Detection and Typing of Human Herpesvirus 6 by Molecular Methods in Specimens from Patients Diagnosed with Encephalitis or Meningitis

Abstract

Human herpesvirus 6 (HHV-6) was detected in specimens from patients hospitalized with symptoms of encephalitis or meningitis. A real-time PCR assay was developed which has a linear dynamic range of 5 to 5 ؋ 10 6 copies of HHV-6 and a sensitivity of five gene copies per reaction. While the assay detects both subtypes, HHV-6A and HHV-6B, it is specific and does not cross-react with a selected specificity panel. A total of 1,482 patient specimens, which were collected between 2003 and 2007, were tested; 26 specimens from 24 patients were found to be positive for HHV-6 by real-time PCR. The HHV-6 detection rate in this population was therefore 1.75%. The majority of the specimens tested (>95%) were cerebrospinal fluid (CSF) specimens. We were able to type 20 of the 26 positive specimens by conventional PCR and sequence analysis; all were HHV-6B. Forty-two percent of the patients were 3 years of age or younger, which may indicate a primary infection in these patients. Given the ages of the remaining patients (from 4 to 81 years), their infections were most probably due to virus reactivations. Where information was available, symptoms of patients included fever (71%), altered mental status (67%), and abnormal CSF profile (75%). Fifty percent of patients of 3 years of age or younger suffered from seizures. The detection of HHV-6 in specimens from patients diagnosed with encephalitis or meningitis, in the absence of a positive PCR result for other agents, strongly suggests a role for HHV-6 in the pathogenesis of these central nervous system diseases.

Molecular detection of viral causes of encephalitis and meningitis in New York State

“…As an internal extraction and RT control and in order to detect PCR inhibition, each clinical specimen was spiked with a known quantity of GFP transcript during the lysis step of the nucleic acid extraction process [Tavakoli et al, 2007;Hull et al, 2008]. The GFP transcript was made as described above from plasmid pTU65 [Chalfie et al, 1994] and subsequently quantified.…”

“…Amplification was carried out in a 25 ml volume. For assays using DNA template (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, adenovirus) or cDNA template (enterovirus, GFP), the reactions contained universal buffer (Applied Biosystems, Foster City, CA), and were incubated at 958C for 10 min followed by 45 cycles of 958C for 15 sec and 608C for 1 min [Tavakoli et al, 2007. For reactions using total nucleic acid template (WNV, EEE) ABI buffer was used and the thermocycling was as above and included an initial step of 488C for 30 min.…”

Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States

“…A triplex real-time assay was assembled based on previously published primer and probe sequences (13). The assay was composed of two targets in the nucleoprotein gene (N gene) of the rabies virus genome and an exogenous internal control comprised of transcript RNA of green fluorescent protein (20). The first rabies virus target primer pair and probe, RABVD1, were taken directly from the Nadin-Davis et al article (13), while the second primer pair and probe, RABVD2-RC, were modified (S. A. Nadin-Davis, personal communication) ( Table 2).…”

Development of a Human Herpesvirus 6 Species-Specific Immunoblotting Assay

“…Details of the nested PCR for determining the genotype of HHV-6 was described in a previous report (28). In brief, the PCR amplifies a 751-bp DNA fragment containing the gene that encodes a putative large tegument protein (U31 gene) using Taq DNA polymerase (AmpliTaq Gold; Perkin-Elmer, Norwalk, CT).…”

“…Methods for the differentiation between HHV-6A and HHV-6B have been developed based on restriction fragment length polymorphism analysis of PCR products, PCR with virus species-specific primers, and Southern blotting with virus speciesspecific probes (3,4,12,28). Subsequently, real-time PCR methods using virus species-specific primers or probes have been introduced for easier discrimination between the two virus species (6).…”