Abstract
Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13–36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5′ end to the NS2–NS3 region followed by JFH1 sequence from Core to the 3′ end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients.
Highlights
RNA viruses are rapidly adapting to their environment
Genetic recombination is the alternative joining of nucleic acids leading to novel combinations of genetic information
Recombination can allow escape from the host immune system and from antiviral treatment, and recombination of live attenuated viral vaccines has led to re-emergence of disease
Summary
The error-prone viral polymerases and the lack of proofreading mechanisms for most RNA viruses lead to high mutation rates. Genetic recombination between viral genomes is an additional mechanism increasing genetic diversity, which has proven to be epidemiologically relevant and allows RNA viruses to adapt to their surroundings [1]. Recombination could allow escape from natural or therapeutically induced immunity [2], or during antiviral treatment constitute an escape mechanism to antiviral compounds with an otherwise high barrier to resistance [3]. Viral recombination has been associated with increased pathogenicity [4], and has caused the emergence of new human pathogens, such as Western equine encephalitis virus [5]. Understanding the nature of viral recombination has general evolutionary implications, and might affect treatment and vaccination for important human pathogens
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