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Viruses 2018, 10(1), 16; doi:10.3390/v10010016

Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes

1
Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, University of London, London WC1E 7HT, UK
2
Institute for Structural and Molecular Biology, Department Biology, Birkbeck College University of London, London WC1E 7HX, UK
*
Author to whom correspondence should be addressed.
Received: 30 November 2017 / Revised: 23 December 2017 / Accepted: 26 December 2017 / Published: 31 December 2017
(This article belongs to the Special Issue Breakthroughs in Viral Replication)
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Abstract

Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for related betaherpesviruses, Roseolovirus, human herpesvirus 6A (HHV-6A) strain U1102. This virus is also naturally “cloned” by germline subtelomeric chromosomal-integration in approximately 1% of human populations, and accurate references are key to understanding pathological relationships between exogenous and endogenous virus. Using whole genome next-generation deep-sequencing Illumina-based methods, we compared the original isolate to tissue-culture passaged and the BACmid-cloned virus. This re-defined the reference genome showing 32 corrections and 5 polymorphisms. Furthermore, minor variant analyses of passaged and BACmid virus identified emerging populations of a further 32 single nucleotide polymorphisms (SNPs) in 10 loci, half non-synonymous indicating cell-culture selection. Analyses of the BAC-virus genome showed deletion of the BAC cassette via loxP recombination removing green fluorescent protein (GFP)-based selection. As shown for HCMV culture effects, select HHV-6A SNPs mapped to genes encoding mediators of virus cellular entry, including virus envelope glycoprotein genes gB and the gH/gL complex. Comparative models suggest stabilisation of the post-fusion conformation. These SNPs are essential to consider in vaccine-design, antimicrobial-resistance, and pathogenesis. View Full-Text
Keywords: human herpesvirus; HHV-6; HHV-6A; Roseolovirus; betaherpesvirus; virus entry complex; gH/gL; gB; deep sequencing; reference genome; cell fusion; glycoprotein structure model human herpesvirus; HHV-6; HHV-6A; Roseolovirus; betaherpesvirus; virus entry complex; gH/gL; gB; deep sequencing; reference genome; cell fusion; glycoprotein structure model
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Tweedy, J.G.; Escriva, E.; Topf, M.; Gompels, U.A. Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes. Viruses 2018, 10, 16.

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