[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.
[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.
No preview · Article · Dec 1999 · Journal of Clinical Virology
[Show abstract][Hide abstract] ABSTRACT: We report two cases of human granulocytic ehrlichiosis (HGE) that occurred in northern California in summer 1998. Patients
had fever, malaise, and myalgia, reported tick bites, had moderate thrombocytopenia, and had normal or slightly elevated liver
enzyme activities. Ehrlichial inclusions were observed in the blood of one patient, and HGE-agent DNA was amplified by PCR
from both patients. Genetically, the strains resembled horse isolates from northern California. The close spatial and temporal
proximity of the two new cases may be due to a nidus of infection in the area or heightened surveillance by local physicians.
[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.
Preview · Article · May 1999 · Journal of Clinical Microbiology
[Show abstract][Hide abstract] ABSTRACT: The 1996 production halt of adenovirus types 4 and 7 vaccines prompted concerns about the resurgence of large respiratory
disease outbreaks among US military basic trainees. This serosurvey was conducted to assess the current susceptibility of
the trainee population to these viruses. A stratified, random sample (n = 303) of trainees' sera was tested using a quantitative colorimetric microneutralization assay to demonstrate antibody titers
considered to provide immunologic protection against each adenovirus type. Results were analyzed for relationships between
susceptibility and 4 demographic factors—gender, race, prior military service, and age. Results showed that 66% and 73% of
trainees were susceptible to serotypes 4 and 7, respectively. Nearly 90% were susceptible to at least one serotype. Susceptibility
was significantly (P < .05) related to lack of prior military service and younger age. Consistent with a serosurvey conducted 20 years ago, these
results demonstrated significant susceptibility to two vaccine-preventable causes of disease. These findings may have civilian
Full-text · Article · Jan 1999 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: A seroprevalence survey to recently proposed adenovirus (AV) serotypes AV 48 and AV 49, isolated primarily from AIDS patients, was conducted among the San Francisco Men's Health Study cohort. This cohort of homosexual, heterosexual, or bisexual HIV-seronegative and -seropositive men from selected San Francisco census tracts has been studied since 1984. The presence or absence of type-specific antibody in 628 serum specimens from 1989 was determined by microneutralization. Thirty of these subjects (26 positive and four negative) were studied longitudinally. Serum specimens taken at 6-month intervals from 1984 to 1993 were tested to characterize antibody response and to document the advent of these new serotypes. Eight subjects were tested against five other AV serotypes for comparison. AV 48 and AV 49 seroprevalence rates were significantly higher in HIV-seropositives, but infection was not limited to the immunocompromised. Sexual preference was not a significant determinant for AV seroprevalence in HIV-seronegatives. However, the extent and duration of the neutralizing antibody response was strikingly different between homosexuals and heterosexuals: an endemic pattern of continuous reexposure over the 9-year period was seen in 90% of 19 homosexuals, while five of six heterosexuals (83%) had an episodic pattern of exposure with antibody decline to undetectable levels. These data suggest that these viruses may be endemic in some part of the homosexual population and that sexual transmission may be the primary source of continuous reexposure.
No preview · Article · Nov 1996 · Journal of Medical Virology
[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: The first full-length hexon protein DNA and deduced amino acid sequences of a subgenus D adenovirus (AV) were determined from candidate AV48 (85-0844). Comprehensive comparison of this sequence with hexon protein sequences from human subgenera A, B, C, D, F, bovine AV3, and mouse AV1 revealed seven discrete hypervariable regions (HVRs) among the 250 variable residues in loops 1 and 2. These regions differed in length between serotypes, from 2 to 38 residues, and contained > 00% of hexon serotype-specific residues among human serotypes. Alignment with the published crystal structure of AV2 established the location and structure of the type-specific regions. Five HVRs were shown to be part of linear loops on the exposed surfaces of the protein, analogous to the serotype-specific loops or "puffs" in picornavirus capsid proteins. The HVRs were supported by a common framework of conserved residues, of which 68 to 75% were hydrophobic. Unique sequences were limited to the seven HVRs, so that one or more of these regions contain the type-specific neutralization epitopes. A neutralizing AV48 hexon-specific antiserum recognized linear peptides that corresponded to six HVRs by enzyme immunoassay. Affinity-purification removal of all peptide-reactive antibodies did not significantly decrease the neutralization titer. Eluted peptide-reactive antibodies did not neutralize. Human antisera that neutralized AV48 did not recognize linear peptides. Purified trimeric native hexon inhibited neutralization, but monomeric heat-denatured hexon did not. We conclude that the AV48 neutralization epitope(s) is complex and conformational.
Preview · Article · Mar 1996 · Journal of Virology
[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.
No preview · Article · Oct 1995 · Journal of Medical Virology
[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.
Preview · Article · Oct 1994 · Journal of Clinical Microbiology
[Show abstract][Hide abstract] ABSTRACT: Neutralizing antibody responses to human immunodeficiency virus type 1 (HIV-1) vary widely and have not been reproducibly associated with prognosis or disease progression. We have found that both low-passage clinical isolates and laboratory-adapted strains of HIV-1 have different sensitivities to neutralization by the same antiserum, depending on the host cell in which the viral stock is prepared. One such isolate (VL069) grown in H9 cells was neutralized by 20 human sera at a geometric mean titer of 1:2,047; this same isolate prepared in peripheral blood mononuclear cell (PBMC) culture was neutralized at a mean titer of < 1:10 by the same sera. Adsorption and mixing experiments indicated that neither antibody to H9 cell components nor blocking by excess viral antigen was responsible for the differences observed. This host cell effect is rapidly reversible upon passage of the virus from PBMCs to H9 cells and back into PBMCs. In contrast, the neutralization characteristics remained remarkably stable over extended culture in PBMCs. Two laboratory strains and five clinical isolates were evaluated in expanded studies of this phenomenon. While the neutralization characteristics of most of the strains studied were affected by the host cell in which the strain was propagated, two of the strains (one clinical isolate and one laboratory strain) appeared antigenically unaffected by their cell of origin. Host cell effect was also evident in neutralization by monoclonal antibodies directed against the CD4-binding region and the V2, V3, and gp41 regions. Possible mechanisms for this host cell effect include (i) mutation during passaging; (ii) selection in different host cells of different subpopulations of the (uncloned) viral stock; and (iii) cell-specific posttranslational modifications. To explore these possibilities, the V3 through V5 region of gp120 was sequenced in preparations made by passing VL069 into H9 cells and into PBMCs; HIVMN grown in CEM-SS cells and in PBMCs was also sequenced. In both cases, a few amino acid changes outside the V3 region were found. Studies are currently under way to assess the significance of these changes.
Preview · Article · Mar 1994 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: To perform a human immunodeficiency virus (HIV) plaque assay in nonadherent host cells, we developed a novel technique in which HIV-infected MT-2 cells were formed into monolayers by centrifugation through molten agarose. Infection, formation of cell monolayers, and enumeration of plaques all took place in 96-well microtiter plates. When this process was preceded by 18 h of incubation of HIV with patient serum samples, neutralizing antibody titers between 1:10 and 1:5,000 could be accurately determined in patient serum samples. In addition to the determination of neutralizing antibody titers (with the use of various serum dilutions and a constant virus concentration), neutralization indices could also be determined with different virus dilutions and a single dilution of patient serum.
Preview · Article · Oct 1990 · Journal of Clinical Microbiology