Quantitative real-time PCR for detection of monkey B virus (Cercopithecine herpesvirus 1) in clinical samples
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.
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Viruses
Herpes simplex 1 (HSV-1) strain KOS (ATCC VR-1493), HSV-2 strain 186 (a kind gift of Dr R. Courtney, The Pennsylvania State University College of Medicine, Hershey, PA), herpesvirus papio 2 (HVP-2) strain X2980 (Levin et al., 1988), simian agent 8 (SA8) strain B264 (ATCC VR-936), B virus laboratory strain E2490 (a kind gift of late Dr R.N. Hull) were used in this study. B virus clinical strains, including 11 rhesus macaque (Macaca mulatta), three Japanese macaque (Macaca fuscata), two
Specificity of gGBV-TaqMan PCR assay for detection of B virus DNA
The PCR primers and probe were selected from the 5′ region of the B virus gG gene, which is significantly different from the HSV-2 homologue and not present in the HSV-1 gG gene (Slomka et al., 1995). To verify that the chosen primers and probe were indeed B virus-specific, PCR amplification was performed using approximately 100 ng of purified B virus, HSV-1, HSV-2, HVP-2, and SA8 genomic DNA per reaction. Only amplification of B virus DNA was detected (Fig. 1A), indicating that the selected
Discussion
In this study, we developed a real-time TaqMan PCR assay for detection of B virus DNA utilizing primers and probe derived from the gG gene sequence. Although the gG gene exhibits considerable sequence variation (Smith et al., 1998), a total of 23 available B virus strains, isolated from humans and three macaque species were successfully amplified using selected gG primers/probe combination, thus confirming high diagnostic value of the developed test. At the same time, no amplification of DNA
Acknowledgements
This work was supported by Public Health Service grant R01 RR03162 and P40 RR05162 from the NIH's National Center for Research Resources.
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