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1.  Recent Emergence and Spread of an Arctic-Related Phylogenetic Lineage of Rabies Virus in Nepal 
Rabies is a zoonotic disease that is endemic in many parts of the developing world, especially in Africa and Asia. However its epidemiology remains largely unappreciated in much of these regions, such as in Nepal, where limited information is available about the spatiotemporal dynamics of the main etiological agent, the rabies virus (RABV). In this study, we describe for the first time the phylogenetic diversity and evolution of RABV circulating in Nepal, as well as their geographical relationships within the broader region. A total of 24 new isolates obtained from Nepal and collected from 2003 to 2011 were full-length sequenced for both the nucleoprotein and the glycoprotein genes, and analysed using neighbour-joining and maximum-likelihood phylogenetic methods with representative viruses from all over the world, including new related RABV strains from neighbouring or more distant countries (Afghanistan, Greenland, Iran, Russia and USA). Despite Nepal's limited land surface and its particular geographical position within the Indian subcontinent, our study revealed the presence of a surprising wide genetic diversity of RABV, with the co-existence of three different phylogenetic groups: an Indian subcontinent clade and two different Arctic-like sub-clades within the Arctic-related clade. This observation suggests at least two independent episodes of rabies introduction from neighbouring countries. In addition, specific phylogenetic and temporal evolution analysis of viruses within the Arctic-related clade has identified a new recently emerged RABV lineage we named as the Arctic-like 3 (AL-3) sub-clade that is already widely spread in Nepal.
Author Summary
Rabies is endemic in most Asian countries and represents a serious public health issue, with an estimated 31,000 people dying each year of this disease. The majority of human cases are transmitted by domestic dogs, which act as the principal reservoir host and vector. However, molecular epidemiology and evolutionary dynamics of the main etiological agent, the rabies virus (RABV), remains largely unappreciated in some regions such as in Nepal. Based on a subset of 24 new Nepalese isolates collected from 2003 to 2011 and representative RABV strains at a global scale, phylogenetic analysis based on the complete nucleoprotein and glycoprotein genes sequences revealed the presence of a surprising wide genetic diversity of RABV circulating in this country. The presence of three different co-existing phylogenetic groups was identified: an Indian subcontinent clade and two different Arctic-like sub-clades within the Arctic-related clade, namely Arctic-like (AL)-1, lineage a (AL-1a), and AL-3. Among these clusters, the AL-3 sub-clade appears as the major Nepalese phylogroup which emerged relatively recently in this country, within the last 30 years. These data has raised some concerns about the exchange of RABV between different countries, and provided key elements for implementation of effective control measures of rabies in Nepal.
PMCID: PMC3836727  PMID: 24278494
2.  Molecular Inferences Suggest Multiple Host Shifts of Rabies Viruses from Bats to Mesocarnivores in Arizona during 2001–2009 
PLoS Pathogens  2012;8(6):e1002786.
In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001–2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T242 in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require additional investigations, using reverse genetics and other approaches.
Author Summary
Host shifts of the rabies virus (RABV) from bats to carnivores are important for our understanding of viral evolution and emergence, and have significant public health implications, particularly for the areas where “terrestrial” rabies has been eliminated. In this study we addressed several rabies outbreaks in carnivores that occurred in the Flagstaff area of Arizona during 2001–2009, and caused by the RABV variant associated with big brown bats (Eptesicus fuscus). Based on phylogenetic analysis we demonstrated that each outbreak resulted from a separate introduction of bat RABV into populations of carnivores. No post-shift changes in viral genomes were detected under the positive selection analysis. Trying to answer the question why certain bat RABV variants are capable for host shifts to carnivores and other variants are not, we developed a convergent evolution analysis, and implemented it for multiple RABV lineages circulating worldwide. This analysis identified several amino acids in RABV proteins which may facilitate host shifts from bats to carnivores. Precise roles of these amino acids require additional investigations, using reverse genetics and animal experimentation. In general, our approach and the results obtained can be used for prediction of host shifts and emergence of other zoonotic pathogens.
PMCID: PMC3380930  PMID: 22737076
3.  High Diversity of Rabies Viruses Associated with Insectivorous Bats in Argentina: Presence of Several Independent Enzootics 
Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory.
We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008.
Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples.
This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection.
Author Summary
In Argentina, successful vaccination and control of terrestrial rabies in the 1980s revealed the importance of the aerial route in RABV transmission. Current distribution of cases shows a predominance of rabies by hematophagous bats in the Northern regions where rabies is a major public health concern; in contrast, in Central and Southern regions where rabies is not a major public health concern, little surveillance is performed. Based on the analysis of insectivorous bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats in these regions ranged from 3.1 to 5.4%. This rate is comparable to other nations such as the United States (9–10%) where insectivorous bats are an important cause of concern for RABV surveillance systems. Antigenic and genetic analysis of a wide collection of rabies strains shows the presence of multiple endemic cycles associated with six bat insectivorous species distributed among an extensive area of the Argentinean territory and several countries of the Americas. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples. Increased public education about the relationship between insectivorous bats and rabies are essential to avoid human cases and potential spread to terrestrial mammals.
PMCID: PMC3348165  PMID: 22590657
4.  Evolutionary History and Phylogeography of Rabies Viruses Associated with Outbreaks in Trinidad 
Bat rabies is an emerging disease of public health significance in the Americas. The Caribbean island of Trinidad experiences periodic outbreaks within the livestock population. We performed molecular characterisation of Trinidad rabies virus (RABV) and used a Bayesian phylogeographic approach to investigate the extent to which outbreaks are a result of in situ evolution versus importation of virus from the nearby South American mainland. Trinidadian RABV sequences were confirmed as bat variant and clustered with Desmodus rotundus (vampire bat) related sequences. They fell into two largely temporally defined lineages designated Trinidad I and II. The Trinidad I lineage which included sequences from 1997–2000 (all but two of which were from the northeast of the island) was most closely related to RABV from Ecuador (2005, 2007), French Guiana (1990) and Venezuela (1993, 1994). Trinidad II comprised sequences from the southwest of the island, which clustered into two groups: Trinidad IIa, which included one sequence each from 2000 and 2007, and Trinidad IIb including all 2010 sequences. The Trinidad II sequences were most closely related to sequences from Brazil (1999, 2004) and Uruguay (2007, 2008). Phylogeographic analyses support three separate RABV introductions from the mainland from which each of the three Trinidadian lineages arose. The estimated dates for the introductions and subsequent lineage expansions suggest periods of in situ evolution within Trinidad following each introduction. These data also indicate co-circulation of Trinidad lineage I and IIa during 2000. In light of these findings and the likely vampire bat origin of Trinidadian RABV, further studies should be conducted to investigate the relationship between RABV spatiotemporal dynamics and vampire bat population ecology, in particular any movement between the mainland and Trinidad.
Author Summary
The Caribbean island of Trinidad experiences periodic rabies virus (RABV) outbreaks within the livestock population. In this study, we inferred the evolutionary history of RABV in the Americas and reconstructed past patterns of RABV geographic spread in order to address the question of whether Trinidadian outbreaks arise from locally maintained RABV or are the result of virus importation from the mainland (presumably via infected bats). Our results provide statistical support for three importation events that gave rise to each of three Trinidadian vampire bat-associated lineages identified in the study. They also indicate limited periods of in situ evolution within Trinidad following each of these introductions. The results also support Mexico and Brazil as major epicenters for the expansion of RABV associated with vampire bats throughout the Americas and consequently to Trinidad. The findings of our study are particularly relevant to local RABV monitoring and control. In addition to justifying vampire bats as the main target for active rabies surveillance and control activities in Trinidad, they suggest that more intense surveillance of regions that lie close to the mainland may be warranted. Finally, in light of these findings, further studies should be conducted to investigate the relationship between RABV spatiotemporal dynamics and vampire bat population ecology.
PMCID: PMC3749974  PMID: 23991230
5.  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 (302T,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.
Author Summary
In this study we provide results of the first active and extensive surveillance effort for rabies virus (RABV) circulation among bats in Guatemala. The survey included multiple geographic areas and multiple species of bats, to assess the broader public and veterinary health risks associated with rabies in bats in Guatemala. RABV was isolated from vampire bats (Desmodus rotundus) collected in two different locations in Guatemala. Sequencing of the isolates revealed a closer relationship to Mexican and Central American vampire bat isolates than to South American isolates. The detection of RABV neutralizing antibodies in 11 species, including insectivorous, frugivorous, and sanguivorous bats, demonstrates viral circulation in both hematophagous and non-hematophagous bat species in Guatemala. The presence of bat RABV in rural communities requires new strategies for public health education regarding contact with bats, improved laboratory-based surveillance of animals associated with human exposures, and novel techniques for modern rabies prevention and control. Additionally, healthcare practitioners should emphasize the collection of a detailed medical history, including questions regarding bat exposure, for patients presenting with clinical syndromes compatible with rabies or any clinically diagnosed progressive encephalitis.
PMCID: PMC4117473  PMID: 25080103
6.  Molecular characterization of Korean rabies virus isolates 
Journal of Veterinary Science  2011;12(1):57-63.
The nucleoprotein (N) and glycoprotein (G) of 11 Korean rabies virus (RABV) isolates collected from animals diagnosed with rabies between 2008 and 2009 were subjected to molecular and phylogenetic analyses. Six isolates originated from domestic animals (cattle and dogs) and five were obtained from wild free-ranging raccoon dogs. The similarities in the nucleotide sequences of the N gene among all Korean isolates ranged from 98.1 to 99.8%, while those of the G gene ranged from 97.9 to 99.3%. Based on the nucleotide analysis of the N and G genes, the Korean RABV isolates were confirmed as genotype I of Lyssavirus and classified into four distinct subgroups with high similarity. Phylogenetic analysis showed that the Korean isolates were most closely related to the non-Korean NeiMeng1025B and 857r strains, which were isolated from rabid raccoon dogs in Eastern China and Russia, respectively. These findings suggest that the Korean RABV isolates originated from a rabid raccoon dog in Northeastern Asia. Genetic analysis of the Korean RABV isolates revealed no substitutions at several antigenic sites, indicating that the isolates circulating in Korea may be pathogenic in several hosts.
PMCID: PMC3053468  PMID: 21368564
characterization; genotype I; molecular epidemiology; rabies virus
7.  Rabies in Iraq: Trends in Human Cases 2001–2010 and Characterisation of Animal Rabies Strains from Baghdad 
Control of rabies requires a consistent supply of dependable resources, constructive cooperation between veterinary and public health authorities, and systematic surveillance. These are challenging in any circumstances, but particularly during conflict. Here we describe available human rabies surveillance data from Iraq, results of renewed sampling for rabies in animals, and the first genetic characterisation of circulating rabies strains from Iraq. Human rabies is notifiable, with reported cases increasing since 2003, and a marked increase in Baghdad between 2009 and 2010. These changes coincide with increasing numbers of reported dog bites. There is no laboratory confirmation of disease or virus characterisation and no systematic surveillance for rabies in animals. To address these issues, brain samples were collected from domestic animals in the greater Baghdad region and tested for rabies. Three of 40 brain samples were positive using the fluorescent antibody test and hemi-nested RT-PCR for rabies virus (RABV). Bayesian phylogenetic analysis using partial nucleoprotein gene sequences derived from the samples demonstrated the viruses belong to a single virus variant and share a common ancestor with viruses from neighbouring countries, 22 (95% HPD 14–32) years ago. These include countries lying to the west, north and east of Iraq, some of which also have other virus variants circulating concurrently. These results suggest possible multiple introductions of rabies into the Middle East, and regular trans-boundary movement of disease. Although 4000 years have passed since the original description of disease consistent with rabies, animals and humans are still dying of this preventable and neglected zoonosis.
Author Summary
Control of rabies requires cooperation between government departments, consistent funding, and an understanding of the epidemiology of the disease obtained through surveillance. Here we describe human rabies surveillance data from Iraq and the results of renewed sampling for rabies in animals. In Iraq, it is obligatory by law to report cases of human rabies. These reports were collated and analysed. Reported cases have increased since 2003, with a marked increase in Baghdad 2009–2010. There is no system for detecting rabies in animals and the strains circulating in Iraq have not previously been characterized. To address this, samples were collected from domestic animals in Baghdad and tested for rabies. Three out of 40 were positive for rabies virus. Comparison of part of the viral genetic sequence with other viruses from the region demonstrated that the viruses from Iraq are more closely related to each other than those from surrounding countries, but diverged from viruses isolated in neighbouring countries approximately 22 (95% HPD 14–32) years ago. Although 4000 years have passed since the original description of disease consistent with rabies, animals and humans are still dying of this preventable and neglected zoonosis.
PMCID: PMC3585036  PMID: 23469303
8.  Molecular epidemiology of livestock rabies viruses isolated in the northeastern Brazilian states of Paraíba and Pernambuco from 2003 - 2009 
BMC Research Notes  2012;5:32.
Limited or no epidemiological information has been reported for rabies viruses (RABVs) isolated from livestock in the northeastern Brazilian states of Paraíba (PB) and Pernambuco (PE). The aim of this study was to clarify the molecular epidemiology of RABVs circulating in livestock, especially cattle, in these areas between 2003 and 2009.
Phylogenetic analysis based on 890 nt of the nucleoprotein (N) gene revealed that the 52 livestock-derived RABV isolates characterized here belonged to a single lineage. These isolates clustered with a vampire bat-related RABV lineage previously identified in other states in Brazil; within PB and PE, this lineage was divided between the previously characterized main lineage and a novel sub-lineage.
The occurrences of livestock rabies in PB and PE originated from vampire bat RABVs, and the causative RABV lineage has been circulating in this area of northeastern Brazil for at least 7 years. This distribution pattern may correlate to that of a vampire bat population isolated by geographic barriers.
PMCID: PMC3285087  PMID: 22243739
9.  Immune Clearance of Attenuated Rabies Virus Results in Neuronal Survival with Altered Gene Expression 
PLoS Pathogens  2012;8(10):e1002971.
Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent “mark” on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these “cured” neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that can persist or progress in the absence of sustained viral antigen.
Author Summary
Rabies is an ancient and fatal neurological disease of animals and humans, caused by infection of the central nervous system (CNS) with Rabies virus (RABV). It is estimated that nearly 55,000 human RABV fatalities occur each year, though this number is likely much higher due to unreported exposures or failure of diagnosis. No treatment has been identified to cure disease after onset of symptoms. Neurovirologists still do not know the cause of rabies' dramatic symptoms and fatality, though it is thought to be due to neuronal loss or dysfunction. Here, we use a novel approach to permanently and genetically tag infected cells so that they can be identified after the infection has been cleared. This allowed us to define neuronal survival time following infection, and to assess neuronal function through gene expression analysis. We found that RABV infection does not lead to loss of neurons, but rather induces a permanent change in gene expression that may be related to the ability of RABV to cause permanent CNS disease. Our study provides evidence that viral infection of the brain can initiate long-term changes that may have consequences for nervous system health, even after the virus has been cleared from the CNS.
PMCID: PMC3469654  PMID: 23071441
10.  Genetic and evolutionary characterization of RABVs from China using the phosphoprotein gene 
Virology Journal  2013;10:14.
While the function of the phosphoprotein (P) gene of the rabies virus (RABV) has been well studied in laboratory adapted RABVs, the genetic diversity and evolution characteristics of the P gene of street RABVs remain unclear. The objective of the present study was to investigate the mutation and evolution of P genes in Chinese street RABVs.
The P gene of 77 RABVs from brain samples of dogs and wild animals collected in eight Chinese provinces through 2003 to 2008 were sequenced. The open reading frame (ORF) of the P genes was 894 nucleotides (nt) in length, with 85-99% (80-89%) amino acid (nucleotide) identity compared with the laboratory RABVs and vaccine strains. Phylogenetic analysis based on the P gene revealed that Chinese RABVs strains could be divided into two distinct clades, and several RABV variants were found to co circulating in the same province. Two conserved (CD1, 2) and two variable (VD1, 2) domains were identified by comparing the deduced primary sequences of the encoded P proteins. Two sequence motifs, one believed to confer binding to the cytoplasmic dynein light chain LC8 and a lysine-rich sequence were conserved throughout the Chinese RABVs. In contrast, the isolates exhibited lower conservation of one phosphate acceptor and one internal translation initiation site identified in the P protein of the rabies challenge virus standard (CVS) strain. Bayesian coalescent analysis showed that the P gene in Chinese RABVs have a substitution rate (3.305x10-4 substitutions per site per year) and evolution history (592 years ago) similar to values for the glycoprotein (G) and nucleoprotein (N) reported previously.
Several substitutions were found in the P gene of Chinese RABVs strains compared to the laboratory adapted and vaccine strains, whether these variations could affect the biological characteristics of Chinese RABVs need to be further investigated. The substitution rate and evolution history of P gene is similar to G and N gene, combine the topology of phylogenetic tree based on the P gene is similar to the G and N gene trees, indicate that the P, G and N genes are equally valid for examining the phylogenetics of RABVs.
PMCID: PMC3548735  PMID: 23294868
Rabies virus; Phosphoprotein gene; Genetic diversity; Molecular evolution
11.  The Phylogeography of Rabies in Grenada, West Indies, and Implications for Control 
In Grenada, West Indies, rabies is endemic, and is thought to be maintained in a wildlife host, the small Indian mongoose (Herpestes auropunctatus) with occasional spillover into other hosts. Therefore, the present study was undertaken to improve understanding of rabies epidemiology in Grenada and to inform rabies control policy. Mongooses were trapped island-wide between April 2011 and March 2013 and examined for the presence of Rabies virus (RABV) antigen using the direct fluorescent antibody test (dFAT) and PCR, and for serum neutralizing antibodies (SNA) using the fluorescent antibody virus neutralization test (FAVN). An additional cohort of brain samples from clinical rabies suspects submitted between April 2011 and March 2014 were also investigated for the presence of virus. Two of the 171 (1.7%) live-trapped mongooses were RABV positive by FAT and PCR, and 20 (11.7%) had SNAs. Rabies was diagnosed in 31 of the submitted animals with suspicious clinical signs: 16 mongooses, 12 dogs, 2 cats and 1 goat. Our investigation has revealed that rabies infection spread from the northeast to the southwest of Grenada within the study period. Phylogenetic analysis revealed that the viruses from Grenada formed a monophyletic clade within the cosmopolitan lineage with a common ancestor predicted to have occurred recently (6–23 years ago), and are distinct from those found in Cuba and Puerto Rico, where mongoose rabies is also endemic. These data suggest that it is likely that this specific strain of RABV was imported from European regions rather than the Americas. These data contribute essential information for any potential rabies control program in Grenada and demonstrate the importance of a sound evidence base for planning interventions.
Author Summary
Rabies, a fatal disease of animals and humans has been endemic in Grenada, West Indies, since the early 1900s. The small Indian mongoose, an introduced animal, is the most likely rabies reservoir, with spillover into domestic animals and humans. To control rabies, large numbers of mongooses were killed in the 1960s/1970s, but this effort did not alter long-term rabies dynamics. Vaccination of dogs, cats and livestock is efficient in protecting these animals, yet is not regularly undertaken. Post-exposure prophylaxis (PEP) in humans is routinely done and no human has died of rabies in Grenada since 1970. However, the threat of rabies and potential to adversely affect the tourism industry, are a burden on the Grenadian government and public. This study has re-evaluated the role of the mongoose in the maintenance of rabies in Grenada, and for the first time, the rabies virus circulating in Grenada has been described. Grenada offers optimal conditions for an oral rabies vaccination (ORV) program, being an island with strict live animal import controls, and a single wildlife rabies reservoir. Although further work is needed before an ORV campaign could be implemented, elimination of rabies from Grenada seems a realistic goal.
PMCID: PMC4199513  PMID: 25330178
12.  ICAM-1-Based Rabies Virus Vaccine Shows Increased Infection and Activation of Primary Murine B Cells In Vitro and Enhanced Antibody Titers In-Vivo 
PLoS ONE  2014;9(1):e87098.
We have previously shown that live-attenuated rabies virus (RABV)-based vaccines infect and directly activate murine and human primary B cells in-vitro, which we propose can be exploited to help develop a single-dose RABV-based vaccine. Here we report on a novel approach to utilize the binding of Intracellular Adhesion Molecule-1 (ICAM-1) to its binding partner, Lymphocyte Function-associated Antigen-1 (LFA-1), on B cells to enhance B cell activation and RABV-specific antibody responses. We used a reverse genetics approach to clone, recover, and characterize a live-attenuated recombinant RABV-based vaccine expressing the murine Icam1 gene (rRABV-mICAM-1). We show that the murine ICAM-1 gene product is incorporated into virus particles, potentially exposing ICAM-1 to extracellular binding partners. While rRABV-mICAM-1 showed 10-100-fold decrease in viral titers on baby hamster kidney cells compared to the parental virus (rRABV), rRABV-mICAM-1 infected and activated primary murine B cells in-vitro more efficiently than rRABV, as indicated by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. ICAM-1 expression on the virus surface was responsible for enhanced B cell infection since pre-treating rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody reduced B cell infection to levels observed with rRABV alone. Furthermore, 100-fold less rRABV-mICAM-1 was needed to induce antibody titers in immunized mice equivalent to antibody titers observed in rRABV-immunized mice. Of note, only 103 focus forming units (ffu)/mouse of rRABV-mICAM-1 was needed to induce significant anti-RABV antibody titers as early as five days post-immunization. As both speed and potency of antibody responses are important in controlling human RABV infection in a post-exposure setting, these data show that expression of Icam1 from the RABV genome, which is then incorporated into the virus particle, is a promising strategy for the development of a single-dose RABV vaccine that requires only a minimum of virus.
PMCID: PMC3906113  PMID: 24489846
13.  Complete Genome Sequence of a Street Rabies Virus Isolated from a Dog in Nigeria 
Genome Announcements  2013;1(1):e00214-12.
A canine rabies virus (RABV) was isolated from a trade dog in Nigeria. Its entire genome was sequenced and found to be closely related to canine RABVs circulating in Africa. Sequence comparison indicates that the virus is closely related to the Africa 2 RABV lineage. The virus is now termed DRV-NG11.
PMCID: PMC3587938  PMID: 23469344
14.  Antibody Quality and Protection from Lethal Ebola Virus Challenge in Nonhuman Primates Immunized with Rabies Virus Based Bivalent Vaccine 
PLoS Pathogens  2013;9(5):e1003389.
We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.
Author Summary
Ebola virus (EBOV) has been associated with outbreaks in human and nonhuman primate populations since 1976. With a fatality rate approaching 90%, EBOV is one of the most lethal infectious diseases in humans. The increased frequency of EBOV outbreaks along with its potential to be used as a bioterrorism agent has dramatically strengthened filovirus vaccine research and development. While there are currently no approved vaccines or post exposure treatments available for human use, several vaccine candidates have shown to protect nonhuman primates from lethal EBOV challenge. Our primary focus is to develop vaccine candidates to protect humans and endangered wildlife species at risk of infection in Africa. Here, we evaluated the efficacy and immunogenicity of our dual vaccines against EBOV and rabies virus (RABV) in rhesus macaques. Our live replication-competent vaccine provided 100% protection following EBOV challenge while the replication-deficient and inactivated candidates provided 50% protection. Interestingly, protection is dependent on the quality of the antibodies rather than the quantity. All three RABV-based EBOV vaccines did induce antibody levels necessary for protection from RABV infection. These results encourage the further development of these novel dual vaccines directed against two of the most lethal viral diseases.
PMCID: PMC3667758  PMID: 23737747
15.  Rabies Virus Infection Induces Type I Interferon Production in an IPS-1 Dependent Manner While Dendritic Cell Activation Relies on IFNAR Signaling 
PLoS Pathogens  2010;6(7):e1001016.
As with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5−/− and RIG-I−/− mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I−/− cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1−/− mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.
Author Summary
Rabies virus (RABV) is a neurotropic RNA virus responsible for the deaths of the at least 40,000 to 70,000 individuals globally each year. However, the innate immune response induced by both wildtype and vaccine strains of RABV is not well understood. In this study, we assessed the pattern recognition receptors involved in the host immune response to RABV in bone marrow derived dendritic cells (DC). Our studies revealed that Toll like receptor (TLR) signaling is not required to induce innate responses to RABV. On the other hand, we see that IPS-1, the adaptor protein for RIG-I like receptor (RLR) signaling, is essential for induction of innate immune responses. Furthermore, we found that RIG-I and Mda-5, both RLRs, are able to induce DC activation and type I interferon production. This finding is significant as we can target unused pattern recognition receptors with recombinant RABV vaccine strains to elicit a varied, and potentially protective, immune response. Lastly, we show that IPS-1 plays an important role in mediating the pathogenicity of RABV and preventing RABV associated paralysis. Overall, this study illustrates that RLRs are essential for recognition of RABV infection and that the subsequent host cell signaling is required to prevent disease.
PMCID: PMC2908622  PMID: 20661430
16.  Immunization of mice with the non-toxic HC50 domain of botulinum neurotoxin presented by rabies virus particles induces a strong immune response affording protection against high-dose botulinum neurotoxin challenge 
Vaccine  2011;29(28):4638-4645.
We previously showed that rabies virus (RABV) virions are excellent vehicles for antigen presentation. Here, a reverse genetic approach was applied to generate recombinant RABV that express a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin type A (BoNT/A) and RABV glycoprotein (G). To promote surface expression and incorporation of HC50/A into RABV virions, the RABV glycoprotein (G) ER translocation sequence, various fragments of RABV ectodomain (ED) and cytoplasmic domain were fused to HC50/A. The HC50/A chimeric proteins were expressed on the surface of cells infected with all of the recombinant RABVs, however, the highest level of surface expression was detected by utilizing 30 amino acids of the RABV G ED (HV50/A-E30). Our results also indicated that this chimeric protein was effectively incorporated into RABV virions. Immunization of mice with inactivated RABV-HC50/A-E30 virions induced a robust anti-HC50/A IgG antibody response that efficiently neutralized circulating BoNT/A in vivo, and protected mice against 1000 fold the lethal dose of BoNT/A.
PMCID: PMC3114282  PMID: 21549784
17.  Intramuscular Inoculation of Mice with the Live-Attenuated Recombinant Rabies Virus TriGAS Results in a Transient Infection of the Draining Lymph Nodes and a Robust, Long-Lasting Protective Immune Response against Rabies 
Journal of Virology  2013;87(3):1834-1841.
A single intramuscular application of the live but not UV-inactivated recombinant rabies virus (RABV) variant TriGAS in mice induces the robust and sustained production of RABV-neutralizing antibodies that correlate with long-term protection against challenge with an otherwise lethal dose of the wild-type RABV. To obtain insight into the mechanism by which live TriGAS induces long-lasting protective immunity, quantitative PCR (qPCR) analysis of muscle tissue, draining lymph nodes, spleen, spinal cord, and brain at different times after TriGAS inoculation revealed the presence of significant copy numbers of RABV-specific RNA in muscle, lymph node, and to a lesser extent, spleen for several days postinfection. Notably, no significant amounts of RABV RNA were detected in brain or spinal cord at any time after TriGAS inoculation. Differential qPCR analysis revealed that the RABV-specific RNA detected in muscle is predominantly genomic RNA, whereas RABV RNA detected in draining lymph nodes is predominantly mRNA. Comparison of genomic RNA and mRNA obtained from isolated lymph node cells showed the highest mRNA-to-genomic-RNA ratios in B cells and dendritic cells (DCs), suggesting that these cells represent the major cell population that is infected in the lymph node. Since RABV RNA declined to undetectable levels by 14 days postinoculation of TriGAS, we speculate that a transient infection of DCs with TriGAS may be highly immunostimulatory through mechanisms that enhance antigen presentation. Our results support the superior efficacy and safety of TriGAS and advocate for its utility as a vaccine.
PMCID: PMC3554143  PMID: 23192867
18.  Laboratory Surveillance of Rabies in Humans, Domestic Animals, and Bats in Madagascar from 2005 to 2010 
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.
PMCID: PMC3170745  PMID: 21991442
19.  Involvement of the Rabies Virus Phosphoprotein Gene in Neuroinvasiveness 
Journal of Virology  2013;87(22):12327-12338.
Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-β) gene (Ifn-β) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.
PMCID: PMC3807887  PMID: 24027304
20.  The origin and phylogeography of dog rabies virus 
The Journal of General Virology  2008;89(Pt 11):2673-2681.
Rabies is a progressively fatal and incurable viral encephalitis caused by a lyssavirus infection. Almost all of the 55 000 annual rabies deaths in humans result from infection with dog rabies viruses (RABV). Despite the importance of rabies for human health, little is known about the spread of RABV in dog populations, and patterns of biodiversity have only been studied in limited geographical space. To address these questions on a global scale, we sequenced 62 new isolates and performed an extensive comparative analysis of RABV gene sequence data, representing 192 isolates sampled from 55 countries. From this, we identified six clades of RABV in non-flying mammals, each of which has a distinct geographical distribution, most likely reflecting major physical barriers to gene flow. Indeed, a detailed analysis of phylogeographic structure revealed only limited viral movement among geographical localities. Using Bayesian coalescent methods we also reveal that the sampled lineages of canid RABV derive from a common ancestor that originated within the past 1500 years. Additionally, we found no evidence for either positive selection or widespread population bottlenecks during the global expansion of canid RABV. Overall, our study reveals that the stochastic processes of genetic drift and population subdivision are the most important factors shaping the global phylogeography of canid RABV.
PMCID: PMC3326349  PMID: 18931062
21.  Rabies Virus Is Recognized by the NLRP3 Inflammasome and Activates Interleukin-1β Release in Murine Dendritic Cells 
Journal of Virology  2013;87(10):5848-5857.
Inflammasome activation is important for the development of an effective host defense against many pathogens, including RNA viruses. However, the mechanism by which the inflammasome recognizes RNA viruses and its role in rabies virus (RABV) pathogenicity and immunogenicity remain poorly defined. To determine the function of the inflammasome in response to RABV infection, we infected murine bone marrow-derived dendritic cells (BMDCs) with RABV. Our results indicate that the infection of BMDCs with RABV induces both the production of pro-interleukin-1β (pro-IL-1β) and its processing, resulting in the secretion of active IL-1β through activation of the NLRP3-, ASC-, and caspase-1-dependent inflammasome. As previously shown for the induction of type I interferon by RABV, the induction of pro-IL-1β also depends upon IPS-1. We demonstrate that both the production of pro-IL-1β and activation of the inflammasome require viral replication. We also demonstrate that increased viral replication in BMDCs derived from IFNAR-deficient mice resulted in significantly more IL-1β release. Additionally, IL-1 receptor-deficient mice show an increase in RABV pathogenicity. Taken together, these results indicate an important role of the inflammasome in innate immune recognition of RABV.
PMCID: PMC3648142  PMID: 23487464
22.  Isolation and Identification of a Novel Rabies Virus Lineage in China with Natural Recombinant Nucleoprotein Gene 
PLoS ONE  2012;7(12):e49992.
Rabies virus (RABV) causes severe neurological disease and death. As an important mechanism for generating genetic diversity in viruses, homologous recombination can lead to the emergence of novel virus strains with increased virulence and changed host tropism. However, it is still unclear whether recombination plays a role in the evolution of RABV. In this study, we isolated and sequenced four circulating RABV strains in China. Phylogenetic analyses identified a novel lineage of hybrid origin that comprises two different strains, J and CQ92. Analyses revealed that the virus 3′ untranslated region (UTR) and part of the N gene (approximate 500 nt in length) were likely derived from Chinese lineage I while the other part of the genomic sequence was homologous to Chinese lineage II. Our findings reveal that homologous recombination can occur naturally in the field and shape the genetic structure of RABV populations.
PMCID: PMC3514186  PMID: 23226506
23.  Enhancement of Blood-Brain Barrier Permeability and Reduction of Tight Junction Protein Expression Are Modulated by Chemokines/Cytokines Induced by Rabies Virus Infection 
Journal of Virology  2014;88(9):4698-4710.
Infection with laboratory-attenuated rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, which has been demonstrated to be an important factor for host survival, since it allows immune effectors to enter the central nervous system (CNS) and clear RABV. To probe the mechanism by which RABV infection enhances BBB permeability, the expression of tight junction (TJ) proteins in the CNS was investigated following intracranial inoculation with laboratory-attenuated or wild-type (wt) RABV. BBB permeability was significantly enhanced in mice infected with laboratory-attenuated, but not wt, RABV. The expression levels of TJ proteins (claudin-5, occludin, and zonula occludens-1) were decreased in mice infected with laboratory-attenuated, but not wt, RABV, suggesting that enhancement of BBB permeability is associated with the reduction of TJ protein expression in RABV infection. RABV neither infects the brain microvascular endothelial cells (BMECs) nor modulates the expression of TJ proteins in BMECs. However, brain extracts prepared from mice infected with laboratory-attenuated, but not wt, RABV reduced TJ protein expression in BMECs. It was found that brain extracts from mice infected with laboratory-attenuated RABV contained significantly higher levels of inflammatory chemokines/cytokines than those from mice infected with wt RABV. Pathway analysis indicates that gamma interferon (IFN-γ) is located in the center of the cytokine network in the RABV-infected mouse brain, and neutralization of IFN-γ reduced both the disruption of BBB permeability in vivo and the downregulation of TJ protein expression in vitro. These findings indicate that the enhancement of BBB permeability and the reduction of TJ protein expression are due not to RABV infection per se but to virus-induced inflammatory chemokines/cytokines.
IMPORTANCE Previous studies have shown that infection with only laboratory-attenuated, not wild-type, rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, allowing immune effectors to enter the central nervous system (CNS) and clear RABV from the CNS. This study investigated the mechanism by which RABV infection enhances BBB permeability. It was found that RABV infection enhances BBB permeability by downregulation of tight junction (TJ) protein expression in the brain microvasculature. It was further found that it is not RABV infection per se but the chemokines/cytokines induced by RABV infection that downregulate the expression of TJ proteins and enhance BBB permeability. Blocking some of these cytokines, such as IFN-γ, ameliorated both the disruption of BBB permeability and the downregulation of TJ protein expression. These studies may provide a foundation for developing therapeutics for clinical rabies, such as medication that could be used to enhance BBB permeability.
PMCID: PMC3993813  PMID: 24522913
24.  Molecular Characterization of Cryptically Circulating Rabies Virus from Ferret Badgers, Taiwan 
Emerging Infectious Diseases  2014;20(5):790-798.
The virus has been circulating in Taiwan for about 100 years.
After the last reported cases of rabies in a human in 1959 and a nonhuman animal in 1961, Taiwan was considered free from rabies. However, during 2012–2013, an outbreak occurred among ferret badgers in Taiwan. To examine the origin of this virus strain, we sequenced 3 complete genomes and acquired multiple rabies virus (RABV) nucleoprotein and glycoprotein sequences. Phylogeographic analyses demonstrated that the RABV affecting the Taiwan ferret badgers (RABV-TWFB) is a distinct lineage within the group of lineages from Asia and that it has been differentiated from its closest lineages, China I (including isolates from Chinese ferret badgers) and the Philippines, 158–210 years ago. The most recent common ancestor of RABV-TWFB originated 91–113 years ago. Our findings indicate that RABV could be cryptically circulating in the environment. An understanding of the underlying mechanism might shed light on the complex interaction between RABV and its host.
PMCID: PMC4012806  PMID: 24751120
Rabies; Melogale moschata subaurantiaca; phylogeography; origin; Taiwan; ferret badger; viruses
25.  Investigating the Role for IL-21 in Rabies Virus Vaccine-induced Immunity 
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R−/−) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R−/− mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R−/− mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R−/− mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.
Author Summary
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving post-exposure treatment. A person, disproportionately a child, dies of rabies every 20 minutes and the cost of rabies prevention exceeds $1 billion US dollars per year. The development of a single-dose human rabies vaccine would greatly reduce the burden of rabies globally by lowering the cost associated with rabies vaccination and saving lives. Understanding how B cells develop to produce protective virus neutralizing antibodies would greatly help to achieve the goal of developing a single-dose vaccine. In this report, we show that IL-21 is critical for the induction of primary vaccine-induced anti-RABV G antibody titers and that the effects of IL-21 are highly dependent on the dose of vaccine administered. In our model of rabies immunogenicity and protection, the lack of IL-21 receptor influenced the detection of B cells in germinal centers in lymph nodes or of plasma cells in bone marrow after immunization with low or high doses of vaccine, respectively. Overall, these preliminary results indicate that IL-21 has the potential to influence B cell development and functions in the context of rabies vaccine-induced immunity and protection.
PMCID: PMC3597479  PMID: 23516660

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