RNA sequencing of the in vivohuman 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 multiplein vivoandin vitrosample 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 inin vivoandin vitrosamples, 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.IMPORTANCEInfection 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.