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We tested for measles, mumps, and rubella viruses in multiple sclerosis by polymerase chain reaction (PCR). Using RNA extracted from 19 multiple sclerosis and 8 control brain specimens, nested PCR was performed after reverse transcription (RT) of the RNA to cDNA using primer pairs directed against two regions in the genomes of measles and mumps viruses and one region in the rubella virus genome. Despite enhanced sensitivity of nested RT PCR, measles, mumps, and rubella viral genomic sequences were not found in any brain specimen.

The controversy over the endemicity of human T cell lymphotropic virus type I (HTLV-I) in Melanesia has been settled recently by the isolation of genetically distinct, highly divergent sequence variants of HTLV-I from unrelated inhabitants of Papua New Guinea and the Solomon Islands. Still at issue, however, is the significance of the high frequency of indeterminate HTLV-I Western blots (defined as reactivity to only gag-encoded proteins) among Melanesians. To investigate whether this indeterminate seroreactivity reflects specific reactivity to the Melanesian HTLV-I variants, 27 seroindeterminate Melanesians from Papua New Guinea and the Solomon Islands were studied for evidence of HTLV-I infection. Although antibodies against Melanesian variant-specific env gene products and variant-specific env gene sequences were detected by Western blot analysis and polymerase chain reaction, respectively, in all 11 HTLV-I Western blot-positive Melanesians, none of the 27 seroindeterminate Melanesians had such variant-specific antibodies or HTLV-I proviral sequences. In addition, attempts to isolate HTLV-I from seroindeterminate individuals were unsuccessful. These data indicate that HTLV-I infection is not the cause of the indeterminate Western blot reactivity seen in Melanesia.

Detection of hantaviruses, the etiological agents of hemorrhagic fever with renal syndrome (HFRS), by virus isolation using experimental animals or cell culture is time-consuming. A more rapid but equally specific method is needed. We used a reverse transcriptase-directed polymerase chain reaction (RT-PCR) to detect hantavirus genomic sequences and compared its sensitivity with conventional virus isolation. RNA, extracted by the guanidinium isothiocyanate-cesium chloride method from hantavirus-infected Vero E6 cells and from tissues of infant mice inoculated intracerebrally with 100 LD50 of hantavirus, was initially reverse transcribed using avian myeloblastosis virus reverse transcriptase. The resulting complementary DNA (cDNA) was used as template to amplify the glycoprotein 2-encoding region of the hantavirus M segment. With this method, Vero E6 cell cultures infected with Hantaan virus strains 76-118 (prototype) and HV114 (an isolate from the urine of an HFRS patient in China) were positive, while control cultures were negative. Brain, lung, and heart tissues from hantavirus-infected mice were positive by RT-PCR at 5, 8, and 11 days after intracerebral inoculation. The specificity of the positive results was confirmed by restriction endonuclease digestion of the amplified fragments with AluI and HpaI. The sensitivity of the RT-PCR was equal to cell culture amplification but required less time. This method is being adapted for detection of hantavirus genomic sequences in clinical specimens and postmortem tissues from patients with HFRS.

RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.