Quantitative real-time PCR for detection of monkey B virus (Cercopithecine herpesvirus 1) in clinical samples

Abstract

A TaqMan based real-time PCR assay was developed for rapid detection and quantitation of herpes B virus (Cercopithecine herpesvirus 1) in clinical samples. The assay utilizes B virus-specific primers and a probe to the non-conserved region of the gG gene to discriminate B virus from closely related alphaherpesviruses. Fifty copies of B virus DNA could be detected with 100% sensitivity with a wide range of quantitation spanning 6 logs. The assay was highly reproducible with intra- and inter-assay coefficients of variation of 0.6 and 2.4%, respectively. Clinical utility of the developed real-time PCR was evaluated by testing genomic DNA prepared from B virus clinical isolates (n=23) and human and monkey clinical specimens (n=62). This novel method was also compared with conventional cell culture with respect to sensitivity and specificity. TaqMan PCR assay was shown to be equally specific and more sensitive than culture method (culture vs. PCR sensitivity 50%) and was able to identify all B virus clinical isolates tested. Fast, reliable assessment of B virus DNA in infected cells and tissues makes real-time PCR assay a valuable tool for diagnosis and management of B virus infections.

Introduction

Zoonosis caused by herpes B virus (Palmer, 1987, Whitley and Hilliard, 2001), heads the list of feared acquired infections in individuals working with macaque monkeys, which are useful as a model for human immunodeficiency virus (HIV)-related investigations. Early diagnosis of this often-fatal zoonosis is associated with reduced morbidity and mortality, and thus, rapid identification of infection following exposure of humans to this agent is critical (Cohen et al., 2002, Davenport et al., 1994, Holmes et al., 1990).

Although virus isolation is still considered the ‘gold standard’ for diagnosis of B virus infections, more rapid and sensitive PCR assays have been developed and applied to detect B virus DNA in clinical specimens (Black and Eberle, 1997, Scinicariello et al., 1993a, Scinicariello et al., 1993b, Slomka et al., 1993). However, none of the current PCR tests is well suited for high-throughput applications because they require multiple post-PCR manipulations, e.g. restriction endonuclease digestion, gel-electrophoresis, and hybridization. While much progress has been made toward the identification of B virus infections in both human and non-human primates, none of the existing assays can quantify viral load in clinical specimens. Quantitation of viral load in cerebrospinal fluid (CSF) samples is potentially valuable for monitoring of the efficacy of drug treatment in B virus-infected individuals.

TaqMan real-time PCR facilitates simultaneous amplification and quantification without any further procedures required for analysis of the amplified products. Contamination risk is reduced and a large number of samples can be processed rapidly. Recently, TaqMan PCR assays have been developed to quantify a variety of human pathogens including herpesviruses (Brengel-Pesce et al., 2002, Ryncarz et al., 1999, Yun et al., 2000), HIV type 1 and 2 (Desire et al., 2001, Schutten et al., 2000), and hepatitis B and C (Mercier et al., 1999).

This paper describes the development and evaluation of TaqMan real-time PCR assay for rapid detection and measurement of B virus DNA in clinical specimens. Specificity and sensitivity of the developed assay were determined by comparative analysis of results obtained by this test versus conventional cell culture for virus identification.