Project description:Roseolovirus, or human herpesvirus 6 (HHV-6) is a ubiquitous human pathogen infecting over 95% of the population by the age of two years. As with other herpesviruses, reactivation of HHV-6 can present with severe complications in immunocompromised individuals. Recent studies have highlighted the importance of herpesvirus-derived micro (mi)RNAs in modulating both cellular and viral gene expression. An initial report, which computed the likelihood of various viruses to encode for miRNAs, did not predict HHV-6 miRNAs. To experimentally screen for small HHV-6 encoded RNAs, we conducted large-scale sequencing of Sup-T-1 cells lytically infected with a laboratory strain of HHV-6B. This revealed an abundant 60-65 nucleotide RNA of unknown function derived from the lytic origin of replication (OriLyt) that gave rise to smaller RNA species of 18-19 nucleotides in length. In addition, we identified four pre-miRNAs, whose mature forms accumulated in Argonaute 2. In contrast to other beta-herpesviruses, HHV-6B miRNAs are expressed from direct repeat regions (DRL and DRR) located at either side of the genome. All miRNAs are conserved in the closely related HHV-6A variant, and one of them is a seed ortholog of the human miR-582-5p. Similar to alpha-herpesvirus miRNAs, they are expressed antisense to immediate early ORFs and thus have the potential to regulate key viral regulators. Small RNA sequencing from total RNA or Ago2 associated small RNAs extracted from HHV-6 infected Sup-T-1 cells
Project description:Limited understanding of the immunopathogenesis of human herpesvirus 6B (HHV-6B) has prevented its acceptance as a pulmonary pathogen after hematopoietic cell transplantation (HCT). We conducted a prospective multicenter study of patients undergoing bronchoalveolar lavage (BAL) for pneumonia after allogeneic HCT. We tested blood and BAL fluid (BALF) for HHV-6B DNA and mRNA transcripts associated with lytic infection and performed RNA-seq on paired blood. Among 116 participants, HHV-6B DNA was detected in 37% of BALs, 49% of which had HHV-6B mRNA detection. We established an HHV-6B DNA threshold (≥2.3 log10copies/ml in BALF) that was highly predictive of HHV-6B mRNA detection and increased risk for death from respiratory failure (adjusted HR, 2.35; 95% CI, 1.08-5.11). Participants with HHV-6B DNA in BALF exhibited distinct host gene expression signatures, notable for enriched interferon signaling pathways in participants clinically diagnosed with idiopathic pneumonia. These data implicate HHV-6B as a pulmonary pathogen after allogeneic HCT.
Project description:Roseolovirus, or human herpesvirus 6 (HHV-6) is a ubiquitous human pathogen infecting over 95% of the population by the age of two years. As with other herpesviruses, reactivation of HHV-6 can present with severe complications in immunocompromised individuals. Recent studies have highlighted the importance of herpesvirus-derived micro (mi)RNAs in modulating both cellular and viral gene expression. An initial report, which computed the likelihood of various viruses to encode for miRNAs, did not predict HHV-6 miRNAs. To experimentally screen for small HHV-6 encoded RNAs, we conducted large-scale sequencing of Sup-T-1 cells lytically infected with a laboratory strain of HHV-6B. This revealed an abundant 60-65 nucleotide RNA of unknown function derived from the lytic origin of replication (OriLyt) that gave rise to smaller RNA species of 18-19 nucleotides in length. In addition, we identified four pre-miRNAs, whose mature forms accumulated in Argonaute 2. In contrast to other beta-herpesviruses, HHV-6B miRNAs are expressed from direct repeat regions (DRL and DRR) located at either side of the genome. All miRNAs are conserved in the closely related HHV-6A variant, and one of them is a seed ortholog of the human miR-582-5p. Similar to alpha-herpesvirus miRNAs, they are expressed antisense to immediate early ORFs and thus have the potential to regulate key viral regulators.
Project description:Purpose: The goal of this study is to determine the expression profile of HHV-6B in whole blood samples from transplant patients with HHV-6B genomes detected in plasma Methods: Viral mRNA profiles in whole blood samples of different transplant patients were generated by deep sequencing using Illumina Hiseq. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation for U38 expression was performed using TaqMan Results: Hierarchical clustering uncovered systematic detection of U38 mRNA transcripts in whole blood samples from all patients with a detectable HHV-6B reactivation in their plasma Conclusions: Our study represents the first detailed analysis of viral transcriptomes associated with HHV-6B reactivation in whole blood samples generated by RNA-seq technology. Our results show that NGS offers a comprehensive and more accurate qualitative evaluation of mRNA content within a whole blood sample.
Project description:Human herpesvirus 6 (HHV-6) A and B are highly ubiquitous betaherpesviruses, infecting the majority of the human population. Like other herpesviruses, our understanding of their protein coding potential is far from complete. Here we use ribosome profiling and RNA-seq to experimentally define the HHV-6 translation products and to follow their temporal expression. We identified hundreds of new open reading frames (ORF)s, including many upstream ORFs (uORF)s and internal ORFs (iORF)s, generating a complete atlas of HHV-6 translation products. Integrating data from human cytomegalovirus we uncover numerous uORFs and iORFs that are conserved between beta herpesviruses and we show uORFs are specifically enriched in late viral genes. We also identified three highly abundant viral long non coding RNAs (lncRNA)s and we show one of these lncRNAs generate a non-polyadenylated stable intron that is conserved between all sequenced beta herpesviruses. Overall, this work uncovers the full complexity of the HHV-6 family genomes and highlights novel features that are conserved between beta herpesviruses, providing a resource for future functional studies.
Project description:The immune response to viral infection represents complex network of dynamic gene and protein interactions. We present here the dynamic gene network of host immune response during human herpesvirus type 6 (HHV-6) infection in a adult T cell leukemia (ATL) cell line. Using a pathway-focused oligonucleotide DNA microarray, we found a possible association between chemokines genes regulating Th1/Th2 balance and genes regulating T-cell proliferation during HHV-6B infection. Keywords: Time course experiment