Molecular diversity of rabies viruses associated with bats in Mexico and other countries of the Americas

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.     

Demonstration of antigenic variation among rabies virus isolates by using monoclonal antibodies to nucleocapsid proteins.

Rabies virus isolates from terrestrial animals in six areas of the United States were examined with a panel of monoclonal antibodies to nucleocapsid proteins. Characteristic differences in immunofluorescence reactions permitted the formation of four antigenically distinct reaction groups from the 231 isolates tested. The geographic distribution of these groups corresponded well with separate rabies enzootic areas recognized by surveillance of sylvatic rabies in the United States. Distinctive reaction patterns were also identified for viral proteins from four infected bat species, and identical patterns were found in eight isolated cases of rabies in terrestrial animals. These findings suggest that monoclonal antibodies can be used to study the prevalence, distribution, and transmission of rabies among wildlife species.     

Antigenic and molecular characterization of bat rabies virus in Europe.

The predominant role of Eptesicus serotinus in the epizootic of bat rabies in Europe was further outlined by the first isolation of the rabies virus from this species in France. The distribution of the virus was studied in naturally infected E. serotinus bats at the time of death and suggested that the papillae of the tongue and the respiratory mucosa may play a role in virus production and excretion. The analysis of 501 French rabies virus isolates from various animal species by antinucleocapsid monoclonal antibodies indicated that transmission of the disease from bats to terrestrial animals is unlikely. The antigenic profile of two isolates from French bats corresponded to that of European bat lyssavirus type 1 (EBL1). Comparisons of 12 different isolates from bats with antinucleocapsid and antiglycoprotein monoclonal antibodies and by direct sequencing of the polymerase chain reaction amplification product of the N gene indicated that EBL1, EBL2, Duvenhage virus (serotype 4 of lyssavirus), and the European fox rabies virus (serotype 1) are phylogenetically distant. They formed four tight genetic clusters named genotypes. EBL1 was shown to be antigenically and genetically more closely related to Duvenhage virus than to EBL2. We propose that EBL1 and EBL2 constitute two distinct genotypes which further serologic characterization will probably classify as new serotypes. We also report a simple method for the rapid characterization of EBL based on the digestion of the polymerase chain reaction product of the N gene by three restriction endonucleases.     

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.     

Phylogeography of rabies virus isolated from herbivores and bats in the Espírito Santo State, Brazil

Rabies is enzootic in the State of Espírito Santo, Brazil. Every year, cattle and horses die from rabies that is transmitted by the vampire bat Desmodus rotundus. This paper describes the spread of the rabies virus by the continuous diffusion model using relaxed random walks with BEAST software. Forty-one (41) sequences of gene G from the rabies virus that was isolated from bats and domestic herbivores from several areas of the state between 2006 and 2010 were analyzed. The phylogenetic tree showed three main clusters as well as two sub-clusters under cluster 2. A spatial analysis showed that three strains of the rabies virus spread independently. In general, central Espírito Santo, which is mountainous, was the area where separation of the virus strains occurred. This physical barrier, however, was overcome at some point in time, as samples from different lineages were found in the same microarea.     

Antigenic and molecular characterization of rabies virus in Argentina

The nucleoprotein genes of 54 human, domestic and wild animals rabies isolates obtained in Argentina between 1995 and 2002 were characterized using monoclonal antibodies and partial gene sequence analysis. The antigenic and genetic diversities of rabies virus in samples from bat and bat-related cases were studied, leading to the identification of five distinct genetic variants. Rabies viruses isolated from vampire bat related cases were very similar to each other, showing 98.9% overall similarity. Specific antigenic variants (AgV) were detected associated with different insectivorous bats species, in samples from Tadarida brasiliensis and Eumops patagonicus bats. In contrast, isolates from Myotis sp. and Histiotus sp. bats could not be matched to any antigenic type. Additionally, bat rabies cases were also detected in southern provinces previously considered rabies-free. Finally, two independent antigenic and genetic variants co-circulating in northern Argentina were found in isolates obtained from dogs and dog-related cases, suggesting two independent cycles of virus transmission. This is the first national coordinated study of antigenic as well as molecular epidemiology of rabies in Argentina. The information presented here will improve our knowledge about rabies epidemiology and therefore, will assist preventing fatal human cases.     

The molecular epidemiology of rabies associated with chiropteran hosts in Mexico

While large-scale dog vaccination campaigns have significantly reduced urban rabies throughout Mexico, reports of sylvatic rabies, including cases of spill-over of bat strains into livestock and humans, are increasing. To improve knowledge of these epidemiological trends, 64 Mexican rabies virus isolates from various host species, have been characterized. Phylogenetic analysis at the viral P locus identified distinct viral strains associated with terrestrial reservoirs (dog, skunk and fox/bobcat) and a variant associated with the insectivorous bat, T. brasiliensis, consistent with prior reports. Of the two distinct clades of viruses associated with the vampire bat reservoir, one comprised just four specimens and formed an outlying group to all other vampire bat rabies isolates including those from South America and the Caribbean, a finding consistent with the early emergence of the vampire bat reservoir in Mexico. Antigenic variation of the vampire bat specimens did not correlate with the main genetic groupings; moreover complete N gene sequence analysis of selected specimens indicated limited variation within the encoded nucleoprotein that could form the basis of antigenic variation. A single isolate recovered from a cat represents a new viral variant not previously identified in North America that probably circulates in a species of insectivorous bat.     

Use of monoclonal antibodies in diagnosis of rabies virus infection and differentiation of rabies and rabies-related viruses

A panel of selected monoclonal antibodies directed against the nucleocapsid antigens of rabies and rabies-related viruses allowed the rapid identification both of antigens of rabies virus origin present in impression smears of brains from infected animals and of three rabies-related viruses (Duvenhage, Lagos bat and Mokola). Within the rabies group, the CVS and Flury HEP strains could also be identified. Analysis of several fixed and street rabies viruses reveals great diversity in the reactivity of the viruses with a panel of monoclonal antibodies directed against glycoprotein antigens. Strains of the same geographical area and species origin displayed similar patterns of reactivity, suggesting an antigenic specificity related to animal species or area. The use of hybridoma monoclonal antibodies should provide a more sophisticated method than presently available for the diagnosis of rabies virus infection.     

Surveillance for European bat lyssavirus in Swiss bats

Most countries in Western Europe are currently free of rabies in terrestrial mammals. Nevertheless, rabies remains a residual risk to public health due to the natural circulation of bat-specific viruses, such as European bat lyssaviruses (EBLVs). European bat lyssavirus types 1 and 2 (EBLV-1 and EBLV-2) are widely distributed throughout Europe, but little is known of their true prevalence and epidemiology. We report that only three out of 837 brains taken from bats submitted to the Swiss Rabies Centre between 1976 and 2009 were found by immunofluorescence (FAT) to be positive for EBLVs. All three positive cases were in Myotis daubentoni, from 1992, 1993 and 2002. In addition to this passive surveillance, we undertook a targeted survey in 2009, aimed at detecting lyssaviruses in live bats in Switzerland. A total of 237 bats of the species M. daubentoni, Myotis myotis, Eptesicus serotinus and Nyctalus noctula were captured at different sites in western Switzerland. Oropharyngeal swabs and blood from each individual were analysed by RT-PCR and rapid fluorescent focus inhibition test (RFFIT), respectively. RNA corresponding to EBLV-2 was detected from oropharyngeal swabs of a single M. daubentoni bat, but no infectious virus was found. Molecular phylogenetic analysis revealed that the corresponding sequence was closely related to the other EBLV-2 sequences identified in previous rabies isolates from Swiss bats (particularly to that found at Geneva in 2002). Three M. daubentoni bats were found to be seropositive by RFFIT. In conclusion, even though the prevalence is low in Switzerland, continuous management and surveillance are required to assess the potential risk to public health.     

Unique characteristics of bat rabies viruses in big brown bats (Eptesicus fuscus)

Rabies virus infection has been documented in several North American bat species, including Eptesicus fuscus. The virus-host relationship between bats and rabies virus (RV) is not well understood. The incidence of non-lethal RV exposure, based on the presence of viral neutralizing antibodies, demonstrates that exposure to RV does not always lead to clinical infection in bats. It is unknown how the route of exposure, rabies virus variant, or health of the bat affects the outcome following exposure. This paper describes the pathogenesis of two big brown bat RV variants in homologous host species. Our study demonstrates that RV variants obtained from the same species of bat from similar geographical areas may result in a diverse clinical progression of disease.     

RT-PCR for detection of all seven genotypes of Lyssavirus genus

The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.     

Molecular epizootiology of rabies associated with terrestrial carnivores in Mexico

Epizootiological patterns of rabies are described, using antigenic and genetic analysis of samples obtained from infected domestic and wild mammals in 20 Mexican states during 1976-2002. Two independent origins are suggested for rabies in Mexican carnivores. One group shares ancestry with canine rabies, while the other group appears to share a common origin with bat rabies in North America. More than 12 sublineages were found in rabid dog populations, suggesting at least six major spatio-temporal foci. Coyote rabies was found as independent enzootic foci that probably emerged via spillover from dog rabies, translocated from major foci in the southcentral and western regions of Mexico. One focus of gray fox rabies was widely distributed in northwestern Mexico, overlapping with a focus in the same species in the southwestern United States. A skunk rabies focus distributed in the northcentral Mexican states appears to share a common origin with bat rabies foci in North America, and is a close relative of southcentral skunk and raccoon rabies in the United States. Two other skunk foci share a common ancestor with canine rabies and were distributed in northwest Mexico and Yucatan.     

Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophus hipposideros) in Slovenia

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Recently, a large number of viruses closely related to members of the genus Coronavirus have been associated with severe acute respiratory syndrome (SARS) and detected in bat species. In this study, samples were collected from 106 live bats of seven different bat species from 27 different locations in Slovenia. Coronaviruses were detected by RT-PCR in 14 out of 36 horseshoe bat (Rhinolophus hipposideros) fecal samples, with 38.8% virus prevalence. Sequence analysis of a 405-nucleotide region of the highly conserved RNA polymerase gene (pol) showed that all coronaviruses detected in this study are genetically closely related, with 99.5–100% nucleotide identity, and belong to group 2 of the coronaviruses. The most closely related virus sequence in GenBank was SARS bat isolate Rp3/2004 (DQ071615) within the SARS-like CoV cluster, sharing 85% nucleotide identity and 95.6% amino acid identity. The potential risk of a new group of bat coronaviruses as a reservoir for human infections is highly suspected, and further molecular epidemiologic studies of these bat coronaviruses are needed.     

Use of anti-glycoprotein monoclonal antibodies to characterize rabies virus in formalin-fixed tissues

Seventy anti-rabies virus monoclonal antibodies (Mabs) were tested for reactivity with rabies and rabies-related viruses in formalin-fixed (FF) tissues. Forty-three of the Mabs were directed against the glycoprotein and 27 were directed against the nucleocapsid as determined by enzyme immunoassays and neutralization tests. Twenty of the anti-glycoprotein Mabs and one of the anti-nucleocapsid Mabs reacted with the rabies challenge virus strain (CVS) in FF tissue. These 21 Mabs were screened against other lyssaviruses in FF tissues: five rabies virus strains (coyote, skunk, raccoon, red bat, and silver-haired bat), and four rabies-related viruses (Australian bat lyssavirus, Duvenhage virus, Lagos bat virus, and Mokola virus). One of the anti-glycoprotein Mabs was reactive with all the virus strains screened. Another of the anti-glycoprotein Mabs reacted with all of the rabies virus strains tested, but not with any of the rabies-related virus strains tested. The remaining Mabs had reactivity patterns that could be useful for characterizing lyssaviruses in FF tissues.     

Rabies in southeast Brazil: a change in the epidemiological pattern

This epidemiological study was conducted using antigenic and genetic characterisation of rabies virus isolates obtained from different animal species in the southeast of Brazil from 1993 to 2007. An alteration in the epidemiological profile was observed. One hundred two samples were tested using a panel of eight monoclonal antibodies, and 94 were genetically characterised by sequencing the nucleoprotein gene. From 1993 to 1997, antigenic variant 2 (AgV-2), related to a rabies virus maintained in dog populations, was responsible for rabies cases in dogs, cats, cattle and horses. Antigenic variant 3 (AgV-3), associated with Desmodus rotundus, was detected in a few cattle samples from rural areas. From 1998 to 2007, rabies virus was detected in bats and urban pets, and four distinct variants were identified. A nucleotide similarity analysis resulted in two primary groups comprising the dog and bat antigenic variants and showing the distinct endemic cycles maintained in the different animal species in this region.     

Cross-reactive antigenicity of nucleoproteins of lyssaviruses recognized by a monospecific antirabies virus nucleoprotein antiserum on paraffin sections of formalin-fixed tissues

Diagnosis of rabies is routinely confirmed by detection of rabies virus antigens in acetone-fixed frozen brain tissues or imprint smears using an immunofluorescence method with commercial antirabies virus antibodies. Since recent molecular analyses disclosed wide heterogeneity in the genome sequences of rabies virus strains and related lyssaviruses, it is necessary to confirm the presence of common epitopes in these lyssaviruses. In this study we confirmed the presence of cross-reactive antigens of various lyssaviruses in paraffin sections of formalin-fixed tissue using a monospecific rabbit antiserum prepared by immunization with a recombinant nucleoprotein of rabies virus. By immunohistochemical application, the antigen was detected predominantly in the cytoplasm of neurons in the brains of mice infected with rabies virus, Duvenhage virus, Mokola virus and European bat lyssavirus-1, while no cross-reaction was observed in uninfected humans and animals including dogs, bats, and raccoons. In addition, we examined one autopsy case that was infected in a rabies-endemic nation and developed the clinical manifestation of rabies after returning to Japan in 1970, and found that the antigen was well preserved in paraffin sections of formalin-fixed tissues. Thus, this suggests that the lyssavirus-specific antigen is recognized by the monospecific antibody against rabies virus nucleoprotein, and that this cross-reactive antigen is detectable on formalin-fixed paraffin-embedded tissues by immunohistochemical analysis.