Characterization of big brown bat (Eptesicus fuscus) rabies virus in a mouse model

Abstract

A majority of human rabies cases in the United States are either imported from countries where dog rabies is endemic or classified as cryptic human cases, where a route of exposure is not known. Notably, essentially all rabies virus (RV) variants associated with cryptic cases of human rabies are maintained in bats. Understanding how RV is maintained in populations of bats and characterizing the diversity of bat RV is thus a high priority problem for public health. Among the knowledge gaps related to bat rabies are understanding the variation in virulence within the population of a single variant and explaining the observation that a substantial number of healthy wild bats have neutralizing antibodies to RV, but no apparent clinical illness. The work described here was designed to address both of those issues. Nine RV isolates were isolated from big brown bats in Colorado and low-passage stocks of each were prepared. These isolates were evaluated for virulence, immunogenicity and salivary gland dissemination to investigate whether there were major differences in these characteristics within this virus population. Inoculated mice were maintained for 12 weeks after virus inoculation to assess mortality and were bled regularly to evaluate their humoral immune responses. Salivary glands from mice that developed clinical rabies were evaluated for virus replication as an indication for potential for further transmission. The dose of RV inoculated had a greater influence on the incubation period and mortality than the individual RV isolate. There was no difference in the humoral immune response in mice between those that were protected and those that succumbed to infection. The only salivary glands that were positive for RV replication were observed from mice in the high dose inoculation groups. Collectively, the results of this experiment indicated that there was low diversity in biologic behavior within the sample of Epitesicus fuscus viruses tested. The humoral immune response of mice to a big brown bat RV variant was explored to address the hypothesis that dose, route or frequency of inoculation may explain the prevalence of neutralizing rabies antibody seen in wild bat populations. Mice were inoculated via intramuscular, intradermal and intranasal routes, with two different low doses of virus and two inoculation schedules. The highest frequency of seroconversion was seen in mice inoculated intramuscularly with the higher of the two doses of RV. Mice that were inoculated intranasally experienced the highest mortality. Mice were rechallenged 3 months following the initial challenge with a high dose of virus intramuscularly to determine if the neutralizing rabies antibodies were protective and if priming of the immune system to RV had occurred in those that failed to seroconvert. The results of this experiment indicate that inoculation of low doses of virus by any of several routes can elicit a detectable humoral immune response without development of disease, which supports the hypothesis that exposure of wild bats to low doses of RV results in seroconversion without clinical disease.