J Clin Microbiol. 2015 Oct;53(10):3155-64. doi: 10.1128/JCM.00483-15. Epub 2015 Jul 22.

Characterization of Hepatitis C Virus Recombination in Cameroon by Use of Nonspecific Next-Generation Sequencing.

Iles JC¹, Njouom R², Foupouapouognigni Y², Bonsall D³, Bowden R⁴, Trebes A⁴, Piazza P⁴, Barnes E³, Pépin J⁵, Klenerman P³, Pybus OG⁶.

Author information

1: Department of Zoology, University of Oxford, Oxford, United Kingdom.
2: Service de Virologie, Centre Pasteur du Cameroun, Yaounde, Cameroon.
3: Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, United Kingdom.
4: Wellcome Trust Centre for Human Genetics, Oxford University, Oxford, United Kingdom.
5: Department of Microbiology and Infectious Diseases, Université de Sherbrooke, Sherbrooke, Canada.
6: Department of Zoology, University of Oxford, Oxford, United Kingdom oliver.pybus@zoo.ox.ac.uk.

Abstract

The importance of recombination in the evolution and genetic diversity of the hepatitis C virus (HCV) is currently uncertain. Only a small number of intergenotypic recombinants have been identified so far, and each has core and envelope genes classified as belonging to genotype 2. Here, we investigated two putative genotype 4/1 recombinants from southern Cameroon using a number of approaches, including standard Sanger sequencing, genotype-specific PCR amplification, and non-HCV-specific Illumina RNA sequencing (RNA-seq). Recombination between genotypes 1 and 4 was confirmed in both samples, and the parental lineages of each recombinant belong to HCV subtypes that are cocirculating at a high prevalence in Cameroon. Using the RNA-seq approach, we obtained a complete genome for one sample, which contained a recombination breakpoint at the E2/P7 gene junction. We developed and applied a new method, called Deep SimPlot, which can be used to visualize and identify viral recombination directly from the short sequence reads created by next-generation sequencing in conjunction with a consensus sequence.