Hepatitis C virus RNA recombination in cell culture Jochen Reiter 1, ,# , Gemma Pérez-Vilaró 2,  , Nicoletta Scheller 1,2,  , Leonardo Bruno Mina 2 , Juana Díez 2 , Andreas Meyerhans 1,3,⇑ 1 Department of Virology, Saarland University, D-66421 Hamburg, Germany; 2 Molecular Virology group; 3 ICREA Infection Biology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain Background & Aims: The Hepatitis C virus (HCV) exhibits large genetic diversity, both on a global scale and at the level of the infected individual. A major underlying mechanism of the observed sequence differences is error-prone virus replication by the viral RNA polymerase NS5B. In addition, based on phyloge- netic comparisons of patient-derived HCV sequences, there is evi- dence of HCV recombination. However, to date little is known about the frequency by which recombination events occur in HCV and under what conditions recombination may become important in HCV evolution. We, therefore, aimed to set up an experimental model system that would allow us to analyze and to characterize recombination events during HCV replication. Methods: A neomycin-selectable, HCV replicon-based recombi- nation detection system was established. HCV replicons were mutated within either the neomycin-phosphotransferase gene or the NS5B polymerase. Upon co-transfection of hepatic cells lines, recombination between the mutated sites is necessary to restore the selectable phenotype. Results: Recombinants were readily detected with frequencies correlating to the distance between the mutations. The recombi- nant frequency normalized to a crossover range of one nucleotide was around 4 Â 10 À8 . Conclusions: An experimental system to select for HCV recombi- nants in cell culture was successfully established. It allowed deriving first estimates of recombinant frequencies. Based on these, recombination in HCV seems rare. However, due to the rapid virus turnover and the large number of HCV-infected liver cells in vivo, it is expected that recombination will be of biological importance when strong selection pressures are operative. Ó 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction The Hepatitis C virus (HCV) is an enveloped, positive strand RNA ((+) RNA) virus of the family of Flaviviridae. Infection occurs pri- marily through exposure to HCV-contaminated blood and remains persistent in the majority of cases. It is estimated that around 170 million individuals are chronically infected world- wide. As persistent HCV infection frequently causes chronic hep- atitis that can progress to liver cirrhosis and liver cell carcinoma, it is a major threat for human health [1,2]. HCV shows a high degree of genetic diversity. At a global scale, six major genotypes or ‘‘clades’’ with a greater than 30% nucleotide divergence have been described [3]. These clades can be further divided into subtypes that differ between 20% and 25% from each other. At the level of the HCV-infected individual, the virus exists as a population of related but genetically distinct virus variants col- lectively named a viral quasispecies [4–8]. By comparing plasma- derived HCV RNA consensus sequences over time, the rate of fixa- tion of mutations was estimated to be around 2 Â 10 À3 per site per year [9]. This is in the typical range for RNA viruses which have RNA-dependent RNA polymerases that lack proof-reading activity [10]. Clinically, the genetic diversity of HCV is of prime importance for antiviral treatment. For example, the rate of sustained response after the standard of care treatment regimen of pegylated inter- feron-a plus ribavirin differs between HCV genotypes, with geno- types 2 and 3 showing a better outcome than genotype 1 [11]. Furthermore, experimental treatments targeting viral enzymes rapidly select viral escape mutants when given as monotherapy, directly illustrating the consequences of the quasispecies nature of HCV in infected individuals [12]. The accumulation of HCV point mutations over time is caused by error-prone replication of the RNA genome by the viral RNA polymerase NS5B. In addition to this so-called genetic drift, HCV recombinants have been detected phylogenetically both as novel circulating recombinant forms and within infected patients [13–21], suggesting that recombination may also play a role in HCV evolution. Recombination is an integral part of the biology of many RNA viruses and indeed a powerful mechanism in virus evolution [22–26]. Within a single replicative step, complex structural changes can be generated that may result in novel viruses with new pathogenic properties and a modified host range [15,22,27,28]. Furthermore, recombination can help to eliminate deleterious segments generated by error prone replication or it may accelerate the appearance of drug resistant variants in individuals undergoing antiviral therapy [22]. Extensive studies Journal of Hepatology 2011 vol. 55 j 777–783 Keywords: Recombination; RNA; HCV; HCV replicon; Virus evolution; Positive- strand RNA virus. Received 30 June 2010; received in revised form 14 December 2010; accepted 20 December 2010; available online 18 February 2011 ⇑ Corresponding author. Address: ICREA Infection Biology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader, 88, Edificio PRBB-3er piso, 08003 Barcelona, Spain. Tel.: +34 933 160 831; fax: +34 933 160 901. E-mail address: andreas.meyerhans@upf.edu (A. Meyerhans).   These authors contributed equally to this work. # Present address: GENEART AG, D-93053 Regensburg, Germany. Research Article