International Bioinformatics Workshop on Virus Evolution and Molecular Epidemiology edition:18 location:Gainesville, Florida, USA date:25-30 August 2013
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes serious sequelae in immunocompromised patients and newborns. The coding capacity of its 235 kbp genome is still not fully comprehended and there is a pressing need to characterize genomic contents in clinically representative strains. We therefore have developed a procedure for the high-throughput generation of full genome sequences from minimally passaged clinical isolates. This procedure relies on low number passaging of clinical strains on human fibroblasts, followed by digestion of cellular DNA and purification of viral DNA. After multiple displacement amplification, highly pure viral DNA was generated. Validation experiments have shown that the consensus sequences derived from these extracts using different next-generation sequencing platforms are representative for the virus population present in the original clinical material. Preliminary results from 25 clinical isolates have revealed extensive genetic variability in several regions of the HCMV genome. In addition, gene-disrupting mutations were identified in a set of 12 genes, including the viral interleukin-10 encoding gene UL111A. Consequently, HCMV coding capacity seems to be variable among clinical isolates and this variation could have clinical significance. Finally, we evaluated the use of the PREDATOR algorithm in Segminator II for the analysis of intrahost population diversity of unpassaged and passaged HCMV isolates. Approximately 4% of the genome was found to display intrahost variability in unpassaged isolates. Furthermore, an inverse relationship between gene-specific inter- and intrahost diversity was noted. Currently, a dataset of 100 complete genome sequences from clinical HCMV strains is being analyzed.