Prevalence of rabies specific antibodies in the Mexican free-tailed bat (Tadarida brasiliensis mexicana) at Lava Cave, New Mexico

Adult female and juvenile Mexican free-tailed bats (Tadarida brasiliensis mexicana) were collected bimonthly at Lava Cave, New Mexico from May through September. The purpose of this study was to examine the prevalence of active rabies infection as well as to determine individual immune status in these hosts. All bats were bled and examined for rabies antibody (total antibody versus IgM) utilizing a modified serum neutralization test. The brains were removed and examined by the fluorescent rabies antibody (FRA) test. No significant difference was observed in the number of adults with rabies neutralizing antibody (total) over the study period. Significant differences in rabies neutralizing antibody (total) were observed among the juveniles sampled during July and August. The number of adults with IgM specific antibody was low (15 of 750, 2%) and did not fluctuate significantly. However, the number of juveniles with IgM antibody did show increased levels in August and September. The number of adults positive by the FRA was low (4 of 750, less than 1%) and did not appear to fluctuate significantly over the study period. The number of juveniles positive by the FRA was three and one-half times higher than observed for the adults (14 of 600, 2%). These results indicate that the Mexican free-tailed bat appears to be exposed to rabies virus shortly after birth as evident by its immune status. The low prevalence (4 of 750, less than 1%) of active infection as determined by the FRA and mouse inoculation and the high prevalence (514 of 750, 69%) of IgG antibody in the adult females indicate that the Mexican free-tailed bat recovers from rabies virus infection.     

Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the persistently infected cells was more efficient in infecting and replicating in naive U937 cells than the virus which was used to establish the persistent infection. These results suggest that macrophages may serve as reservoirs of infection in vivo, sequestering virus which may subsequently be activated from its persistent state, resulting in clinical infection and death.     

Susceptibility and Pathogenesis of Little Brown Bats (Myotis lucifugus) to Heterologous and Homologous Rabies Viruses

Rabies virus (RABV) maintenance in bats is not well understood. Big brown bats (Eptesicus fuscus), little brown bats (Myotis lucifugus), and Mexican free-tailed bats (Tadarida brasiliensis) are the most common bats species in the United States. These colonial bat species also have the most frequent contact with humans and domestic animals. However, the silver-haired bat (Lasionycteris noctivagans) RABV is associated with the majority of human rabies virus infections in the United States and Canada. This is of interest because silver-haired bats are more solitary bats with infrequent human interaction. Our goal was to determine the likelihood of a colonial bat species becoming infected with and transmitting a heterologous RABV. To ascertain the potential of heterologous RABV infection in colonial bat species, little brown bats were inoculated with a homologous RABV or one of two heterologous RABVs. Additionally, to determine if the route of exposure influenced the disease process, bats were inoculated either intramuscularly (i.m.) or subcutaneously (s.c.) with a homologous or heterologous RABV. Our results demonstrate that intramuscular inoculation results in a more rapid progression of disease onset, whereas the incubation time in bats inoculated s.c. is significantly longer. Additionally, cross protection was not consistently achieved in bats previously inoculated with a heterologous RABV following a challenge with a homologous RABV 6 months later. Finally, bats that developed rabies following s.c. inoculation were significantly more likely to shed virus in their saliva and demonstrated increased viral dissemination. In summary, bats inoculated via the s.c. route are more likely to shed virus, thus increasing the likelihood of transmission.     

Rabies virus infects mouse and human lymphocytes and induces apoptosis.

Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response.     

Seasonal variation in age,sex, and reproductive status of Mexican free-tailed bats

In North America, Mexican free-tailed bats (Tadarida brasiliensis mexicana) consume vast numbers of insects contributing to the economic well-being of society. Mexican free-tailed bats have declined due to historic guano mining, roost destruction, and bioaccumulation of organochlorine pesticides. Long-distance migrations and dense congregations at roosts exacerbate these declines. Wind energy development further threatens bat communities worldwide and presents emerging challenges to bat conservation. Effective mitigation of bat mortality at wind energy facilities requires baseline data on the biology of affected populations. We collected data on age, sex, and reproductive condition of Mexican free-tailed bats at a cave roost in eastern Nevada located 6 km from a 152-MW industrial wind energy facility. Over 5 years, we captured 46,353 Mexican free-tailed bats. Although just over half of the caught individuals were nonreproductive adult males (53.6%), 826 pregnant, 892 lactating, 10,101 post-lactating, and 4327 nonreproductive adult females were captured. Juveniles comprised 11.5% of captures. Female reproductive phenology was delayed relative to conspecific roosts at lower latitudes, likely due to cooler temperatures. Roost use by reproductive females and juvenile bats demonstrates this site is a maternity roost, with significant ecological and conservation value. To our knowledge, no other industrial scale wind energy facilities exist in such proximity to a heavily used bat roost in North America. Given the susceptibility of Mexican free-tailed bats to wind turbine mortality and the proximity of this roost to a wind energy facility, these data provide a foundation from which differential impacts on demographic groups can be assessed.     

Bat rabies in urban centers in Chile

One hundred and five rabies isolates obtained from domestic animals and insectivorous bats in Chile between 1977 and 1998 were molecularly characterized by limited sequence analysis of their nucleoprotein genes. These isolates were compared with viruses isolated from known domestic and wildlife rabies reservoirs in the Americas to identify potential reservoirs of rabies in Chile. The phylogenetic analyses showed that none of the Chilean isolates segregated with viruses from the terrestrial reservoirs. No non-rabies lyssaviruses were found in this study. The Chilean samples were not related to viruses of the sylvatic cycle maintained by the common vampire bat (Desmodus rotundus) in Latin America. Five genetic variants were identified from insectivorous bats in Chile. The Brazilian free-tailed bat (Tadarida brasiliensis) was identified as the reservoir for the rabies genetic variant most frequently isolated in the country between 1977 and 1998. The close association of a group of viruses obtained from a domestic dog (Canis familiaris), Brazilian free-tailed bats, and a red bat (Lasiurus borealis) with viruses maintained by Lasiurus spp. in North America implicated species of this genus as the possible reservoirs of this particular genetic variant in Chile. Reservoirs for the other three variants remain unknown.     

狂犬病毒糖蛋白DNA疫苗的研制及其免疫效果的观察

构建了含有狂犬病毒（RV）CVS株糖蛋白（GP）基因的重组质粒pCMVCVSRG,将其转染至鼠NIH3T3细胞中,用间接免疫荧光法和APAAP法均证实RVGP能在真核细胞中表达。分别将合RV不同毒株的GP基因的质粒（DNA疫苗）及空白载体质粒（对照组）免疫小鼠,仅DNA疫苗免疫的小鼠产生了中和抗体。以RV攻击后,DNA疫苗免疫组小鼠的存活率与对照组相比,差异有极显著性意义（P＜0．01）;不同的启动子（CMV或SV40）与不同GP基因（来源于CVS株或ERA株）对DNA疫苗的免疫效果无明显影响。在注射120d后．用PCR方法仍可检测出RVGP基因。结果表明：狂犬病DNA疫苗能够诱生低水平的中和抗体和记忆性B淋巴细胞,并能保护小鼠抵抗RV的攻击。该疫苗能在体内稳定存在。狂犬病DNA疫苗的研制为狂犬病免疫开辟了一条新途径,并可为防治其他疾病的DNA疫苗的研制奠定基础。     

Immune sera and antiglycoprotein monoclonal antibodies inhibit in vitro cell-to-cell spread of pathogenic rabies viruses.

Although the cell-to-cell spread of many viruses in vitro is inhibited by antibody, the effect of antibody on such spread of rabies viruses is uncertain. Thus, we examined the effects of anti-rabies virus immune sera and monoclonal antibodies (MAbs) on the in vitro spread of pathogenic rabies viruses in neuronal and nonneuronal cells. Both anti-rabies virus immune sera and neutralizing antiglycoprotein MAbs inhibited the cell-to-cell spread of street rabies virus, challenge virus standard, and ERA rabies viruses in cultures of neuroblastoma cells and of nonneuronal BHK-21 and chicken embryo-related cells. Furthermore, the cell-to-cell spread of virus was inhibited by greater than or equal to 75% with less than 1 IU/ml of human antirabies immunoglobulin. Nonneutralizing antinucleocapsid MAbs did not inhibit viral spread. After the immune serum was removed from the monolayers, virus spread rapidly to uninfected cells. Thus, antibody controlled the cell-to-cell spread of the virus but did not eliminate it from the cultures. Because antibody was more effective in inhibiting viral spread in fibroblast and epithelioid cells than in neuroblastoma cells infected at a high multiplicity of infection, we suggest that the inhibition of viral cell-to-cell spread by antibody in vivo would more likely occur at an initial site of exposure and before nerves are infected.     

Era vaccine-derived cases of rabies in wildlife and domestic animals in Ontario, Canada, 1989-2004

A vaccination program for the control of terrestrial rabies in the province of Ontario, Canada, began in 1989. During the period between 1989 and 2004, over 13 million baits containing the live, attenuated rabies virus ERA-BHK21 were distributed across the province, with the aim of immunizing foxes by the oral route. Animals recovered from bait distribution areas were assayed by fluorescent antibody test for rabies virus infection. Immunoreactivity with a panel of monoclonal antibodies that discriminate between ERA and rabies virus variants known to circulate in Ontario, and molecular genetic analyses were used to identify animals infected with ERA. Nine cases of ERA variant rabies were identified over the 16-yr period of study; these did not appear to be stratified by species, year of discovery, or location of capture. The ERA-positive animals were found across the province in eight counties, all of which had been baited in the year of case discovery. The nine ERA-positive cases included four red foxes (Vulpes vulpes), two raccoons (Procyon lotor), two striped skunks (Mephitis mephitis), and one bovine calf (Bos taurus). Molecular phylogenetic analyses of the partial N gene sequences generated from these isolates indicated that these nine cases were due to infection with the ERA variant. The glycoprotein sequences were predicted from G gene sequencing of all nine field isolates and two laboratory stock ERA viruses. This revealed some heterogeneity at residue 120 (either arginine or histidine) in both field and laboratory stocks as well as a few other mutations in field isolates. The significance of this heterogeneity remains unclear. Our data demonstrate that the ERA vaccine distributed in Ontario carried residual pathogenicity; however, there does not appear to be any evidence of ERA establishment in wildlife populations over the 16-yr period. These results are consistent with previous reports of the rare detection of ERA vaccine-induced rabies and with laboratory studies of ERA pathogenicity.     

Experimental rabies virus infection of big brown bats (Eptesicus fuscus)

A captive colony of adult Big Brown Bats (Eptesicus fuscus) was experimentally infected with a rabies virus (RABV) variant isolated from the salivary glands of a naturally infected Big Brown Bat and passaged once through murine neuroblastoma cell culture. Bats were divided into 11 groups, which were composed of one to three noninfected and one to three infected individuals each. Twenty of 38 animals were infected intramuscularly into both left and right masseter muscles; they received a total of 10(3.2) median mouse intracerebral lethal dose (MICLD50) of Big Brown Bat RABV variant. Experimental outcome after viral exposure was followed in the bats for 140 days postinoculation (PI). Of 20 infected bats, 16 developed clinical rabies, and the mean incubation period was 24 days (range: 13-52 days). Three infected bats never seroconverted and succumbed early to infection (13 days). Four infected bats that survived until the end of the experiment without any signs of disease maintained detectable antibody titers until the third month PI, peaking between days 13 and 43, and consequent drop-off below the threshold for detection occurred by day 140. Limited excretion of virus in saliva of infected bats during the clinical course of disease was observed in two individuals on days 13 and 15 PI (<24 hr prior to onset of clinical illness). No bat-to-bat transmission of RABV to noninfected bats was detected.     

The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System

Background

The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.

Methodology/Principal Findings

Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus–neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.

Conclusions/Significance

The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues.     

Rabies in insectivorous bats of western Canada, 1979 to 1983

A total of 1,745, 362, and 536 bats collected in Alberta, British Columbia, and Saskatchewan, respectively, was tested for rabies virus between 1979 and 1983. Only one (0.1%) of 769 bats collected at random from buildings was infected with rabies virus in contrast to 95 (5%) of 1,874 symptomatic, rabies-suspect bats submitted for testing. The pattern of infection in the rabies-suspect bats was similar in Alberta and Saskatchewan, but differed in British Columbia. Rabies was diagnosed in four species of bats in each of Alberta and Saskatchewan, but in seven species in British Columbia. Annual prevalence in rabies-suspect bats was similar in colonial species within each province. Rabies was found rarely in suspect little brown bats (Myotis lucifugus) (less than 1%). In suspect big brown bats (Eptesicus fuscus), the prevalence was low in Saskatchewan (3%), moderate in Alberta (10%), and high in British Columbia (25%). Big brown bats accounted for over 55% of the rabid bats detected in each province. Annual prevalence reported in silver-haired bats (Lasionycteris noctivagans) and hoary bats (Lasiurus cinereus) was variable in all three provinces. Rabies is enzootic in northern insectivorous bats.     

Overexpression of cytochrome C by a recombinant rabies virus attenuates pathogenicity and enhances antiviral immunity

The pathogenicity of individual rabies virus strains appears to correlate inversely with the extent of apoptotic cell death they induce and with the expression of rabies virus glycoprotein, a major inducer of an antiviral immune response. To determine whether the induction of apoptosis by rabies virus contributes to a decreased pathogenicity by stimulating antiviral immunity, we have analyzed these parameters in tissue cultures and in mice infected with a recombinant rabies virus construct that expresses the proapoptotic protein cytochrome c. The extent of apoptosis was strongly increased in primary neuron cultures infected with the recombinant virus carrying the active cytochrome c gene [SPBN-Cyto c(+)], compared with cells infected with the recombinant virus containing the inactive cytochrome c gene [SPBN-Cyto c(-)]. Mortality in mice infected intranasally with SPBN-Cyto c(+) was substantially lower than in SPBN-Cyto c(-)-infected mice. Furthermore, virus-neutralizing antibody (VNA) titers were significantly higher in mice immunized with SPBN-Cyto c(+) at the same dose. The VNA titers induced by these recombinant viruses paralleled their protective activities against a lethal rabies virus challenge infection, with SPBN-Cyto c(+) revealing an effective dose 20 times lower than that of SPBN-Cyto c(-). The strong increase in immunogenicity, coupled with the marked reduction in pathogenicity, identifies the SPBN-Cyto c(+) construct as a candidate for a live rabies virus vaccine.     

Suppression of cell-mediated immunity by street rabies virus infection.

An attempt to define a severe suppression of cell-mediated immunity by street rabies virus infection was undertaken by using the mice lethally and peripherally infected with a street rabies virus (1088 strain). The cell-mediated cytotoxic (CMC) activity of the spleen cells from those mice once slightly increased until day 4 after infection but declined rapidly thereafter until their death on days 10 to 12 after infection. In parallel with a decrease of CMC response of the spleen cells from 1088-infected mice, proliferative response to Con A, IL-2 activity in the culture supernatants of Con A-induced proliferation, responsiveness to exogenously added IL-2 and to Con A to express IL-2R, of those cells became suppressed, and the marked decrease of the total number of spleen cells was observed. Selective depletion of CD4+ and CD8+ cells in the spleens, abnormalities of IL-1 and E-type prostaglandins (PGE2) production or production of inhibitory component able to block IL-2 activity by spleen cells were not observed and these factors did not appear to be associated with the suppression of proliferative response to Con A. However, an apparent association of CD8+ cells in the suppression of differentiation of pre-cytotoxic lymphocytes (CTL) into CTL was demonstrated in the co-culture experiments of the spleen cells from 1088-infected mice with spleen cells of mice infected with an attenuated rabies virus (ERA strain) which can induce higher levels of CMC response. There was no evidence of the productive replication of rabies virus in thymus and spleen of 1088-infected mice. The relationship of these observations to current theories on virus-induced immunosuppression was discussed.     

Bat Rabies in Guatemala

Rabies in bats is considered enzootic throughout the New World, but few comparative data are available for most countries in the region. As part of a larger pathogen detection program, enhanced bat rabies surveillance was conducted in Guatemala, between 2009 and 2011. A total of 672 bats of 31 species were sampled and tested for rabies. The prevalence of rabies virus (RABV) detection among all collected bats was low (0.3%). Viral antigens were detected and infectious virus was isolated from the brains of two common vampire bats (Desmodus rotundus). RABV was also isolated from oral swabs, lungs and kidneys of both bats, whereas viral RNA was detected in all of the tissues examined by hemi-nested RT-PCR except for the liver of one bat. Sequencing of the nucleoprotein gene showed that both viruses were 100% identical, whereas sequencing of the glycoprotein gene revealed one non-synonymous substitution (302_T,S). The two vampire bat RABV isolates in this study were phylogenetically related to viruses associated with vampire bats in the eastern states of Mexico and El Salvador. Additionally, 7% of sera collected from 398 bats demonstrated RABV neutralizing antibody. The proportion of seropositive bats varied significantly across trophic guilds, suggestive of complex intraspecific compartmentalization of RABV perpetuation.     

Target Cells of Cytotoxic T Lymphocytes Directed to the Individual Structural Proteins of Rabies Virus

Target cells of cytotoxic T lymphocytes (CTL) directed to the individual structural proteins (except for the large polymerase (L) protein) of rabies virus were established by expressing only the respective protein in murine neuroblastoma (NA) and murine macrophage (J774-1) cell lines. Mice infected with the ERA strain of rabies virus developed CTL responses to all of these rabies virus proteins. The cytotoxic activity was abrogated by pretreatment of the effector cells with anti-CD8 monoclonal antibody (MAb) and complement but not with anti-CD4 MAb. Cell lysis by CTL was blocked in the presence of anti-major histocompatibility complex (MHC) class 1 antibodies in J774-1 cell lines. Rabies virus-infected cells express these proteins at the surface, which can be recognized and lysed by the respective CTL. Mice immunized with β-propiolactone-inactivated virus induced a CTL response against glycoprotein but not against internal viral components. This assay system might be useful for further analysis of the possible contribution of these proteins in the cell-mediated immune protection against rabies.     

Induction of rabies virus-specific T-helper cells by synthetic peptides that carry dominant T-helper cell epitopes of the viral ribonucleoprotein.

The T-helper cell response to the internal proteins of rabies virus was investigated. The rabies virus nucleoprotein was shown to be a major target antigen for T-helper cells that cross-react between rabies and rabies-related viruses. T-helper cells were assayed in vitro by testing virus-induced lymphocytes for lymphokine secretion in response to antigen. Immunodominant T-helper cell epitopes of the viral nucleoprotein were identified in vitro by using synthetic peptides delineated from the amino acid sequence of the nucleoprotein. The response to synthetic peptides were under Ir gene control. Antigenic peptides were tested in vivo for stimulation of rabies virus-specific T-helper cells. Inoculation of mice with peptides bearing immunodominant T-helper cell epitopes resulted in an accelerated and enhanced neutralizing antibody response upon booster immunization with inactivated rabies virus.     

Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.     

Eastern equine encephalomyelitis virus in experimentally infected bats.

Colonial bats (Myotis supp. and Eptesicus sp.) were infected with eastern equine encephalomyelitis virus by subcutaneous inoculation or by the bite of infected mosquitoes. Bats were maintained in an environment simulating conditions encountered in hibernacula or in summer maternal colonies. Virus was detected in the blood of hibernating bats at irregular intervals over a 42-day observation period; viremia perhaps was influenced by the amount of disturbance (arousal) involved in the blood sampling process. Target organs included brown fat, spleen, lung, kidneys, pancreas, and liver. Neutralizing antibody was not detected in sera collected from these bats between days 4 and 42 post-inoculation. In nonhibernating bats, virus was recovered from mammary glands, brown fat, pancreas, lungs, kidneys, and liver, in addition to blood. Attempts to infect bats orally or to transmit virus to suckling mice by the bite of viremic bats were unsuccessful. Virus was transmitted from viremic chickens to E. fuscus by the bite of Culiseta melanura and Aedes aegypti.     

Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro.

We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not challenged with street rabies virus had anti-rabies virus activity. In vivo studies illustrated that mice inoculated in the footpad with preincubated mixtures of anti-N sera and virus were protected. In addition, anti-N sera inoculated into the site of virus challenge protected mice. The antiviral activity of anti-N sera was also demonstrated in vitro. Infectious virus was not detected in cultures 24 h following infection with virus that had been preincubated with anti-N sera. At later time points, infectious virus was detected, but inhibition of viral production was consistently > or = 99% compared with control cultures. The protective and antiviral inhibitory activity of the anti-N sera was identified as anti-N antibody by several methods. First, absorption of anti-N sera with goat anti-mouse immunoglobulin serum, but not normal goat serum, removed the activity. Second, radioimmuno-precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sucrose density gradient-fractionated anti-N sera showed that antiviral activity was present only in the fraction containing anti-N antibody. Finally, absorption of anti-N sera with insect cells infected with a baculovirus expressing the N protein removed the protective activity. These data indicate that anti-N antibody is a component of the resistance to rabies virus infections.