Detection of smallpox virus DNA by LightCycler PCR

A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [T(m)], 56.40 degrees C), monkeypox virus (T(m), 56.24 degrees C), and vaccinia virus (T(m), 56.72 degrees C), including the Dryvax vaccine strain, from smallpox virus (T(m), 62.45 degrees C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.     

Molecular evidence and clinical significance of herpesvirus coinfection in the central nervous system.

A total of 60 cerebrospinal fluid (CSF) specimens from patients manifesting symptoms resembling viral central nervous system (CNS) disease were examined for the presence of herpes simplex virus (HSV), human herpesvirus 6 (HHV-6), Epstein-Barr virus (EBV), cytomegalovirus, varicella-zoster virus, Borrelia burgdorferi, and Tropheryma whippelii DNA by PCR. Of 30 specimens which were selected on the basis of HSV DNA positivity, 2 were concomitantly positive for HHV-6 DNA and 1 was positive for EBV DNA. In the three specimens positive for more than one herpesvirus, amplicons generated with virus-specific primer sets hybridized specifically to the corresponding virus-specific probe. Sequence analysis of the two amplified DNA fragments demonstrated that they were derived from distinct herpesviruses. Of 22 patients with clinically diagnosed encephalitis, 2 of 3 patients coinfected with HSV and HHV-6 died, compared to 1 of 19 (5%) patients infected with only HSV. Of 30 CSF specimens that were negative for HSV DNA, EBV DNA was detected in one sample. These data indicated the presence of DNA specific for two distinct herpesviruses in the same CSF specimen, providing molecular evidence that coinfection with this group of viruses may occur in the CNS.