et al., 1993) and during experimental transmission of 0001-5972 # 1999 SGM HBH