No abstract

A consensus primer PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) and which uses degenerate primers in a nested format was developed. Primers were designed to target sequences coding for highly conserved amino acid motifs covering a region of approximately 800 bp. The assay was applied to 22 species of herpesviruses (8 human and 14 animal viruses), with PCR products obtained for 21 of 22 viruses. In the process, 14 previously unreported amino acid-coding sequences from herpesviral DNA polymerases were obtained, including regions of human herpesviruses 7 and 8. The 50 to 60 amino acid-coding sequences recovered in the present study were determined to be unique to each viral species studied, with very little sequence variation between strains of a single species when studied. Template dilution studies in the presence of human carrier DNA demonstrated that six human herpesviruses (herpesviruses 1, 2, 3, 4, 5, and 6B) could be detected at levels at or below 100 genome equivalents per 100 ng of carrier DNA. These data suggest that consensus primer PCR targeted to herpesviral DNA polymerase may prove to be useful in the detection and identification of known herpesviruses in clinical samples and the initial characterization of new herpesviral genomes.

Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in >70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.Multiple sclerosis (MS) is a disease of young adults that is characterized clinically by a variable relapsing and remitting course and pathologically by the progressive accumulation of plaques of demyelination within the white matter of the central nervous system. In normal white matter, the axons of neurons are surrounded by myelin sheaths, made from the cell membranes of oligodendrocytes. In MS plaques, the myelin sheaths are destroyed, leaving the naked axons intact but impaired in their conduction of action potentials. The currently held view is that an autoimmune inflammatory reaction against components of myelin results in destruction of oligodendrocytes. The demyelinating lesions in MS are found throughout the central nervous system, with a predilection for the periventricular white matter, optic nerve, brainstem, spinal cord, and cerebellum, resulting in a disease that is pleiomorphic in its clinical presentation.In spite of the substantial evidence that autoimmunemediated demyelination plays a major role in the progression of MS, epidemiologic studies suggest that an infectious agent may also be involved (1). Prior reports have suggested that viral infection of cells within the central nervous system may initiate the events leading to focal demyelination (2), and a number of viruses have been implicated in the pathogenesis of MS (3). Despite extensive investigation, however, none of these associations has been confirmed (4), and the issue of viral involvement in the pathogenesis of MS remains unresolved.To search for an MS-associated pathogen, we used representational difference analysis (RDA) (5). In RDA, successive rounds of subtractive hybridization and PCR amplification enriched for DNA sequences that were present in a DNA preparation from diseased tissue (MS brain) but absent from control DNA (n...

SummaryThe Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signaturetag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.