Immunohistochemical recognition of a wide spectrum of lyssavirus in formalin-fixed tissues by one monoclonal antibody

Automated indirect immunoperoxidase (avidin-biotin complex) staining using monoclonal antibody #5DF12-3B6, directed against rabies N-protein, was used to detect rabies antigen in tissue samples from animals either naturally or experimentally infected with rabies. This monoclonal antibody recognized all 16 strains of rabies virus tested, as well as rabies-related lyssaviruses including Duvenhage, Lagos Bat, and Mokola. The sample infected with Mokola virus initially showed only weak staining, however, deletion of protease digestion resulted in stronger stain uptake. The test was sensitive and specific, correctly identifying rabies antigen in all but one of the samples tested (37/38), and no apparent staining in any of the negative samples tested (23/23). Tissues from 16 mammalian species were tested, including one rabies infected human tissue sample. The utility of the immunoperoxidase staining method described in this study lies in the ability of one monoclonal to recognize a broad spectrum of lyssaviruses in formalin-fixed tissues.

Raccoon poxvirus live recombinant feline panleukopenia virus VP2 and rabies virus glycoprotein bivalent vaccine.

A raccoon poxvirus (RCNV) recombinant for immunizing against feline panleukopenia and rabies was developed by homologous recombination with a chimeric plasmid for insertional inactivation of the RCNV thymidine kinase gene. The recombinant, RCN-FPV/VP2-rabG, coexpressed the feline panleukopenia virus (FPV) VP2 protein and the rabies virus spike glycoprotein (rabG) under oppositely oriented vaccinia virus P11 promoters. Cats vaccinated subcutaneously with the recombinant showed relatively high neutralizing antibody responses against rabies virus and FPV, and protection against an otherwise virulent FPV challenge with no drop in white blood cell count. Because of containment constraints, no rabies virus challenges were done, but the high concentrations (> 8 IU) of rabies neutralizing antibodies were consistent with levels that usually indicate an ability to counter the infection.

The epidemiology of bat rabies in New York State, 1988-92.

In 1993 New York and Texas each reported a human rabies case traced to a rare variant of rabies virus found in an uncommon species of bat. This study examined the epidemiology of bat rabies in New York State. Demographic, species, and animal-contact information for bats submitted for rabies testing from 1988-92 was analysed. The prevalence of rabies in 6810 bats was 4.6%. Nearly 90% of the 308 rabid bats identified to species were the common big brown bat (Eptesicus fuscus), which comprised 62% of all submissions. Only 25 submissions were silver-haired bats (Lasionycterus noctivagans), the species associated with the two 1993 human cases of rabies, and only two of these bats were positive. Rabies was most prevalent in female bats, in bats submitted because of human [corrected] contact, and in animals tested during September and October. These results highlight the unusual circumstances surrounding the recent human rabies cases in the United States. A species of bat rarely encountered by humans, and contributing little to the total rabies cases in bats, has been implicated in the majority of the indigenously acquired human rabies cases in the United States. The factors contributing to the transmission of this rare rabies variant remain unclear.

Development and evaluation of an in vitro virus isolation procedure as a replacement for the mouse inoculation test in rabies diagnosis.

A tissue culture virus isolation procedure for rabies street strain virus on mouse neuroblastoma cells is described. Parameters for the optimum sensitivity of the procedure were determined to include a minimum 4-day incubation of virus in tissue culture and the use of diethylaminoethyl-dextran for increased cell susceptibility. The in vitro procedure performed well in a comparison with the fluorescent-antibody test and the mouse inoculation test (MIT) on weakly positive brain tissue. Decomposed specimens and virus inhibitors present in brain suspensions were found to interfere with the in vitro procedure. A Formalin-methanol fixative was found to be superior on plastic 96-well plates to previously used fixatives. A 2-year clinical trial of the procedure in parallel with the MIT demonstrated the practicality of the procedure. Accordingly, the New York State rabies diagnostic laboratory has replaced the MIT with the in vitro procedure as a backup for the fluorescent-antibody test in the routine diagnosis of rabies.

Comparison of sensitivity of BHK-21 and murine neuroblastoma cells in the isolation of a street strain rabies virus.

The sensitivities of BHK-21 (C-13) and murine neuroblastoma (C-1300; clone NA) cells for the isolation of small quantities of a street strain rabies virus were compared. Suspensions of brain from mice sacrificed prior to the onset of clinical signs of rabies were used to stimulate weakly positive diagnostic specimens. The results of cell culture isolation were compared with those of the direct fluorescent-antibody test and virus isolation in weanling mice. Neuroblastoma cells were more sensitive to the street strain rabies virus than were BHK-21 cells. Neuroblastoma cell virus isolation, the mouse inoculation test, and the fluorescent-antibody test all showed comparable sensitivity.

Experimentally induced rabies in four cats inoculated with a rabies virus isolated from a bat.

Four cats were inoculated IM with rabies virus isolated from the salivary gland of a naturally infected big brown bat (Eptesicus fuscus). The 4 cats developed clinical signs of rabies after a median incubation period of 42 days. The median duration of clinical illness was 5 days. Results of fluorescent antibody evaluation, mouse inoculation, and tissue culture isolation indicated large differences in virus concentrations in various areas of the CNS of individual cats. These differences also were observed between cats. Rabies virus was isolated from the salivary glands and saliva of 2 cats; urinary bladder was the only other nonneural tissue found infected. Our observations indicated that cat rabies can be caused by bat rabies virus; that cats thus infected have infectious saliva during aggressive behavior and can therefore transmit the disease; and that adequate specimens of hippocampus, cerebellum, and brain stem are essential for reliable determination of rabies infection. The findings support recommendations for regular rabies vaccination of cats, even in areas of rabies-free terrestrial mammals.

Tissue culture technique for routine isolation of street strain rabies virus.

A tissue culture test for the primary isolation of street strain rabies virus from the brains of suspect animals was evaluated. It was found to be reliable and comparable in sensitivity to the standard mouse inoculation technique. The test, which yields final results in 48 h, was performed in BHK-21 cells on tissue culture chamber slides. The addition of diethylaminoethyl dextran to the cell suspension before seeding the slide promoted the subsequent viral invasiveness of positive test specimens. The method described may be considered as a substitute for the mouse inoculation test which is currently used as a backup to the fluorescent antibody test in the diagnosis of rabies.

Naturally occurring rabies virus and neutralizing antibody in two species of insectivorous bats of new york state.

Seven colonies of Eptesicus fuscus , the big brown bat, and five colonies of Myotis lucifugus , the little brown bat, in New York State were sampled for rabies virus and virus-neutralizing antibody. Eight of 278 E. fuscus were found to have virus, while 18 of 187 had antibody titers of ≥1:8. One of 333 M. lucifugus yielded virus, while three of 127 had antibody. These data demonstrate the presence of rabies virus as well as immunity to rabies in some insectivorous bats of New York State. Evaluation of these findings in relation to the epizootiology of the disease in bats requires further investigation.

A micromethod for measuring rabies-neutralizing antibody.

Plasma samples collected in micro-hematocrit tubes were shown to have rabies-neutralizing antibody titers comparable to serum samples. Micro-hematocrit tube sampling makes possible the monitoring of antibody levels in very small animals without causing serious injury.

Production of rabies fluorescent conjugate by immunization of rabbits with purified rabies antigen.

Fluorescein-labelled antirabies virus conjugate was produced by hyperimmunizing rabbits with a purified rabies virus (CVS-27). The conjugate had a working dilution of >/=1: 500 in the rabies fluorescent antibody test. Stability under lyophilization was satisfactory.

Standardization and quantitation of immunofluorescence in the rabies fluorescent-antibody test.

The standardization and quantitation of immunofluorescence with a Farrand microphotometer in the rabies fluorescent-antibody technique were determined. The system is useful in maintaining the quality of examinations for rabies in a public health laboratory since quantitation of reagent, equipment performance, and specificity of reaction can be evaluated. An analysis of the results demonstrates the high degree of reproducibility and the accuracy of the techniques described.

Pantropism of rabies virus in free-ranging rabid red fox Vulpes fulva.

The fluorescent antibody and mouse inoculation tests were employed to study the pantropism of rabies virus in tissue from free-ranging red fox (Vulpes fulva) submitted for rabies diagnosis. Viral antigen was found in various organs and tissues of the body. The distribution of antigen within tissues is discussed in the light of the pathogenesis and potential excretion of the virus.