Beyond viruses: clinical profiles and etiologies associated with encephalitis.

BACKGROUND: Encephalitis is a complex syndrome, and its etiology is often not identified. The California Encephalitis Project was initiated in 1998 to identify the causes and further describe the clinical and epidemiologic characteristics of encephalitis. METHODS: A standardized report form was used to collect demographic and clinical data. Serum, cerebrospinal fluid, and respiratory specimens were obtained prospectively and were tested for the presence of herpesviruses, arboviruses, enteroviruses, measles, respiratory viruses, Chlamydia species, and Mycoplasma pneumoniae. The association between an identified infection and encephalitis was defined using predetermined, organism-specific criteria for confirmed, probable, or possible causes. RESULTS: From 1998 through 2005, a total of 1570 patients were enrolled. Given the large number of patients, subgroups of patients with similar clinical characteristics and laboratory findings were identified. Ten clinical profiles were described. A confirmed or probable etiologic agent was identified for 16% of cases of encephalitis: 69% of these agents were viral; 20%, bacterial; 7%, prion; 3%, parasitic; and 1%, fungal. An additional 13% of cases had a possible etiology identified. Many of the agents classified as possible causes are suspected but have not yet been definitively demonstrated to cause encephalitis; these agents include M. pneumoniae (n=96), influenza virus (n=22), adenovirus (n=14), Chlamydia species (n=10), and human metapneumovirus (n=4). A noninfectious etiology was identified for 8% of cases, and no etiology was found for 63% of cases. CONCLUSIONS: Although the etiology of encephalitis remains unknown in most cases, the recognition of discrete clinical profiles among patients with encephalitis should help focus our efforts toward understanding the etiology, pathogenesis, course, and management of this complex syndrome.

Molecular epidemiology of enzootic rabies in California.

BACKGROUND: 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). OBJECTIVES: To characterize the enzootic strains of rabies virus in California wildlife for epidemiological investigation of transmission to non-reservoir animals and humans. STUDY DESIGN: 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. RESULTS AND CONCLUSIONS: 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.

Seroepidemiology of new AIDS-associated adenoviruses among the San Francisco Men's Health Study.

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.

Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues.

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.

Persistent infection of YAC-1 cells by coxsackievirus B3.

Persistent infection (PI) of YAC-1 cells by coxsackievirus B3 (CBV-3) was characterized. CBV-3 PIs were maintained for 7 months or more, although in two other cases cells were cured of virus at 6 and 6.5 months of PI. The titre of infectious virus peaked during the first week of the infection and then gradually decreased. The proportion of cells producing infectious centres increased to 100% by 48 h after infection, remained at that level up to the seventh day, and then rapidly decreased. Susceptibility to PI by CBV-3 varied widely among 40 clones from uninfected YAC-1 cells as judged by the yield of infectious virus at 6 weeks post-infection. None of the clones was completely lysed by the virus. Clones were not obtained from cells infected for 2 or 7 days. Of six clones obtained from cells infected for 14 days and 24 clones from cells infected for 6 weeks, none was producing virus and all were resistant to reinfection by CBV-3. Six of the clones were serially subcultured and all remained resistant for as long as they were maintained (5 months). During the course of the PI, viral variants which produced smaller plaques and required a longer incubation period for the development of visible plaques replaced the original viral population. Thus the PI involved a carrier culture with a large proportion of resistant cells. The resistant state did not require the continued presence of virus.

Persistent infection of mouse fibroblasts with Coxsackievirus.

Infection of fibroblast cell lines initiated from BALB/c or NFR mice with coxsackievirus B3 (CBV-3) or B4 (CBV-4) resulted in infections which persisted for a limited number of subpassages of the infected cells in most cases, but for over a year in one case. In all instances primary acute infections were characterized by cytopathology and release of infectious virus progeny. Viral antigen could be detected during the acute phase of infection, but not in subcultured infected cells. Infectious center assays showed that every cell was infected during the acute phase of infection, but that from the first subcultivation on, the numbers of cells which were able to initiate infection were greatly reduced. The long term persistent CBV-3 infection was characterized by wide fluctuations in titers of virus released into the supernatant fluids. Interferon did not appear to play a role in maintenance of the persistent infection. Information derived from studies on mechanisms of CBV persistence in the in vitro model may help to elucidate the role of CBV in chronic human diseases such as myocarditis.

Coxsackievirus B3 persistence and myocarditis in NFR nu/nu and +/nu mice.

NFR nude (nu/nu) and euthymic (+/nu) littermates were infected with coxsackievirus B3 (CBV-3) and assayed for virus persistence in the heart, pancreas and spleen, for development of myocarditis and for antibody production. The virus grew to higher titer and persisted longer in hearts of nu/nu mice. In both types of mice there was comparable myocarditis with a mononuclear cell inflammatory response, and there was some evidence of chronic lesions for up to 21 days in +/nu and 28 days in nu/nu mice. Antibody of the IgM class was present at 7 days in both strains of mice. Thereafter, +/nu mice produced high titers of virus-specific IgG antibody, while nu/nu mice produced little or no viral IgG antibody. The persistence of virus for up to 28 days in NFR nude mice is longer than has been reported previously, and offers an opportunity for further study of the role of T-cells in virus persistence and myocarditis.

Quantitative colorimetric microneutralization assay for characterization of adenoviruses.

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.

Comparison of methods to detect enteroviral genome in frozen and fixed myocardium by polymerase chain reaction.

BACKGROUND: Understanding the pathogenesis of viral myocarditis is linked to the availability of sensitive assays to detect viral genome in clinical material. Recent advances in molecular techniques permit detection of viral-specific RNA in tissue samples. We describe here a comparison of different methods for RNA extraction, reverse transcription, and gene amplification of Coxsackievirus B3 virus in fixed and frozen mouse myocardium. EXPERIMENTAL DESIGN: Homogenized Coxsackie B3 virus-infected myocardium was assayed for virus titer and formalin fixed, paraffin-embedded sections from the same heart were subjected to RNA extraction by 3 different methods. Optimal conditions were determined for a one-step assay combining reverse transcription and the polymerase chain reaction to detect enteroviral genome in RNA extracted by these different methods. The presence of amplifiable cDNA was confirmed by amplification of porphobilinogen deaminase mRNA as a positive control. RESULTS: Extraction of RNA from paraffin-embedded myocardium after overnight digestion with proteinase K (200 micrograms/ml) and 0.5% sodium dodecyl sulfate was the most efficient of the three methods compared. With our optimized polymerase chain reaction assay, in which the cDNA synthesis and amplification steps are combined, we detected as little as 10 TCID50 of virus from frozen viral stocks and tissue homogenates and 1.0 TCID50 of virus from fixed tissue. CONCLUSIONS: This sensitive polymerase chain reaction assay will facilitate examination of archival clinical samples to establish a retrospective diagnosis of enterovirus infection.

Adenovirus serotype evolution is driven by illegitimate recombination in the hypervariable regions of the hexon protein.

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.

Monoclonal antibodies for study of antigenic variation in coxsackievirus type B4: association of antigenic determinants with myocarditic properties of the virus.

Monoclonal antibodies were produced to a field strain ( Mil ) of group B coxsackievirus type 4 (CBV-4), and to the prototype JVB strain. Nine were neutralizing antibodies and four were non-neutralizing antibodies with virus-specific activity in indirect immunofluorescence (IF) staining. On the basis of reactivity with the panel of monoclonal antibodies, nine different strains of CBV-4 were found to fall into five distinct antigenic groups. Antigenic variants were produced by using the neutralizing monoclonal antibodies to select variants from the Mil virus stock which were no longer susceptible to the selecting antibody. A high frequency of antigenic variation was seen. By using the variants in cross-neutralization and IF tests with the monoclonal antibodies, it was possible to identify five tentative antigenic sites functional in neutralization; one site appeared to be complex and possibly to consist of overlapping epitopes. Reactivity of the monoclonal antibodies was similar, but not necessarily identical, by neutralization and by IF staining. The antigenic variants were found to differ from the parent Mil strain, and from one another, in their myocarditic and cardiotropic properties in a murine model. Two of the variants produced more extensive cardiac pathology, and two produced higher virus titres in the heart than was produced by the parent strain. One variant was notable for extensive production of necrotic lesions in the myocardium. Four of the variants showed less histopathology and three produced less virus in the heart than was produced by the parent strain.

Differing cardiotropic and myocarditic properties of group B type 4 coxsackievirus strains.

A murine model system for evaluation of myocarditic and cardiotropic properties of strains of group B, type 4 coxsackievirus (CBV-4) was developed in male BALB/c mice 4 weeks of age. Differing cardiotropic and myocarditic properties could be identified among field strains within the CBV-4 serotype. These properties were consistent for the virus strains, and were independent of the infecting virus dose. Virus replication in the heart appeared to be essential for development of myocarditis, but some infected hearts with high levels of infectious virus did not show myocarditis. Two of the myocarditic strains showed different histopathology in infected hearts; with one strain (Mil) the myocarditis was characterized by a marked inflammatory reaction with occasional accompanying myofiber necrosis. With the other strain (Bol), necrosis was the predominant finding, with a much lesser degree of inflammation. These findings suggest that there may be various pathogenic or immunopathogenic mechanisms by which CBV-4 can produce myocarditis.

Coxsackievirus B3 persistence and myocarditis in N:NIH(S) II nu/nu and +/nu mice.

N:NIH(S) II nu/nu (athymic) and +/nu (euthymic) mice were inoculated with coxsackievirus B3 (CBV-3) and examined at various times after infection for virus titres in the heart, myocarditis and serum neutralizing antibodies. Virus was recovered from the hearts of nu/nu mice for up to 94 days post-inoculation, but was not recovered from the hearts of any +/nu mice beyond 14 days. Inflammatory cell infiltration and necrosis were present in the hearts of +/nu mice at all harvest times (7, 14, 21 and 28 days). Inflammation and necrosis did not become evident in nu/nu mice until 14 days post-inoculation, and was the present in mice from each harvest until the end of the experiment (94 days). In athymic mice, myocarditis showed a strong correlation with persistence of CBV-3 in the heart. In N:NIH(S) II mice, the presence (+/nu) or absence (nu/nu) of a thymus had a major influence on the clearance of virus from the heart and on the development of myocarditis.

Strain variation in adenovirus serotypes 4 and 7a causing acute respiratory disease.

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.

Simplified microneutralization test for serotyping adenovirus isolates.

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.

Prevalence of antibodies to adenovirus serotypes 4 and 7 among unimmunized US Army trainees: results of a retrospective nationwide seroprevalence survey.

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 implications.

Adult adenovirus infections: loss of orphaned vaccines precipitates military respiratory disease epidemics. For the Adenovirus Surveillance Group.

Adenovirus vaccines have greatly reduced military respiratory disease morbidity since the 1970s. However, in 1995, for economic reasons, the sole manufacturer of these vaccines ceased production. A population-based adenovirus surveillance was established among trainees with acute respiratory illness at 4 US military training centers as the last stores of vaccines were depleted. From October 1996 to June 1998, 1814 (53.1%) of 3413 throat cultures for symptomatic trainees (78% men) yielded adenovirus. Adenovirus types 4, 7, 3, and 21 accounted for 57%, 25%, 9%, and 7% of the isolates, respectively. Unvaccinated trainees were much more likely than vaccinated trainees to be positive for types 4 or 7 (odds ratio [OR] = 28.1; 95% CI, 20.2-39.2). Two training centers experienced epidemics of respiratory disease affecting thousands of trainees when vaccines were not available. Until a new manufacturer is identified, the loss of orphaned adenovirus vaccines will result in thousands of additional preventable adenovirus infections.