Dataset Information

Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay.

ABSTRACT: The human herpesviruses HHV-6A and HHV-6B have been associated with various neurologic disorders partly due to the detection of elevated viral DNA levels in patients compared to controls. However the reported frequency of these viruses varies widely, likely reflecting differences in PCR methodologies used for detection. Digital droplet PCR (ddPCR) is a third generation PCR technology that enables the absolute quantification of target DNA molecules. Mounting evidence of the biological differences between HHV-6A and HHV-6B has led to their recent reclassification as separate species. As it is now especially relevant to investigate each virus, our objectives were to first design a multiplex HHV-6A and HHV-6B ddPCR assay and then to investigate the incidence of HHV-6A and HHV-6B coinfection in samples from healthy donors and patients with MS, a disease in which HHV-6 is thought to play a role. In our assessment of healthy donors, we observed a heretofore-underappreciated high frequency of coinfection in PBMC and serum, and found that our assay precisely detects both HHV-6A and HHV-6B chromosomally integrated virus, which has important implications in clinical settings. Interestingly, upon comparing the saliva from MS patients and healthy donors, we detected a significantly elevated frequency of coinfection in MS saliva; increased detection of HHV-6A in MS patients is consistent with other studies suggesting that this viral species (thought to be more neurotropic than HHV-6B) is more prevalent among MS patients compared to healthy donors. As the biology and disease associations between these two viral species differ, identifying and quantifying both species of HHV-6 may provide clinically relevant information, as well as enhance our understanding of the roles of each in health and disease.

SUBMITTER: Leibovitch EC

PROVIDER: S-EPMC3963908 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay.

Leibovitch Emily C EC Brunetto Giovanna S GS Caruso Breanna B Caruso Breanna B Fenton Kaylan K Ohayon Joan J Reich Daniel S DS Jacobson Steven S

PloS one 20140324 3

The human herpesviruses HHV-6A and HHV-6B have been associated with various neurologic disorders partly due to the detection of elevated viral DNA levels in patients compared to controls. However the reported frequency of these viruses varies widely, likely reflecting differences in PCR methodologies used for detection. Digital droplet PCR (ddPCR) is a third generation PCR technology that enables the absolute quantification of target DNA molecules. Mounting evidence of the biological differences ...[more]

PMID: 24663487

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Project description:Human betaherpesviruses 6A and 6B (HHV-6A and HHV-6B) are highly prevalent in human populations. The genomes of these viruses can be stably integrated at the telomeres of human chromosomes and be vertically transmitted (inherited chromosomally integrated HHV-6A/HHV-6B, iciHHV-6A/iciHHV-6B). We reconstructed the population structures of HHV-6A and HHV-6B, showing that HHV-6A diverged less than HHV-6B genomes from the projected common ancestral population. Thus, HHV-6B genomes experienced stronger drift, as also supported by calculation of nucleotide diversity and Tajima's D. Analysis of ancestry proportions indicated that HHV-6A exogenous viruses and iciHHV-6A derived most of their genomes from distinct ancestral sources. Conversely, ancestry proportions were similar in exogenous HHV-6B viruses and iciHHV-6B. In line with previous indications, this suggests the distinct exogenous viral populations that originated iciHHV-6B in subjects with European and Asian ancestry are still causing infections in the corresponding geographic areas. Notably, for both iciHHV-6A and iciHHV-6B, we found that European and American sequences tend to have high proportions of ancestry from viral populations that experienced considerable drift, suggesting that they underwent one or more bottlenecks followed by population expansion. Finally, analysis of HHV-6B exogenous viruses sampled in Japan indicated that proportions of ancestry components of most of these viruses are different from the majority of those sampled in the USA. More generally, we show that, in both viral species, both integrated and exogenous viral genomes have different ancestry components, partially depending on geographic location. It would be extremely important to determine whether such differences account for the diversity of HHV-6A/HHV-6B-associated clinical symptoms and epidemiology. Also, the sequencing of additional exogenous and integrated viral genomes will be instrumental to confirm and expand our conclusions, which are based on a relatively small number of genomes, sequenced with variable quality, and with unequal sampling in terms of geographic origin.

Project description:Within the family Herpesviridae, sub-family β-herpesvirinae, and genus Roseolovirus, there are only three human herpesviruses that have been described: HHV-6A, HHV-6B, and HHV-7. Initially, HHV-6A and HHV-6B were considered as two variants of the same virus (i.e., HHV6). Despite high overall genetic sequence identity (~90%), HHV-6A and HHV-6B are now recognized as two distinct viruses. Sequence divergence (e.g., >30%) in key coding regions and significant differences in physiological and biochemical profiles (e.g., use of different receptors for viral entry) underscore the conclusion that HHV-6A and HHV-6B are distinct viruses of the β-herpesvirinae. Despite these viruses being implicated as causative agents in several nervous system disorders (e.g., multiple sclerosis, epilepsy, and chronic fatigue syndrome), the mechanisms of action and relative contributions of each virus to neurological dysfunction are unclear. Unresolved questions regarding differences in cell tropism, receptor use and binding affinity (i.e., CD46 versus CD134), host neuro-immunological responses, and relative virulence between HHV-6A versus HHV-6B prevent a complete characterization. Although it has been shown that both HHV-6A and HHV-6B can infect glia (and, recently, cerebellar Purkinje cells), cell tropism of HHV-6A versus HHV-6B for different nerve cell types remains vague. In this study, we show that both viruses can infect different nerve cell types (i.e., glia versus neurons) and different neurotransmitter phenotypes derived from differentiated human neural stem cells. As demonstrated by immunofluorescence, HHV-6A and HHV-6B productively infect VGluT1-containing cells (i.e., glutamatergic neurons) and dopamine-containing cells (i.e., dopaminergic neurons). However, neither virus appears to infect GAD67-containing cells (i.e., GABAergic neurons). As determined by qPCR, expression of immunological factors (e.g., cytokines) in cells infected with HHV-6A versus HHV6-B also differs. These data along with morphometric and image analyses of infected differentiated neural stem cell cultures indicate that while HHV-6B may have greater opportunity for transmission, HHV-6A induces more severe cytopathic effects (e.g., syncytia) at the same post-infection end points. Cumulatively, results suggest that HHV-6A is more virulent than HHV-6B in susceptible cells, while neither virus productively infects GABAergic cells. Consistency between these in vitro data and in vivo experiments would provide new insights into potential mechanisms for HHV6-induced epileptogenesis.

Project description: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.

Dataset Information

Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay.

Publications

Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay.

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