Phylodynamics of vampire bat‐transmitted rabies in Argentina

Common vampire bat populations distributed from Mexico to Argentina are important rabies reservoir hosts in Latin America. The aim of this work was to analyse the population structure of the rabies virus (RABV) variants associated with vampire bats in the Americas and to study their phylodynamic pattern within Argentina. The phylogenetic analysis based on all available vampire bat‐related N gene sequences showed both a geographical and a temporal structure. The two largest groups of RABV variants from Argentina were isolated from northwestern Argentina and from the central western zone of northeastern Argentina, corresponding to livestock areas with different climatic, topographic and biogeographical conditions, which determined their dissemination and evolutionary patterns. In addition, multiple introductions of the infection into Argentina, possibly from Brazil, were detected. The phylodynamic analysis suggests that RABV transmission dynamics is characterized by initial epizootic waves followed by local enzootic cycles with variable persistence. Anthropogenic interventions in the ecosystem should be assessed taking into account not only the environmental impact but also the potential risk of disease spreading through dissemination of current RABV lineages or the emergence of novel ones associated with vampire bats.     

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.     

Bioecological Drivers of Rabies Virus Circulation in a Neotropical Bat Community

Introduction

In addition to the commonly accepted importance of the vampire bat in the maintenance and transmission of the rabies virus (RABV) in South America, RABV infection of other species is widely evidenced, challenging their role in the viral cycle.

Methodology / Principles findings

To identify the bioecological drivers of RABV circulation in neotropical bat communities, we conducted a molecular and serological survey on almost 1,000 bats from 30 species, and a 4-year longitudinal survey in two colonies of vampire bats in French Guiana. RABV was molecularly detected in a common vampire and in a frugivorous bat. The sequences corresponded to haematophagous bat-related strains and were close to viruses circulating in the Brazilian Amazon region. Species’ seroprevalence ranged from 0 to 20%, and the risk of seropositivity was higher in bats with a haematophagous diet, living in monospecific colonies and in dense forests. The longitudinal survey showed substantial temporal fluctuations, with individual waves of seroconversions and waning immunity. The high prevalences observed in bat communities, in most habitats and in species that do not share the same microhabitats and bioecological patterns, the temporal variations, and a rather short period of detectable antibodies as observed in recaptured vampires suggest (i) frequent exposure of animals, (ii) an ability of the infected host to control and eliminate the virus, (iii) more relaxed modes of exposure between bats than the commonly assumed infection via direct contact with saliva of infected animals, all of which should be further investigated.

Conclusions / significance

We hypothesize that RABV circulation in French Guiana is mainly maintained in the pristine forest habitats that may provide sufficient food resources to allow vampire bats, the main prevalent species, to survive and RABV to be propagated. However, on the forest edge and in disturbed areas, human activities may induce more insidious effects such as defaunation. One of the ecological consequences is the disappearance of resources for tertiary or secondary consumers. Populations of vampires may then shift to alternative resources such as cattle, domestic animals and humans. Therefore, a good forest status, allowing both a dilution effect in highly rich bat communities and the maintenance of large populations of medium-sized and large mammals used as prey by vampires, should prevent their migration to anthropized areas.     

Molecular Inferences Suggest Multiple Host Shifts of Rabies Viruses from Bats to Mesocarnivores in Arizona during 2001–2009

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.     

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.     

High diversity of rabies viruses associated with insectivorous bats in Argentina: presence of several independent enzootics

Background

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.

Methods

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.

Results

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.

Conclusions

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.     

A comparative study of rabies virus isolates from hematophagous bats in Brazil

The Brazilian chiropteran fauna consists of 167 species; of which, three are hematophagous: the common vampire bat (Desmodus rotundus), the white-winged vampire bat (Diaemus youngi), and the hairy-legged vampire bat (Diphylla ecaudata). The aim of this study was to describe the isolation of Rabies virus from common and hairy-legged vampire bats and to report the first comparative antigenic and genetic studies of isolates from these bats. Antigenic and genetic typing of both isolates identified them as antigenic variant 3 (AgV3), the variant frequently isolated from common vampire bats. Phylogenetic analysis showed 99.3% identity between the isolates. This is the first time since 1934 that Rabies virus has been isolated from hairy-legged vampire bats in Brazil. Our analysis provides evidence that the existence of rabies-positive isolates from hairy-legged vampire bats may be the result of an interspecific rabies transmission event from common vampire bats and suggests that roost cohabitation may occur.     

Using DNA metabarcoding for simultaneous inference of common vampire bat diet and population structure

Metabarcoding diet analysis has become a valuable tool in animal ecology; however, co‐amplified predator sequences are not generally used for anything other than to validate predator identity. Exemplified by the common vampire bat, we demonstrate the use of metabarcoding to infer predator population structure alongside diet assessments. Growing populations of common vampire bats impact human, livestock and wildlife health in Latin America through transmission of pathogens, such as lethal rabies viruses. Techniques to determine large‐scale variation in vampire bat diet and bat population structure would empower locality‐ and species‐specific projections of disease transmission risks. However, previously used methods are not cost‐effective and efficient for large‐scale applications. Using bloodmeal and faecal samples from common vampire bats from coastal, Andean and Amazonian regions of Peru, we showcase metabarcoding as a scalable tool to assess vampire bat population structure and feeding preferences. Dietary metabarcoding was highly effective, detecting vertebrate prey in 93.2% of the samples. Bats predominantly preyed on domestic animals, but fed on tapirs at one Amazonian site. In addition, we identified arthropods in 9.3% of samples, likely reflecting consumption of ectoparasites. Using the same data, we document mitochondrial geographic population structure in the common vampire bat in Peru. Such simultaneous inference of vampire bat diet and population structure can enable new insights into the interplay between vampire bat ecology and disease transmission risks. Importantly, the methodology can be incorporated into metabarcoding diet studies of other animals to couple information on diet and population structure.     

Ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control

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.     

Using host traits to predict reservoir host species of rabies virus

Wildlife are important reservoirs for many pathogens, yet the role that different species play in pathogen maintenance frequently remains unknown. This is the case for rabies, a viral disease of mammals. While Carnivora (carnivores) and Chiroptera (bats) are the canonical mammalian orders known to be responsible for the maintenance and onward transmission of rabies Lyssavirus (RABV), the role of most species within these orders remains unknown and is continually changing as a result of contemporary host shifting. We combined a trait-based analytical approach with gradient boosting machine learning models to identify physiological and ecological host features associated with being a reservoir for RABV. We then used a cooperative game theory approach to determine species-specific traits associated with known RABV reservoirs. Being a carnivore reservoir for RABV was associated with phylogenetic similarity to known RABV reservoirs, along with other traits such as having larger litters and earlier sexual maturity. For bats, location in the Americas and geographic range were the most important predictors of RABV reservoir status, along with having a large litter. Our models identified 44 carnivore and 34 bat species that are currently not recognized as RABV reservoirs, but that have trait profiles suggesting their capacity to be or become reservoirs. Further, our findings suggest that potential reservoir species among bats and carnivores occur both within and outside of areas with current RABV circulation. These results show the ability of a trait-based approach to detect potential reservoirs of infection and could inform rabies control programs and surveillance efforts by identifying the types of species and traits that facilitate RABV maintenance and transmission.     

Focus: Zoonotic Disease: Rabies in Costa Rica – Next Steps Towards Controlling Bat-Borne Rabies After its Elimination in Dogs

Rabies is an acute, progressive encephalitis caused by a lyssavirus, with the highest case fatality of any conventional infectious disease. More than 17 different lyssaviruses have been described, but rabies virus is the most widely distributed and important member of the genus. Globally, tens of thousands of human fatalities still occur each year. Although all mammals are susceptible, most human fatalities are caused by the bites of rabid dogs, within lesser developed countries. A global plan envisions the elimination of human rabies cases caused via dogs by the year 2030. The combination of prophylaxis of exposed humans and mass vaccination of dogs is an essential strategy for such success. Regionally, the Americas are well on the way to meet this goal. As one example of achievement, Costa Rica, a small country within Central America, reported the last autochthonous case of human rabies transmitted by a dog at the end of the 1970s. Today, rabies virus transmitted by the common vampire bat, Desmodus rotundus, as well as other wildlife, remains a major concern for humans, livestock, and other animals throughout the region. This review summarizes the historical occurrence of dog rabies and its elimination in Costa Rica, describes the current occurrence of the disease with a particular focus upon affected livestock, discusses the ecology of the vampire bat as the primary reservoir relevant to management, details the clinical characteristics of recent human rabies cases, and provides suggestions for resolution of global challenges posed by this zoonosis within a One Health context.     

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.     

Isolation and Identification of a Novel Rabies Virus Lineage in China with Natural Recombinant Nucleoprotein Gene

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.     

Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil

Background

Vampire bats are important rabies virus vectors, causing critical problems in both the livestock industry and public health sector in Latin America. In order to assess the epidemiological characteristics of vampire bat-transmitted rabies, the authors conducted phylogenetic and geographical analyses using sequence data of a large number of cattle rabies isolates collected from a wide geographical area in Brazil.

Methods

Partial nucleoprotein genes of rabies viruses isolated from 666 cattle and 18 vampire bats between 1987 and 2006 were sequenced and used for phylogenetic analysis. The genetic variants were plotted on topographical maps of Brazil.

Results

In this study, 593 samples consisting of 24 genetic variants were analyzed. Regional localization of variants was observed, with the distribution of several variants found to be delimited by mountain ranges which served as geographic boundaries. The geographical distributions of vampire-bat and cattle isolates that were classified as the identical phylogenetic group were found to overlap with high certainty. Most of the samples analyzed in this study were isolated from adjacent areas linked by rivers.

Conclusion

This study revealed the existence of several dozen regional variants associated with vampire bats in Brazil, with the distribution patterns of these variants found to be affected by mountain ranges and rivers. These results suggest that epidemiological characteristics of vampire bat-related rabies appear to be associated with the topographical and geographical characteristics of areas where cattle are maintained, and the factors affecting vampire bat ecology.

     

PERMANENT GENETIC RESOURCES: Development of polymorphic microsatellite loci for the common vampire bat, Desmodus rotundus (Chiroptera: Phylostomidae)

The common vampire bat (Desmodus rotundus) is one of three haematophagous species of bats and the only species in this genus. These New World bats prey on mammals and create significant economic impacts through transmission of rabies in areas where livestock are prevalent. Furthermore, in some portions of their range, it is not uncommon for them to prey upon humans. It is critical to the management of this species and for understanding the spread of bat rabies that detailed studies of D. rotundus population structure be conducted. To further such studies, we have characterized 12 microsatellite loci for this species.     

Vampire bats exhibit evolutionary reduction of bitter taste receptor genes common to other bats

The bitter taste serves as an important natural defence against the ingestion of poisonous foods and is thus believed to be indispensable in animals. However, vampire bats are obligate blood feeders that show a reduced behavioural response towards bitter-tasting compounds. To test whether bitter taste receptor genes (T2Rs) have been relaxed from selective constraint in vampire bats, we sampled all three vampire bat species and 11 non-vampire bats, and sequenced nine one-to-one orthologous T2Rs that are assumed to be functionally conserved in all bats. We generated 85 T2R sequences and found that vampire bats have a significantly greater percentage of pseudogenes than other bats. These results strongly suggest a relaxation of selective constraint and a reduction of bitter taste function in vampire bats. We also found that vampire bats retain many intact T2Rs, and that the taste signalling pathway gene Calhm1 remains complete and intact with strong functional constraint. These results suggest the presence of some bitter taste function in vampire bats, although it is not likely to play a major role in food selection. Together, our study suggests that the evolutionary reduction of bitter taste function in animals is more pervasive than previously believed, and highlights the importance of extra-oral functions of taste receptor genes.     

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.