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Analysis of ganciclovir-resistant human herpesvirus 6B clinical isolates using quenching probe PCR methodology.

ABSTRACT: Quenching probe PCR (QP-PCR) analysis was used to determine the frequency of ganciclovir (GCV) resistance among clinical isolates of human herpesvirus 6B (HHV-6B) obtained from patients with primary viral infection and viral reactivation. Forty-two HHV-6B clinical isolates were repeatedly recovered from 15 hematopoietic stem cell transplant (HSCT) recipients, and 20 isolates were recovered from 20 exanthem subitum (ES) patients. Of the 15 HSCT recipients, 9 received GCV during the observation period; however, none of the ES patients were treated with GCV. Two established laboratory strains, Z29 and HST, were used as standards in this study. Regions 1 and 2 of the U69 gene of all of the clinical isolates demonstrated the same melting temperature as regions 1 and 2 of the Z29 strain. For region 3, the melting temperatures of all clinical isolates fell between the melting temperature of the plasmid containing the A462D single nucleotide polymorphism (SNP) and the melting temperature of the Z29 strain, and the melting temperatures profiles of all clinical isolates were similar to the melting temperature profile of the Japanese HST strain. As expected, none of the 20 clinical isolates recovered from the ES patients and the 14 isolates recovered from the HSCT recipients who did not receive GCV treatment carried the six known SNPs associated with GCV resistance. Interestingly, these six SNPs were not detected in the 28 clinical isolates recovered from the 9 HSCT recipients who received GCV. Additional sequence analysis of the U69 gene from the 15 representative isolates from the 15 HSCT recipients identified other SNPs. These SNPs were identical to those identified in the HST strain. Therefore, the rate of emergence of GCV-resistant HHV-6B strains appears to be relatively low, even in HSCT recipients treated with GCV.

SUBMITTER: Hiramatsu H

PROVIDER: S-EPMC4394808 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Analysis of ganciclovir-resistant human herpesvirus 6B clinical isolates using quenching probe PCR methodology.

Hiramatsu Hiroyuki H Suzuki Ryota R Yamada Shigeki S Ihira Masaru M Isegawa Yuji Y Kawamura Yoshiki Y Matsuoka Erina E Miura Hiroki H Yoshikawa Tetsushi T

Antimicrobial agents and chemotherapy 20150217 5

Quenching probe PCR (QP-PCR) analysis was used to determine the frequency of ganciclovir (GCV) resistance among clinical isolates of human herpesvirus 6B (HHV-6B) obtained from patients with primary viral infection and viral reactivation. Forty-two HHV-6B clinical isolates were repeatedly recovered from 15 hematopoietic stem cell transplant (HSCT) recipients, and 20 isolates were recovered from 20 exanthem subitum (ES) patients. Of the 15 HSCT recipients, 9 received GCV during the observation pe ...[more]

PMID: 25691643

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Project description:The "omics" revolution of recent years has simplified the study of RNA transcripts produced during viral infection and under specific defined conditions. In the quest to find new and differentially expressed transcripts during the course of human Herpesvirus 6B (HHV-6B) infection, we made use of large-scale RNA sequencing to analyze the HHV-6B transcriptome during productive infection of human Molt-3 T-cells. Analyses were performed at different time points following infection and specific inhibitors were used to classify the kinetic class of each open reading frame (ORF) reported in the annotated genome of HHV-6B Z29 strain. The initial search focussed on HHV-6B-specific reads matching new HHV-6B transcripts. Differential expression of new HHV-6B transcripts were observed in all samples analyzed. The presence of many of these new HHV-6B transcripts were confirmed by RT-PCR and Sanger sequencing. Many of these transcripts represented new splice variants of previously reported ORFs, including some transcripts that have yet to be defined. Overall, our work demonstrates the diversity and the complexity of the HHV-6B transcriptome.IMPORTANCERNA sequencing (RNA-seq) is an important tool for studying RNA transcripts, particularly during active viral infection. We made use of RNA-seq to study human Herpesvirus 6B (HHV-6B) infection. Using six different time points, we were able to identify the presence of differentially spliced genes at 6, 9, 12, 24, 48 and 72 hours post-infection. Determination of the RNA profiles in the presence of cycloheximide (CHX) or phosphonoacetic acid (PAA) also permitted identification of the kinetic class of each ORF described in the annotated GenBank file. We also identified new spliced transcripts for certain genes and evaluated their relative expression over time. These data and next-generation sequencing (NGS) of the viral DNA have led us to propose a new version of the HHV-6B Z29 GenBank annotated file, without changing ORF names in order to facilitate trace back and correlate our work with previous studies on HHV-6B.

Project description:BACKGROUND: Human herpesvirus-6 (HHV-6) is a beta-herpesvirus with 90% seroprevalence that infects and establishes latency in the central nervous system. Two HHV-6 variants are known: HHV-6A and HHV-6B. Active infection or reactivation of HHV-6 in the brain is associated with neurological disorders, including epilepsy, encephalitis, and multiple sclerosis. In a preliminary study, we found HHV-6B DNA in resected brain tissue from patients with mesial temporal lobe epilepsy (MTLE) and have localized viral antigen to glial fibrillary acidic protein (GFAP)-positive glia in the same brain sections. We sought, first, to determine the extent of HHV-6 infection in brain material resected from MTLE and non-MTLE patients; and second, to establish in vitro primary astrocyte cultures from freshly resected brain material and determine expression of glutamate transporters. METHODS AND FINDINGS: HHV-6B infection in astrocytes and brain specimens was investigated in resected brain material from MTLE and non-MTLE patients using PCR and immunofluorescence. HHV-6B viral DNA was detected by TaqMan PCR in brain resections from 11 of 16 (69%) additional patients with MTLE and from zero of seven (0%) additional patients without MTLE. All brain regions that tested positive by HHV-6B variant-specific TaqMan PCR were positive for viral DNA by nested PCR. Primary astrocytes were isolated and cultured from seven epilepsy brain resections and astrocyte purity was defined by GFAP reactivity. HHV-6 gp116/54/64 antigen was detected in primary cultured GFAP-positive astrocytes from resected tissue that was HHV-6 DNA positive-the first demonstration of an ex vivo HHV-6-infected astrocyte culture isolated from HHV-6-positive brain material. Previous work has shown that MTLE is related to glutamate transporter dysfunction. We infected astrocyte cultures in vitro with HHV-6 and found a marked decrease in glutamate transporter EAAT-2 expression. CONCLUSIONS: Overall, we have now detected HHV-6B in 15 of 24 patients with mesial temporal sclerosis/MTLE, in contrast to zero of 14 with other syndromes. Our results suggest a potential etiology and pathogenic mechanism for MTLE.

Dataset Information

Analysis of ganciclovir-resistant human herpesvirus 6B clinical isolates using quenching probe PCR methodology.

Publications

Analysis of ganciclovir-resistant human herpesvirus 6B clinical isolates using quenching probe PCR methodology.

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