[Show abstract][Hide abstract] ABSTRACT: A simplified microneutralization procedure is described that uses an empirically determined virus challenge dose, a single dilution of antiserum, and observation of cytopathic effect to determine the adenovirus serotype. The simplified test has faster turnaround time and was 96% concordant with a confirmatory test using serial dilutions of type-specific sera. This method will find utility in high-volume serotyping work.
Journal of Clinical Microbiology 09/2001; 39(8):2984-6. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular characterization of rabies virus has been used to trace spillover transmission from reservoir species to non-reservoir animals and humans (molecular epidemiology), and to monitor emergence of specific strains of the virus into new species and geographic areas (molecular surveillance).
To characterize the enzootic strains of rabies virus in California wildlife for epidemiological investigation of transmission to non-reservoir animals and humans.
Molecular characterization was performed on rabies strains from 213 bats, 276 terrestrial animals and one human case, by RT-PCR amplification of the viral nucleocapsid (N) gene followed by Dde I digestion and restriction endonuclease analysis (REA). Brain material from 88 terrestrial animals and 74 bats was stained with a panel of 20 monoclonal antibodies (MABs) directed against the N protein. In order to characterize rabies from very small quantities of brain tissue a nested RT-PCR was developed and evaluated for sensitivity of rabies detection.
Enzootic terrestrial rabies in California is confined to the Central Valley, the western slope of the Sierra Nevada, and the Central and Northern Pacific Coast Ranges. No terrestrial reservoirs were identified south of the Tehachapi Mountains or east of the Sierra Nevada. Bat strains accounted for rabies in terrestrial animals in these regions. Among terrestrial rabies strains REA identified ten genotypes that segregated geographically and were associated with skunk and fox populations from distinct locations. Co-circulation of three genotypes occurred in Placer County, which had the highest incidence of rabies in skunks. In regions with terrestrial enzootic rabies, the strain from that region accounted for 73% of spillover cases. Bat strains accounted for the remaining 27%. Among terrestrial animals MABs identified three predominant patterns. In rabies strains from bats REA identified ten major and two minor patterns primarily associated with genus and species of bat. Sharing of strains between species was observed. An additional sixteen unclassified REA bat patterns were observed in only one or two individuals of various species. MABs identified four major patterns in bats associated with genus and species of bat with considerable variability. The bat strains most frequently detected in spillover cases throughout California were from the California myotis (Myotis californicus) and Mexican free-tailed bat (Tadarida brasiliensis). Nested RT-PCR increased the detection level of rabies virus 100,000-fold, to 0.03 TCID50.
Journal of Clinical Virology 12/1999; 14(3):207-19. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to determine the suitability of vaccine strains established in the 1960s for a new vaccine, a comprehensive study of strain variation of adenovirus serotype 4 (AV 4) and AV 7 was undertaken. A 1,500-bp region of the hexon gene containing the AV neutralization epitopes from prototype, vaccine, and community-acquired strains and from wild-type strains from military personnel that cause acute respiratory disease (ARD) was sequenced and analyzed. The whole hexon gene from prototype strains, vaccine strains, and selected isolates was sequenced. AV 7 and AV 7a were found to have distinct genotypes, and all vaccine and wild-type strains recovered from 1963 to 1997 had the AV 7a genotype. There was no significant strain variation in the neutralization epitopes of the AV 7a genotype over a 42-year period. The evolution of AV 4 was more complex, with continuous genetic drift punctuated by replacement with a new strain. The current strain of AV 4, which has been in circulation since 1995, is significantly different from the AV 4 prototype and the vaccine strains. Genetic differences were confirmed to be antigenic differences by neutralization tests, which define the new strain as an AV 4 variant. A type-specific PCR for AV 4, AV 7/7a, and AV 21 was developed, and this PCR facilitated the rapid identification of isolates from outbreaks of ARD.
Journal of Clinical Microbiology 05/1999; 37(4):1107-12. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The origin of AIDS-associated adenoviruses (AV 43-AV 49) was investigated by examining evolutionary relationships among 18 serologically related subgenus D serotypes and 3 intermediates and determining the mutation rate of a single serotype, AV 48, among clinical isolates from AIDS patients over a 6-year period. Nucleotide sequence of conserved and seven hypervariable regions (HVRs) of the hexon protein, the pVI core protein signal peptide, and noncoding region between the two genes was determined. Among AV 48 isolates the base misincorporation rate was 3.2 per 10,000 bases over 6 years. A 6-bp deletion occurred in one isolate between short direct repeats in HVR 7. Among subgenus D serotypes mutation rates were extremely low in the pVI peptide, the 5' hexon noncoding region, and first 187 bases of hexon protein. Small 2- and 3-bp deletions between short direct repeats in a polypurine stretch in the noncoding region occurred in 3 strains. Mutation increased with proximity to the HVRs. Within HVR 1, 2, 4, 5, and 7 variability consisted of extensive intrachromosomal illegitimate recombination, including deletions between short direct repeats, insertions and duplications in repetitive polypurine stretches, and numerous base substitutions. All serotypes and intermediates differed by at least one illegitimate recombination event, with one exception. We conclude that AV serotype evolution is driven by illegitimate recombination events (antigenic shift), concommitant with single base mutation (antigenic drift), and that the HVRs are "hot spots" for both. These events could be explained by slippage-misalignment of the AV DNA polymerase in repetitive polypurine stretches during single-strand DNA replication. This mutability in the surface regions of the major viral coat protein confers a distinct survival advantage to this family of viruses.
[Show abstract][Hide abstract] ABSTRACT: A mixture of adenoviruses 31 and 49 was isolated from the brain of an AIDS patient with encephalitis. Adenovirus hexon protein was detected in neurons by indirect immunofluorescence. By restriction endonuclease analysis both adenovirus 31 and 49 were shown to be new genotypes. This is the first report of the isolation of a mixture of adenoviruses from adenovirus encephalitis and the first association of adenovirus 49, a new candidate serotype, with encephalitis.
Journal of Medical Virology 11/1995; 47(2):168-71. · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A microneutralization assay that automates the reading and interpretation of viral infectivity and neutralization data was developed for the characterization of adenoviruses (AV). Virus, serum, and cells were added to 96-well plates, incubated for 7 days, and stained with vital stain. The A550 of solubilized dye was read in a plate reader interfaced to a personal computer which analyzed the results. Correlation of A550 values with visual observation of cytopathic effect was extremely good (r = 0.977625). Clinical isolates of 17 AV from 11 patients were characterized by colorimetric neutralization assay. Prototype AV titers tested were comparable to those determined by tube methods. Prototype homotypic antiserum titers were comparable to or greater than those determined by standard tube neutralization.
Journal of Clinical Microbiology 10/1994; 32(9):2331-4. · 4.07 Impact Factor