Boeckh, M. | Search bioRxiv

bioRxiv2018

RNA sequencing of the in vivo human herpesvirus 6B transcriptome to identify targets for clinical assays distinguishing between latent and active infections

Human herpesvirus 6B (HHV-6B) DNA is frequently detected in human samples, especially after hematopoietic cell transplantation (HCT). Diagnostic assays distinguishing HHV-6B reactivation from latency are limited, and this has contributed to confusion in research and made the design of clinical approaches to diagnose and treat HHV-6-associated diseases challenging. We used RNA sequencing to characterize and compare the HHV-6B transcriptome in multiple in vivo and in vitro sample types, including 1) whole blood from HCT recipients with and without HHV-6B plasma viremia; 2) tumor tissue samples from subjects with large B cell lymphoma infected with HHV-6B; 3) lymphoblastoid cell lines from subjects with inherited chromosomally integrated HHV-6B or latent infection with HHV-6B; and 4) HHV-6B Z29 infected SupT1 CD4+ T cells. We demonstrated substantial overlap in the HHV-6B transcriptome observed in in vivo and in vitro samples, although there was variability in the breadth and quantity of gene expression across samples. No HHV-6B transcripts were detected in whole blood samples from subjects without plasma HHV-6B viremia. The HHV-6B viral polymerase gene U38 was the only HHV-6B transcript detected in all RNA-seq data sets and was one of the most highly expressed genes. Using a novel reverse transcription PCR assay targeting HHV-6B U38, we identified U38 messenger RNA in all tested whole blood samples from patients with concurrent HHV-6B viremia, indicating its utility as a diagnostic assay for HHV-6B replication. This study demonstrates the feasibility of pathogen transcriptome analyses in HCT recipients to identify better targets for diagnostic, and potentially therapeutic, applications.\n\nIMPORTANCEInfection with human herpesvirus 6B (HHV-6B), a DNA virus, occurs early in life, results in chronic viral latency in diverse cell types, and affects the population at large. Additionally, HHV- 6B can integrate into germline chromosomes, resulting in individuals with viral DNA in every nucleated cell. Given that PCR to detect viral DNA is the mainstay for diagnosing HHV-6B infection, the characteristics of HHV-6B infection complicate efforts to distinguish between latent and active viral infection, particularly in immunocompromised patients who have frequent HHV- 6B reactivation. In this study, we used RNA sequencing to characterize the HHV-6B gene expression profile in multiple sample types, and our findings identified evidence-based targets for diagnostic tests that distinguish between latent and active viral infection.

genomics↗

bioRxiv2017

Genomic and proteomic analysis of Human herpesvirus 6 reveals distinct clustering of acute versus inherited forms and reannotation of reference strain

Human herpesvirus-6A and -6B (HHV-6) are betaherpesviruses that reach >90% seroprevalence in the adult population. Unique among human herpesviruses, HHV-6 can integrate into the subtelomeric regions of human chromosomes; when this occurs in germ line cells it causes a condition called inherited chromosomally integrated HHV-6 (iciHHV-6). To date, only two complete genomes are available for HHV-6B. Using a custom capture panel for HHV-6B, we report near-complete genomes from 61 isolates of HHV-6B from active infections (20 from Japan, 35 from New York state, and 6 from Uganda), and 64 strains of iciHHV-6B (mostly from North America). We also report partial genome sequences from 10 strains of iciHHV-6A. Although the overall sequence diversity of HHV-6 is limited relative to other human herpesviruses, our sequencing identified geographical clustering of HHV-6B sequences from active infections, as well as evidence of recombination among HHV-6B strains. One strain of active HHV-6B was more divergent than any other HHV-6B previously sequenced. In contrast to the active infections, sequences from iciHHV-6 cases showed reduced sequence diversity. Strikingly, multiple iciHHV-6B sequences from unrelated individuals were found to be completely identical, consistent with a founder effect. However, several iciHHV-6B strains intermingled with strains from active pediatric infection, consistent with the hypothesis that intermittent de novo integration into host germline cells can occur during active infection Comparative genomic analysis of the newly sequenced strains revealed numerous instances where conflicting annotations between the two existing reference genomes could be resolved. Combining these findings with transcriptome sequencing and shotgun proteomics, we reannotated the HHV-6B genome and found multiple instances of novel splicing and genes that hitherto had gone unannotated. The results presented here constitute a significant genomic resource for future studies on the detection, diversity, and control of HHV-6.\n\nAuthor SummaryHHV-6 is a ubiquitous large DNA virus that is the most common cause of febrile seizures and reactivates in allogeneic stem cell patients. It also has the unique ability among human herpesviruses to be integrated into the genome of every cell via integration in the germ line, a condition called inherited chromosomally integrated (ici)HHV-6, which affects approximately 1% of the population. To date, very little is known about the comparative genomics of HHV-6. We sequenced 61 isolates of HHV-6B from active infections, 64 strains of iciHHV-6B, and 10 strains of iciHHV-6A. We found geographic clustering of HHV-6B strains from active infections. In contrast, iciHHV-6B had reduced sequence diversity, with many identical sequences of iciHHV-6 found in individuals not known to share recent common ancestry, consistent with a founder effect from a remote common ancestor with iciHHV-6. We also combined our genomic analysis with transcriptome sequencing and shotgun proteomics to correct previous misannotations of the HHV-6 genome.

microbiology↗