The study of rabies virus infection in bats can be challenging due to quarantine requirements, husbandry concerns, genetic differences among animals, and lack of medical history. To date, all rabies virus (RABV) studies in bats have been performed in wild caught animals. Determining the RABV exposure history of a wild caught bat based on the presence or absence of viral neutralizing antibodies (VNA) may be misleading. Previous studies have demonstrated that the presence of VNA following natural or experimental inoculation is often ephemeral. With this knowledge, it is difficult to determine if a seronegative, wild caught bat has been previously exposed to RABV. The influence of prior rabies exposure in healthy, wild caught bats is unknown. To investigate the pathogenesis of RABV infection in bats born in captivity (naïve bats), naïve bats were inoculated intramuscularly with one of two Eptesicus fuscus rabies virus variants, EfV1 or EfV2. To determine the host response to a heterologous RABV, a separate group of naïve bats were inoculated with a Lasionycteris noctivagans RABV (LnV1). Six months following the first inoculation, all bats were challenged with EfV2. Our results indicate that naïve bats may have some level of innate resistance to intramuscular RABV inoculation. Additionally, naïve bats inoculated with the LnV demonstrated the lowest clinical infection rate of all groups. However, primary inoculation with EfV1 or LnV did not appear to be protective against a challenge with the more pathogenic EfV2.
During the past decade, incidence of human infection with rabies virus (RABV) spread by the common vampire bat (Desmodus rotundus) increased considerably in South America, especially in remote areas of the Amazon rainforest, where these bats commonly feed on humans. To better understand the epizootiology of rabies associated with vampire bats, we used complete sequences of the nucleoprotein gene to infer phylogenetic relationships among 157 RABV isolates collected from humans, domestic animals, and wildlife, including bats, in Peru during 2002–2007. This analysis revealed distinct geographic structuring that indicates that RABVs spread gradually and involve different vampire bat subpopulations with different transmission cycles. Three putative new RABV lineages were found in 3 non–vampire bat species that may represent new virus reservoirs. Detection of novel RABV variants and accurate identification of reservoir hosts are critically important for the prevention and control of potential virus transmission, especially to humans.
rabies; molecular epidemiology; bats; Peru; viruses; zoonoses; vampire bats
The currently recommended intradermal regimen for post-exposure prophylaxis spreads over a month period which many times lead to low compliance from the patients. There is a need to introduce and evaluate short course regimens to overcome this problem. This study was conducted to evaluate the immunogenicity and safety of a “new one week intradermal regimen” for rabies post-exposure prophylaxis. A total of 80 healthy adult volunteers were enrolled and allocated randomly either to purified chick embryo cell (PCECV) rabies vaccine or purified verocell rabies vaccine (PVRV), 40 in each group. Each subject received intradermally one of the vaccines, using the one week regimen (4–4-4). Blood samples were collected on Days 0, 7, 14, 28,180 and 365 for estimation of rabies virus neutralizing antibody (RVNA) concentration. The sera samples were analyzed by rapid fluorescent focus inhibition test (RFFIT). All subjects in both the groups had adequate RVNA concentration of ≥ 0.5 IU/mL from day 14 to till day 180 and the difference of geometric mean concentrations between the two groups was not significant (p > 0.606). Further to assess the immunological memory produced by this new regimen, a “single visit four site” intradermal booster vaccination was given to those who did not have adequate RVNA concentration on day 365. This resulted in a quick and enhanced RVNA concentration in these subjects thus denoting a successful anamnestic response. The incidence of adverse events was 8.3% in PCECV group and 1.6% in PVRV group (p = 0.001) and the regimen was well tolerated without any dropouts. In conclusion, the new “one week intradermal regimen” is immunogenic and safe for rabies post-exposure prophylaxis and needs to be further evaluated in persons exposed to rabies.
rabies; purified chick embryo cell rabies vaccine; purified vero cell rabies vaccine; randomized controlled trial; intradermal rabies vaccination
The frequent occurrence of ferret badger-associated human rabies cases in southeast China highlights the lack of laboratory-based surveillance and urges revisiting the potential importance of this animal in rabies transmission. To determine if the ferret badgers actually contribute to human and dog rabies cases, and the possible origin of the ferret badger-associated rabies in the region, an active rabies survey was conducted to determine the frequency of rabies infection and seroprevalence in dogs and ferret badgers.
A retrospective survey on rabies epidemics was performed in Zhejiang, Jiangxi and Anhui provinces in southeast China. The brain tissues from ferret badgers and dogs were assayed by fluorescent antibody test. Rabies virus was isolated and sequenced for phylogenetic analysis. The sera from ferret badgers and dogs were titrated using rabies virus neutralizing antibodies (VNA) test.
The ferret badgers presented a higher percentage of rabies seroconversion than dogs did in the endemic region, reaching a maximum of 95% in the collected samples. Nine ferret badger-associated rabies viruses were isolated, sequenced, and were phylogenetically clustered as a separate group. Nucleotide sequence revealed 99.4-99.8% homology within the ferret badger isolates, and 83-89% homology to the dog isolates in the nucleoprotein and glycoprotein genes in the same rabies endemic regions.
Our data suggest ferret badger-associated rabies has likely formed as an independent enzootic originating from dogs during the long-term rabies infestation in southeast China. The eventual role of FB rabies in public health remains unclear. However, management of ferret badger bites, rabies awareness and control in the related regions should be an immediate need.
Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans.
Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS.
Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection.
Inexorable lethality is still commonly attributed to rabies infection, although there is increasing evidence for non-lethal infection and even recovery from clinical rabies in various animal species and humans. This paper reports non-lethal infection in dogs. The striking difference between dogs that survived a wt RABV infection and dogs that succumbed to the infection is that the surviving dogs showed high level of VNA in the serum and in the CSF, as well as mild immune cell accumulation in the CNS, whereas dogs that succumbed to disease showed little or no VNA in the serum or in the CSF and developed severe CNS inflammation. Considering the role of VNA in clearing the virus from the CNS, production of VNA within the CNS or infiltration of VNA from the periphery into the CNS across the blood-brain barrier appears to be important for clearing the virus from CNS thereby preventing a lethal rabies infection.
An outbreak of bovine rabies occurred on a ranch when cattle were bitten by vampire bats. Microscopic lesions showed a nonsuppurative encephalitis with intracytoplasmic inclusion bodies. Immunohistochemistry demonstrated viral antigen in the brain, and monoclonal antibodies identified a serotype 1 (vampire strain) of the rabies virus.
To assess the immunogenicity of intradermal (ID) booster doses of Purified Chick Embryo Cell rabies vaccine (PCECV, Rabipur) administered to Thai schoolchildren one, three and five years after a primary ID pre-exposure (PrEP) vaccination series.
In this follow-up study of a randomized, open-label, phase II clinical trial, two simulated post-exposure booster doses of PCECV were administered on days 0 and 3 intradermally to 703 healthy schoolchildren, one, three or five years after primary vaccination with either two or three ID doses of 0.1 mL PCECV. Blood was drawn immediately before and 7, 14 and 365 days after the first booster dose to determine rabies virus neutralizing antibody (RVNA) concentrations.
An anamnestic response of approximately 30-fold increase in RVNA concentrations was demonstrated within 14 days after booster. All children (100%) developed adequate RVNA concentrations above 0.5 IU/mL. No vaccine related serious adverse events were seen in any of the vaccinees.
ID rabies PrEP with PCECV is safe and immunogenic in schoolchildren and the anamnestic response to a two booster dose vaccination series was found to be adequate one, three, and five years after a two- or three-dose primary PrEP vaccination series.
The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (107 PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals.
Despite extensive culling of common vampire bats in Latin America, lethal human rabies outbreaks transmitted by this species are increasingly recognized, and livestock rabies occurs with striking frequency. To identify the individual and population-level factors driving rabies virus (RV) transmission in vampire bats, we conducted a longitudinal capture–recapture study in 20 vampire bat colonies spanning four regions of Peru. Serology demonstrated the circulation of RV in vampire bats from all regions in all years. Seroprevalence ranged from 3 to 28 per cent and was highest in juvenile and sub-adult bats. RV exposure was independent of bat colony size, consistent with an absence of population density thresholds for viral invasion and extinction. Culling campaigns implemented during our study failed to reduce seroprevalence and were perhaps counterproductive for disease control owing to the targeted removal of adults, but potentially greater importance of juvenile and sub-adult bats for transmission. These findings provide new insights into the mechanisms of RV maintenance in vampire bats and highlight the need for ecologically informed approaches to rabies prevention in Latin America.
culling; disease thresholds; longitudinal; Lyssavirus; chiroptera; Desmodus
Background. Rabies virus (RABV) has circulated in Madagascar at least since the 19th century. Objectives. To assess the circulation of lyssavirus in the island from 2005 to 2010. Materials and Methods. Animal (including bats) and human samples were tested for RABV and other lyssavirus using antigen, ribonucleic acid (RNA), and antibodies detection and virus isolation. Results. Half of the 437 domestic or tame wild terrestrial mammal brains tested were found RABV antigen positive, including 54% of the 341 dogs tested. This percentage ranged from 26% to 75% across the period. Nine of the 10 suspected human cases tested were laboratory confirmed. RABV circulation was confirmed in 34 of the 38 districts sampled. No lyssavirus RNA was detected in 1983 bats specimens. Nevertheless, antibodies against Lagos bat virus were detected in the sera of 12 among 50 Eidolon dupreanum specimens sampled. Conclusion. More than a century after the introduction of the vaccine, rabies still remains endemic in Madagascar.
The USDA, Wildlife Services cooperative oral rabies vaccination (ORV) program uses a live vaccinia virus-vectored (genus Orthopoxvirus) vaccine, Raboral V-RG® (V-RG), to vaccinate specific wildlife species against rabies virus in several regions of the U.S. Several naturally occurring orthopoxviruses have been found in North America, including one isolated from asymptomatic raccoons (Procyon lotor). The effect of naturally occurring antibodies to orthopoxviruses on successful V-RG vaccination in raccoons is the focus of this study.
Overall, raccoons pre-immunized (n = 10) with a recombinant raccoonpox virus vaccine (RCN-F1) responded to vaccination with V-RG with lower rabies virus neutralizing antibody (VNA) titers than those which were not pre-immunized (n = 10) and some failed to seroconvert for rabies VNA to detectable levels.
These results suggest that the success of some ORV campaigns may be hindered where raccoonpox virus or possibly other orthopoxvirus antibodies are common in wildlife species targeted for ORV. If these areas are identified, different vaccination strategies may be warranted.
Two human rabies cases caused by a bat-associated virus variant were identified in September 2001 in Costa Rica, after a 31-year absence of the disease in persons. Both patients lived in a rural area where cattle had a high risk for bat bites, but neither person had a definitive history of being bitten by a rabid animal. Characterization of the rabies viruses from the patients showed that the reservoir was the hematophagous Vampire Bat, Desmodus rotundus, and that a sick cat was the vector.
human rabies; bat; Costa Rica; dispatch
Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.
In French Guiana, from 1984 to 2011, 14 animal rabies cases and 1 human rabies case (2008) were diagnosed. In January 2011, vampire-bat attacks occurred in 2 isolated villages. In mid-January, a medical team from the Cayenne Centre for Anti-Rabies Treatment visited the sites to manage individuals potentially exposed to rabies and, in April, an anti-rabies vaccination campaign for dogs was conducted. Twenty individuals were bitten by bats in 1 month, most frequently on the feet. The median time to start management was 15 days. The complete Zagreb vaccination protocol (2 doses on day 0 and 1 dose on days 7 and 21) was administered to 16 patients, 12 also received specific immunoglobulins. The antibody titration was obtained for 12 patients (different from those who received immunoglobulins). The antibody titers were ≥0.5 EU/mL for all of them. The serology has not been implemented for the 12 patients who received immunoglobulins. Accidental destruction of a vampire-bat colony could be responsible for the attacks. The isolation and absence of sensitization of the populations were the main explanations for the management difficulties encountered. Sensitization programs should be conducted regularly.
Rabies is a disease almost invariably fatal in humans once the first clinical signs appear. In French Guiana bats represent the virus reservoir, especially vampire bats. From 1984 to 2011, 14 animal rabies cases and 1 human rabies case (2008) were diagnosed. In case of bat bite, anti-rabies immunoglobulins (RIG) and rabies vaccine must be rapidly administrated. The specific rabies management is exclusively performed by Centre for Anti-Rabies Treatment (CART), located at the Institut Pasteur in Cayenne, the prefecture of French Guiana, and 6 Anti-Rabies Treatment Outposts distributed along the coastal edge and along the two main rivers. Only a CART physician can administer RIG. In January 2011, vampire-bat attacks occurred in 2 isolated villages. In mid-January, a medical team from the CART visited the sites to manage individuals potentially exposed to rabies and, in April, an anti-rabies vaccination campaign for dogs was conducted. The most relevant contribution of this study is to underline difficulties to provide rabies post-exposure prophylaxis to remote populations exposed to bat rabies in the Amazonian region and to show the lack of awareness of these rural populations concerning rabies and the risk associated to vampire bats.
After a human rabies outbreak in southeastern Peru, we collected bats to estimate the prevalence of rabies in various species. Among 165 bats from 6 genera and 10 species, 10.3% were antibody positive; antibody prevalence was similar in vampire and nonvampire bats. Thus, nonvampire bats may also be a source for human rabies in Peru.
Rabies virus; rabies; viruses; outbreak investigation; reservoir; vampire bats; Peru; dispatch
Six hundred and twenty-eight insectivorous bats originating from seven provinces were submitted to this Institute for rabies diagnosis between August 1, 1963 and December 31, 1967. Brain tissue was examined by the fluorescent antibody technique and the mouse infectivity test was carried out with brain, salivary gland, interscapular adipose tissue and kidney samples. Rabies virus was detected in 44 bats, 29 of which were from Ontario, 12 from British Columbia and three from Manitoba. Most of the positive cases were diagnosed in summer months. Seven species were represented among the specimens found to be rabid; there were 32 big brown bats, three hoary bats, three silver-haired bats, two little brown bats, one eastern pipistrelle, one Keen myotis and one red bat. Another bat which was not identified also proved to be infected with rabies.
Syrian hamsters were injected with inactivated, attenuated, and virulent rabies virus (RV), and the antibody response was quantified by a neutralization test and the immunoglobulin class of the virus antibody was characterized by indirect fluorescent microscopy. Serum antibodies to RV were found to be predominantly of the immunoglobulin G2 (IgG2) class, although IgG1 anti-RV also were detected in high-titered sera obtained after secondary challenge. Brain extracts of hamsters inoculated intracerebrally with RV contained only IgG2 anti-RV. IgA and IgM anti-RV were not detected. The preferential IgG2 response to RV is in marked contrast to the isolated IgG1 response detected after inoculation of hamsters with soluble purified protein antigens.
Bat rabies and its transmission to humans and other species in Mexico were investigated. Eighty-nine samples obtained from rabid livestock, cats, dogs, and humans in Mexico were studied by antigenic typing and partial sequence analysis. Samples were further compared with enzootic rabies associated with different species of bats in the Americas. Patterns of nucleotide variation allowed the definition of at least 20 monophyletic clusters associated with 9 or more different bat species. Several lineages associated with distinctive antigenic patterns were found in rabies viruses related to rabies in vampire bats in Mexico. Vampire bat rabies virus lineages associated with antigenic variant 3 are widely spread from Mexico to South America, suggesting these lineages as the most likely ancestors of vampire bat rabies and the ones that have been moved by vampire bat populations throughout the Americas. Rabies viruses related to Lasiurus cinereus, Histiotus montanus, and some other not yet identified species of the genus Lasiurus were found circulating in Mexico. Long-range dissemination patterns of rabies are not necessarily associated with migratory bat species, as in the case of rabies in Desmodus rotundus and Histiotus montanus. Human rabies was associated with vampire bat transmission in most cases, and in one case, rabies transmission from free-tailed bats was inferred. The occurrence of rabies spillover from bats to domestic animals was also demonstrated. Genetic typing of rabies viruses allowed us to distinguish trends of disease dissemination and to address, in a preliminary fashion, aspects of the complex evolution of rabies viruses in different host-reservoir species.
The metadata of 10 published studies and 3 vaccine trial reports comprising of 19 vaccine cohorts from four countries conducted over a period of 23 years (1986–2009) was used for metaanalysis. The vaccines studied were purified chick embryo cell vaccine (Rabipur, India and Germany), purified vero cell rabies vaccine (Verorab, France; Indirab, India) and human diploid cell vaccine (MIRV, France). The potency of these vaccines varied from 0.55 IU to 2.32 IU per intradermal dose of 0.1 ml per site. The vaccines were administered to 1,011 subjects comprising of 19 cohorts and using five different ID regimens. The immunogenicity was measured by assays of rabies virus neutralizing antibody (RVNA) titres using rapid fluorescent focus inhibition test (RFFIT) [15 cohorts] and mouse neutralization test (MNT) [4 cohorts]. The statistical analysis of the data was done by Karl Pearson's correlation coefficient to measure the relationship between antigenicity and immunogenicity. It was revealed that, there was no significant linear relationship between antigenicity and immunogenicity of rabies vaccines when administered by intradermal route (p > 0.230 and p > 0.568).
rabies vaccines; intradermal route; antigenicity; immunogenicity; metaanalysis
Rabies and rabies-related virus strains were studied by using a panel of monoclonal antibodies directed against either nucleocapsid proteins or cell surface antigens of Mokola virus (Mok-3). Each strain was used in parallel to infect cultured cells and mice. Then, the patterns of reactivity of the different monoclonal antibodies were determined by the immunofluorescent-antibody staining procedure. On cells, the monoclonal antibodies differentiated fixed rabies virus strains (serotype 1) from rabies-related virus strains. The seven fixed strains (CVS, PV4, PM, Flury LEP and HEP, ERA, and SAD) reacted identically. The previous serotype groupings (serotype 2, Lagos-bat virus; serotype 3, Mokola virus; serotype 4, Duvenhage virus) established with anti-rabies monoclonal antibodies were confirmed, except for that of Lagos-bat Kindia, which appeared to be related to the African subtype of the Duvenhage serotype (Duv-2). Within the Mokola (Mok-1, -2, -3, and -5 and Umhlanga) and the Lagos-bat (Lag-1 and -2, Zimbabwe, Pinetown, and Dakar) serotypes, each strain appeared to be distinct. The African subtype of the Duvenhage serotype reacted differently from the European subtype. Within the Duvenhage serotype, subtypes Duv-4, -5, and -6 and Denmark reacted identically, while subtypes Duv-1, -2, and -3 and German Democratic Republic appeared to be distinct. The monoclonal antibodies specific for the cell surface antigens were also used in neutralization tests with all the strains. Two of them neutralized the infectivity of Mokola virus.
Previous studies have investigated rabies virus (RABV) epizootiology in Brazilian free-tailed bats (Tadarida brasiliensis) in natural cave roosts. However, little is known about geographic variation in RABV exposure, or if the use of man-made roosts by this species affects enzootic RABV infection dynamics within colonies. We sampled rabies viral neutralizing antibodies in bats at three bridge and three cave roosts at multiple time points during the reproductive season to investigate temporal and roost variation in RABV exposure. We report seropositive bats in all age and sex classes with minimal geographic variation in RABV seroprevalence among Brazilian free-tailed bat colonies in south-central Texas. While roost type was not a significant predictor of RABV seroprevalence, it was significantly associated with seasonal fluctuations, suggesting patterns of exposure that differ between roosts. Temporal patterns suggest increased RABV seroprevalence after parturition in cave colonies, potentially related to an influx of susceptible young, in contrast to more uniform seroprevalence in bridge colonies. This study highlights the importance of life history and roost ecology in understanding patterns of RABV seroprevalence in colonies of the Brazilian free-tailed bat.
Brazilian free-tailed bat; Epizootiology; Rabies virus; Roost ecology
Vampire bat related rabies harms both livestock industry and public health sector in central Brazil. The geographical distributions of vampire bat-transmitted rabies virus variants are delimited by mountain chains. These findings were elucidated by analyzing a high conserved nucleoprotein gene. This study aims to elucidate the detailed epidemiological characters of vampire bat-transmitted rabies virus by phylogenetic methods based on 619-nt sequence including unconserved G-L intergenic region.
The vampire bat-transmitted rabies virus isolates divided into 8 phylogenetic lineages in the previous nucleoprotein gene analysis were divided into 10 phylogenetic lineages with significant bootstrap values. The distributions of most variants were reconfirmed to be delimited by mountain chains. Furthermore, variants in undulating areas have narrow distributions and are apparently separated by mountain ridges.
This study demonstrates that the 619-nt sequence including G-L intergenic region is more useful for a state-level phylogenetic analysis of rabies virus than the partial nucleoprotein gene, and simultaneously that the distribution of vampire bat-transmitted RABV variants tends to be separated not only by mountain chains but also by mountain ridges, thus suggesting that the diversity of vampire bat-transmitted RABV variants was delimited by geographical undulations.
Our previous studies indicated that recombinant rabies viruses (rRABV) expressing chemokines or cytokines (including GM-CSF) could enhance the immunogenicity by recruiting and/or activating dendritic cells (DC). In this study, bacterial flagellin was cloned into the RABV genome and recombinant virus LBNSE-Flagellin was rescued. To compare the immunogenicity of LBNSE-Flagellin with recombinant virus expressing GMCSF (LBNSE-GMCSF), mice were immunized with each of these rRABVs by intramuscular (i.m.) or oral route. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. The i.m.-immunized mice were bled at three weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with 50 LD50 challenge virus standard (CVS-24). Orally immunized mice were boosted after three weeks and then bled and challenged one week after the booster immunization. It was found that both LBNSE-GMCSF and LBNSE-Flagellin recruited/activated more DCs and B cells in the periphery, stimulated higher levels of adaptive immune responses (VNA), and protected more mice against challenge infection than the parent virus LBNSE in both the i.m. and the orally immunized groups. Together, these studies suggest that recombinant RABV expressing GM-CSF or flagellin are more immunogenic than the parent virus in both i.m. and oral immunizations.
Although current postexposure prophylaxis rabies virus (RV) vaccines are effective, ~40,000–70,000 rabies-related deaths are reported annually worldwide. The development of effective formulations requiring only 1–2 applications would significantly reduce mortality. We assessed in mice and nonhuman primates the efficacy of replication-deficient RV vaccine vectors that lack either the matrix (M) or phosphoprotein (P) gene. A single dose of M gene–deficient RV induced a more rapid and efficient anti-RV response than did P gene–deficient RV immunization. Furthermore, the M gene–deleted RV vaccine induced 4-fold higher virus-neutralizing antibody (VNA) levels in rhesus macaques than did a commercial vaccine within 10 days after inoculation, and at 180 days after immunization rhesus macaques remained healthy and had higher-avidity antibodies, higher VNA titers, and a more potent antibody response typical of a type 1 T helper response than did animals immunized with a commercial vaccine. The data presented in this article suggest that the M gene–deleted RV vaccine is safe and effective and holds the potential of replacing current pre- and postexposure RV vaccines.
The inflexibility of existing serological techniques for detection of rabies in surveillance constrains the benefit to be gained from many current control strategies. We analysed 304 serum samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors bearing the CVS-11 envelope glycoprotein. Compared with the widely used gold standard fluorescent antibody virus neutralisation assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody titres (r = 0.915) were observed with the pseudotype assay. To increase the assay's surveillance specificity in Africa we incorporated the envelope glycoprotein of local viruses, Lagos bat virus, Duvenhage virus or Mokola virus and also cloned the lacZ gene to provide a reporter element. Neutralisation assays using pseudotypes bearing these glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they pose to human health. Importantly, the CVS-11 pseudotypes were highly stable during freeze–thaw cycles and storage at room temperature. These results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections.
Rabies virus; Lyssavirus; Africa; Pseudotype