Copy Number Heterogeneity, Large Origin Tandem Repeats, and Interspecies Recombination in Human Herpesvirus 6A (HHV-6A) and HHV-6B Reference Strains

Abstract

Quantitative PCR is a diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of human herpesvirus 6A/B (HHV-6A/B) is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B 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 Inc. Eleven of 17 (65%) HHV-6A/B 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 1,254 bp and 983 bp, respectively. The average copy number measured was between 5 and 10 times the number of copies of the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and a >5.5-kb region from HHV-6B, from U41 to U43, that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-to-U24 region and the U94/U95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different laboratories on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate that 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 accuracy 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-6A/B biology and the standard selection process.

Human Herpesvirus 6 and Malignancy: A Review

“…The use of animal models could be especially useful in this area, but to date, few such models exist for the study of HHV-6. In going forward, it will be necessary to standardize reference materials ( 111 ), emphasize collaboration among laboratories, and perform in vitro experiments to characterize the transforming potential of HHV-6, including further analysis of the functions of the viral DR-7 gene, as well as any effects the pair of viruses may have on other oncogenic agents.…”

“…Long-distance rearrangements between U12 to U20, U73 to R3, U86 to U95, and the U91-to-U100/DR intergenic region are represented by curved dashed lines, and the estimated viral subpopulation containing the respective deletion is indicated by the percentage. From Greninger et al ( 111 ).…”

Ultrasensitive Capture of Human Herpes Simplex Virus Genomes Directly from Clinical Samples Reveals Extraordinarily Limited Evolution in Cell Culture

“…Culture of the polyomaviruses BK and JC viruses (BKV and JCV, respectively) is often performed in simian virus 40 (SV40) large-T antigen immortalized cell lines, allowing near-complete loss of the BKV and JCV large-T antigen via transcomplementation, representing loss of one-third of the viral genome ( 14 , 15 ). Culture adaptation of human herpesvirus 6A/B results in large tandem repeats in the origin of replication and other regions that are not found in low-passage-number clinical isolates and likely helps to accelerate viral replication in vitro ( 16 – 18 ). Similarly, laboratory passage of human cytomegalovirus (CMV), Epstein-Barr virus, and varicella-zoster virus can result in surprisingly large deletions comprising multiple genes and kilobases ( 19 – 22 ).…”

LAVA: a streamlined visualization tool for longitudinal analysis of viral alleles

“…However, LAVA automatically detects this situation, and both prints a warning to the console and colors points corresponding to complex mutations distinctly. Sequence variations such as copy number changes, recombination, or large deletions and insertions that escape the bwa-mem aligner may also be missed (35). Due to the nature of its visualization, LAVA also does not display overlapping genes properly and instead shows them side-by-side.…”