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Copy number heterogeneity, large origin tandem repeats, and interspecies recombination in HHV-6A and HHV-6B reference strains

View ORCID ProfileAlexander L. Greninger, View ORCID ProfilePavitra Roychoudhury, Negar Makhsous, View ORCID ProfileDerek Hanson, Jill Chase, Gerhard Krueger, Hong Xie, Meei-Li Huang, Lindsay Saunders, Dharam Ablashi, David M. Koelle, Linda Cook, Keith R. Jerome
doi: https://doi.org/10.1101/193805
Alexander L. Greninger
Department of Laboratory Medicine, University of Washington, Seattle, WA, USAFred Hutchinson Cancer Research Institute, Seattle, WA, USA
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  • For correspondence: agrening@uw.edu
Pavitra Roychoudhury
Department of Laboratory Medicine, University of Washington, Seattle, WA, USAFred Hutchinson Cancer Research Institute, Seattle, WA, USA
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  • ORCID record for Pavitra Roychoudhury
Negar Makhsous
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Derek Hanson
Department of Medicine, University of Washington, Seattle, WA, USA
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Jill Chase
HHV-6 Foundation, Santa Barbara, CA, USA
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Gerhard Krueger
Department of Pathology and Laboratory Medicine, University of Houston, Houston,TX, USA
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Hong Xie
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Meei-Li Huang
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Lindsay Saunders
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Dharam Ablashi
HHV-6 Foundation, Santa Barbara, CA, USA
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David M. Koelle
Department of Laboratory Medicine, University of Washington, Seattle, WA, USAFred Hutchinson Cancer Research Institute, Seattle, WA, USADepartment of Medicine, University of Washington, Seattle, WA, USADepartment of Global Health, University of Washington, Seattle, WA, USA
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Linda Cook
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
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Keith R. Jerome
Department of Laboratory Medicine, University of Washington, Seattle, WA, USAFred Hutchinson Cancer Research Institute, Seattle, WA, USA
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Abstract

Quantitative PCR is the diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of HHV-6 is important for detection of viral reactivation and inherited chromosomally integrated HHV-6 in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies. 11 of 17 (65%) HHV6 candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1254 bp and 983 bp, respectively. Copy number measured between 4-10X copies relative to the rest of the genome. We also report the first interspecies recombinant HHV-6 strain with a HHV-6A GS backbone and >5.5kb region from HHV-6B Z29 from U41-U43 that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at UL1/UL2, U87, and U89, as well as deletion in the U12-U24 region and U94/95 genes. HHV-6 strains derived from cord blood mononuclear cells from different labs on different continents revealed no copy number differences throughout the viral genome. These data indicate large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae.

Importance Anything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very usefulness through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well-known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6 biology and the standard selection process.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted September 25, 2017.
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Copy number heterogeneity, large origin tandem repeats, and interspecies recombination in HHV-6A and HHV-6B reference strains
Alexander L. Greninger, Pavitra Roychoudhury, Negar Makhsous, Derek Hanson, Jill Chase, Gerhard Krueger, Hong Xie, Meei-Li Huang, Lindsay Saunders, Dharam Ablashi, David M. Koelle, Linda Cook, Keith R. Jerome
bioRxiv 193805; doi: https://doi.org/10.1101/193805
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Copy number heterogeneity, large origin tandem repeats, and interspecies recombination in HHV-6A and HHV-6B reference strains
Alexander L. Greninger, Pavitra Roychoudhury, Negar Makhsous, Derek Hanson, Jill Chase, Gerhard Krueger, Hong Xie, Meei-Li Huang, Lindsay Saunders, Dharam Ablashi, David M. Koelle, Linda Cook, Keith R. Jerome
bioRxiv 193805; doi: https://doi.org/10.1101/193805

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