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.

Effect of clarithromycin on experimental rhinovirus-16 colds: a randomized, double-blind, controlled trial.

PURPOSE: Macrolide antibiotics are frequently prescribed to patients with symptoms of a common cold. Despite their lack of proven antiviral activity, macrolide antibiotics may have anti-inflammatory actions, such as inhibition of mucus secretion and production of interleukins 6 and 8 by epithelial cells. Because the symptoms of rhinovirus colds are attributed to the inflammatory response to infection, we studied the effects of treatment with clarithromycin on the symptomatic and inflammatory response to nasal inoculation with rhinovirus. SUBJECTS AND METHODS: We performed a prospective, double-blind, controlled trial in 24 healthy subjects who were seronegative for antibodies to rhinovirus-16. Subjects were randomly assigned to receive either clarithromycin (500 mg) or trimethoprim-sulfamethoxazole (800/160 mg, as a control antibiotic) twice a day for 8 days, beginning 24 hours before inoculation with rhinovirus-16. RESULTS: All 12 subjects in each group were infected and developed symptomatic colds. The groups did not differ in the intensity of cold symptoms (median [25th to 75th percentile] score in the clarithromycin group of 25 [5 to 33] versus 21 [11 to 26] in the trimethoprim-sulfamethoxazole group, P = 0.86), weight of nasal secretions (25 g [8 to 56 g] versus 12 g [5 to 28 g], P = 0.27), or decline in nasal peak flow during the 8 days following viral inoculation. In both groups, similar and significant increases from baseline were observed in the numbers of total cells and neutrophils, and in the concentrations of interleukins 6 and 8, in nasal lavage fluid during the cold. The changes that we observed did not differ from those in an untreated historical control group. CONCLUSIONS: We conclude that clarithromycin treatment has little or no effect on the severity of cold symptoms or the intensity of neutrophilic nasal inflammation in experimental rhinovirus-16 colds.

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.

Oxygen cost of ventilation in the resting horse.

The purpose of this study was to develop a technique to measure the oxygen cost of ventilation and the values of ventilatory parameters in seven normal horses rebreathing carbon dioxide (CO2). All the horses responded to increased inspiratory levels of CO2 by increasing their tidal volume (VT) and frequency of breathing (Vf). The mean (SE) oxygen cost litre-1 of ventilation, measured at rates of ventilation greater than 200 litres min-1 was 1.7 (0.04) ml litre-1, similar to that of normal human subjects ventilating submaximally. It was concluded that the CO2 rebreathing test is a practical, non-invasive means of measuring the oxygen cost of breathing and the ventilatory response to CO2 in horses.

Use of high-speed cinematography and computer generated gait diagrams for the study of equine hindlimb kinematics.

High-speed cinematography with computer aided analysis was used to study equine hindlimb kinematics. Eight horses were filmed at the trot or the pace. Filming was done from the side (lateral) and the back (caudal). Parameters measured from the lateral filming included the heights of the tuber coxae and tailhead, protraction and retraction of the hoof and angular changes of the tarsus and stifle. Abduction and adduction of the limb and tarsal height changes were measured from the caudal filming. The maximum and minimum values plus the standard deviations and coefficients of variations are presented in tabular form. Three gait diagrams were constructed to represent stifle angle versus tarsal angle, metatarsophalangeal height versus protraction-retraction (fetlock height diagram) and tuber coxae and tailhead height versus stride (pelvic height diagram). Application of the technique to the group of horses revealed good repeatability of the gait diagrams within a limb and the diagrams appeared to be sensitive indicators of left/right asymmetries.

Effects of lesion size and location on equine articular cartilage repair.

The mechanisms and completeness of equine articular cartilage repair were studied in ten horses over a nine month period. Large (15 mm square) and small (5 mm square) full-thickness lesions were made in weight bearing and nonweight bearing areas of the radiocarpal, middle carpal and femoropatellar joints. The horses were euthanized in groups of two 1, 2.5, 4, 5 and 9 months later. Gross pathology, microradiography, and histopathology were used to evaluate qualitative aspects of articular repair. Computer assisted microdensitometry of safranin-O stained cartilage sections was used to quantitate cartilage matrix proteoglycan levels. Structural repair had occurred in most small defects at the end of nine months by a combination of matrix flow and extrinsic repair mechanisms. Elaboration of matrix proteoglycans was not complete at this time. Statistically better healing occurred in small weight bearing lesions, compared to large or nonweight bearing lesions. Synovial and perichondrial pannus interfered with healing of osteochondral defects that were adjacent to the cranial rim of the third carpal bone. Clinical and experimental experience suggests that these lesions are unlikely to heal, whereas similar lesions in the radiocarpal and femoropatellar joints had satisfactory outcomes. Observations made in this study support the use of early postoperative ambulation, passive flexion of operated joints, and recuperative periods of up to a year for large cartilage defects.

Attempts to restore abduction of the paralyzed equine arytenoid cartilage. II. Nerve implantation (pilot study).

The purpose of this project was to attempt restoration of abduction of a recently experimentally denervated left dorsal cricoarytenoid muscle by implanting a transected nerve-end into the paralyzed muscle. In six ponies the cut end of the second cervical nerve was implanted into a slit made in the left dorsal cricoarytenoid muscle. The nerve end was secured in place with one 5-0 polypropylene suture connecting the epineurium to the epimysium. The left recurrent laryngeal nerve was transected during this procedure. All six ponies showed signs of complete left laryngeal hemiplegia immediately after surgery. Postoperatively all ponies were evaluated qualitatively on a monthly basis by subjective examination for evidence of abduction of the arytenoid cartilages on endoscopy and quantitatively by measurement of the cross sectional area of the left and right half of the rima glottidis. Subjective endoscopic evidence of partial abduction was seen in four of the six ponies six months postoperatively. Measurement of the cross sectional area of the rima glottidis revealed a total loss of 38% of the area immediately postoperatively. There were no significant changes in cross sectional areas of the rima glottidis between the immediate postoperative evaluation to the six months postoperative evaluation. Gross postmortem examination revealed partial dorsal cricoarytenoid muscle atrophy as evidenced by a 24-55% decrease in muscle mass compared to the right dorsal cricoarytenoid muscle. Histopathological studies revealed regions with clusters of large muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Two new candidate adenovirus serotypes.

Two new adenovirus (AV) subgenus D serotypes, which we propose as AV candidates 48 and 49, are described. Both AV 48 and 49 were unique by serum neutralization and the pattern of restriction endonuclease fragments. By the hemagglutination inhibition assay there was a significant reciprocal cross between AV 48 and 44 and one-way crosses between AV 48 and AV 37 sera and between AV 30 and AV 49 sera. Eleven AV 48 and 5 AV 49 isolates, mainly from AIDS patients, have been identified at the Viral and Rickettsial Disease Laboratory.

Spectrum of monoclonal antibodies to coxsackievirus B-3 includes type- and group-specific antibodies.

Fifteen monoclonal antibodies (MAbs) made to coxsackievirus B-3 were tested against a panel of enteroviruses by indirect immunofluorescence. The MAbs included seven which reacted with coxsackievirus B-3 only, five which reacted with more than one enterovirus included in the panel, one which reacted with broad enteroviral specificity and did not react with any heterologous virus (group specific), and two which reacted with all enteroviruses tested and at least one heterologous virus. The group-specific MAb identified 44 of 45 clinical isolates as enteroviruses, while the two broadly reactive MAbs reacted with all 45 of the clinical isolates. These MAbs are potentially important diagnostic reagents for grouping and typing enteroviruses by indirect immunofluorescence.

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.

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.

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.

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.

Coxsackievirus B-3 selection of virus resistant Buffalo green monkey kidney cells and chromosome analysis of parental and resistant cells.

Following coxsackievirus B-3 (CBV-3) infection and lysis of highly susceptible Buffalo green monkey kidney (BGMK) cells, there was a regrowth of cells. Cultures of regrown cells were established and they continued to release infectious CBV-3 for up to 20 weeks. The parental BGMK cells were susceptible to CBV-3, CBV-4 and poliovirus type 2 induced cytopathic effect (CPE), while the cured cells were resistant to CBV-3 and CBV-4 but not to poliovirus type 2. Attachment of CBV-3 was restricted on cured BGMK cells but not on parental cells. Chromosome analysis showed that the cured cells originated from the BGMK cell line and that they were missing two marker chromosomes present in the parental cells.

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.

Characterization of echovirus 22 variants.

Ten presumptive enterovirus isolates which could not be neutralized by type specific antisera to any prototype enterovirus were related to echovirus 22 using molecular, biologic and serologic methods. Viral protein fingerprinting and PCR first suggested that these strains were variants of echovirus 22. Three of the strains were echovirus 22 prime strains, i.e., antiserum made to the variant strain neutralized the variant and the prototype strain. The other strains were neutralized by antiserum to the prime strains. Unlike typical enteroviruses, echovirus 22 and 23 prototype viruses and 7 of the 10 variants were heat stable at 50 degrees C in H2O for 1 h.

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.