Evolução do diagnóstico virológico de raiva humana no Instituto Pasteur de São Paulo, Brasil (1970-2020): análise de dados / Evolution of the virological diagnosis of human rabies at the Pasteur Institute of São Paulo, Brazil (1970-2020): data analysis

O diagnóstico laboratorial precoce de raiva humana deve ser realizado por testes apropriados, visto que a aplicação de protocolos de tratamento médico em indivíduos internados depende dos resultados laboratoriais. O presente estudo analisou os dados referentes aos 560 casos suspeitos de raiva humana submetidos ao diagnóstico virológico no IP-SP entre os anos de 1970 e 2020. Houve um avanço das metodologias laboratoriais, especialmente as moleculares, que passaram a ser essenciais, possibilitando o tratamento de indivíduos expostos, bem como a determinação da fonte de infecção dos casos, fato fundamental para a efetividade de ações de controle em regiões vulneráveis à disseminação da doença. Intervenções no ciclo urbano da raiva, por meio de vacinação de cães e gatos e encaminhamento de amostras para diagnóstico, diminuiram os casos transmitidos por cães, principalmente no Sudeste. Em contrapartida, no mesmo período foi observado um aumento exponencial de casos relacionados ao ciclo silvestre nas regiões Norte (32%) e Nordeste (53,3%), tendo os morcegos como principais transmissores (72%), seguidos dos primatas não humanos (6%) e dos canídeos silvestres (1%). Esses resultados demonstraram a importância do aprimoramento do diagnóstico laboratorial, que é parte essencial na condução de estratégias de controle, bem como de tratamento de indivíduos expostos.

Early laboratory diagnosis of human rabies should be performed by appropriate tests, since the application of medical treatment protocols in hospitalized individuals depends on laboratory results. The present study analyzed the data referring to the 560 suspected cases of human rabies submitted to virological diagnosis in the IP-SP from 1970 to 2020. There has been an advance in laboratory methodologies, especially molecular ones, which have become essential, enabling the treatment of exposed individuals, as well as allowing the determination of the source of infection of cases, a fundamental fact for the effectiveness of control actions in regions vulnerable to the spread of the disease. Interventions in the urban cycle of rabies, through vaccination of dogs and cats and referral of samples for diagnosis decreased the cases transmitted by dogs, especially in the Southeast, on the other hand, an exponential increase of cases was observed in the same period, in the North (32%) and Northeast (53.3%) regions, with cases related to the wild cycle, with bats as the main transmitters (72%), followed by non-human primates (6%), and wild canids (1%). Our results demonstrated the importance of improving laboratory diagnosis, which is an essential part of conducting control strategies as well as the treatment of exposed individuals.

Epidemiologia de doenças zoonóticas em cães e gatos atendidas em hospital veterinário da região de Araçatuba-SP / Epidemiology of zoonotic diseases in dogs and cats treated at a veterinary hospital in the region of Araçatuba-SP

A epidemiologia é uma ferramenta de análise de doenças para explicação de fatores de casualidade aliados aos fatores ambientais, norteando, assim, medidas de prevenção. O objetivo foi calcular as taxas de incidência de zoonoses em cães e gatos e identificar fatores de predisposição acerca de idade, raça e mês de ocorrência de animais atendidos em um hospital veterinário de Araçatuba, estado de São Paulo, entre 2017 e 2019. A população canina apresentou taxa de incidência de leishmaniose visceral de 12,3%, 17,6% e 18,1% em 2017, 2018 e 2019, respectivamente. Também foi detectada giardíase em cães em 2018, com uma taxa de incidência de 1,2%. A maioria dos cães acometidos com leishmaniose visceral e giardíase tinha entre 1 a 3 e ≥ a 8 anos de idade, respectivamente, sem raça definida, sendo a maioria diagnosticada nos meses de outubro a março. Em felinos, foram detectadas, em 2017 e 2018, esporotricose e criptococose, respectivamente, com taxa de incidência de 3,3% para ambas, e em 2019 foi detectada giardíase, com incidência de 3%. Os felinos com esporotricose tinham de 1 a 3 anos, sem raça definida, e foram diagnosticados em fevereiro de 2017. Em 2018, os felinos com criptococose eram sem raça definida, tinham entre 4 a 7 e ≥ a 8 anos com diagnóstico em dezembro, e, em 2019, um felino com giardíase, sem raça definida, com idade de 1 a 3 anos, foi diagnosticado no mês de março. Esses dados demonstram a necessidade de um projeto de educação em saúde pública com tutores de cães e gatos sobre zoonoses

Epidemiology is a tool for analyzing diseases to explain causal factors combined with environmental factors, thus guiding prevention. The objective was to calculate the incidence rates of zoonoses in dogs and cats and identify predisposing factors regarding age, breed and month of occurrence of animals treated at the veterinary hospital of Araçatuba-SP, between 2017 and 2019. The population canine presented an incidence rate of Visceral Leishmaniasis (VS) of 12.3%, 17.6% and 18.1 in 2017, 2018 and 2019, respectively. Giardiasis has also been detected in dogs in 2018, with an incidence rate of 1.2%. The majority of dogs affected with Visceral Leishmaniasis and Giardiasis were between 1 to 3 and ≥ 8 years of age, respectively, without defined breed, with the majority being diagnosed in the months of October to March. In felines, it was detected in 2017 and 2018, Sporotrichosis and Cryptococcosis, respectively, with an incidence rate of 3.3% for both and in 2019 Giardiasis was detected with an incidence of 3%. Felines with Sporotrichosis had to 1 to 3 years old, mixed breed and diagnosed in February 2017. In 2018, felines with Cryptococcosis were without defined race, between 4 to 7 and ≥ 8 years old diagnosed in December, and in 2019 a feline with Giardiasis, of no defined breed, aged 1 to 3 years old, diagnosed in March. These data demonstrate the need for a public health education project with dog and cat owners about zoonoses

No Evidence of Rabies Exposure in Wild Marmosets (Callithrix jacchus) of Northeast Brazil.

Rabies transmitted by wildlife is the main source of human rabies mortality in Latin America and considered an emerging disease. The common marmoset Callithrix jacchus of Brazil is the only known primate reservoir of rabies worldwide. We tested whether alive free-ranging C. jacchus were exposed to rabies in four northeast states that have previously reported rabies-positive dead C. jacchus (Pernambuco and Bahia) or not (Paraíba and Rio Grande do Norte). Our results show no evidence of rabies antibodies or infection in the sampled C. jacchus, suggesting that apparently healthy marmosets are not widely exposed to rabies over their natural range.

In vitro effects of bufotenine against RNA and DNA viruses.

Bufotenine, an alkaloid that can be found in plant extracts and skin secretions of amphibians, is reported to have potential antiviral activity. The present study evaluated the antiviral activity of bufotenine against different genetic lineages of rabies virus (RABV, a single-stranded, negative-sense RNA virus), canine coronavirus (CCoV, a positive-sense RNA virus) and two double-stranded DNA viruses (two strains of herpes simplex virus type 1/HSV-1 [KOS and the acyclovir-resistant HSV-1 strain 29R] and canine adenovirus 2, CAV-2). The maximal non-toxic bufotenine concentrations in Vero and BHK-21 cells were determined by MTT assays. The antiviral activity of bufotenine against each virus was assessed by examination of reductions in infectious virus titres and plaque assays. All experiments were performed with and without bufotenine, and the results were compared. Bufotenine demonstrated significant RABV inhibitory activity. No antiviral action was observed against CCoV, CAV-2 or HSV-1. These findings indicate that the antiviral activity of bufotenine is somewhat linked to the particular infectious dose used and the genetic lineage of the virus, although the mechanisms of its effects remain undetermined.

Human embryonic kidney (HEK-293) cell line: An alternative for rabies virus diagnosis and research.

Rabies is a serious public health problem in developing countries and is caused by Rabies lyssavirus (RABV), a neurotropic RNA virus. The gold standard test for rabies diagnosis is the direct fluorescent antibody test (DFAT). Nevertheless, a confirmatory method is recommended, such as rabies tissue culture infection test (RTCIT). Several cell lines have been tested for RTCIT, and the murine neuroblastoma (Neuro-2a) cell line has been shown to be the most permissive for infection. The human embryonic kidney (HEK-293) cell line was recently thought as an option, due to neuronal protein expression and easy maintenance. In the present work, we evaluated the susceptibility of HEK-293 cell line to RTCIT compared to Neuro-2a. We used a total of 93 brain samples, 48 negatives and 45 positives for RABV previously tested by DFAT or RT-PCR and by RTCIT in Neuro-2a. Of the positive samples, 43 were positive in the traditional RTCIT using Neuro-2a. Two protocols of HEK-293 cell line to RTCIT were tested (with and without virus adsorption) with different incubations times: 24, 48 and 72 h. The highest positive rate in HEK-293 (41 positive samples) resulted from the adsorption protocol with 72 h incubation period, in contrast to 43 positive samples with the traditional RTCIT with Neuro-2a. No satisfactory results were observed using the protocol without adsorption, regardless of the incubation time. Despite the slightly higher sensitivity of Neuro-2a cells, the use of the HEK-293 cells still offers positive aspects, such as, more rapid results, with the advantage of fast and easy growth over Neuro-2a cell line. Therefore, our findings confirm that HEK-293 cells are susceptible to RABV and can be an alternative for RTCIT.

In vitro efects of bufotenine against RNA and DNA viruses

Bufotenine, an alkaloid that can be found in plant extracts and skin secretions of amphibians, is reported to have potential antiviral activity. The present study evaluated the antiviral activity of bufotenine against different genetic lineages of rabies virus (RABV, a single-stranded, negative-sense RNA virus), canine coronavirus (CCoV, a positive-sense RNA virus) and two double-stranded DNA viruses (two strains of herpes simplex virus type 1/HSV-1 [KOS and the acyclovir-resistant HSV-1 strain 29R] and canine adenovirus 2, CAV-2). The maximal non-toxic bufotenine concentrations in Vero and BHK-21 cells were determined by MTT assays. The antiviral activity of bufotenine against each virus was assessed by examination of reductions in infectious virus titres and plaque assays. All experiments were performed with and without bufotenine, and the results were compared. Bufotenine demonstrated significant RABV inhibitory activity. No antiviral action was observed against CCoV, CAV-2 or HSV-1. These findings indicate that the antiviral activity of bufotenine is somewhat linked to the particular infectious dose used and the genetic lineage of the virus, although the mechanisms of its effects remain undetermined.

Analysis of rabies diagnosis in dogs and cats in the state of São Paulo, Brazil

The genetic lineage of rabies virus (RABV) associated with dogs has not been found in the state of São Paulo since 1998, and all cases of rabies in domestic animals reported since then have involved the RABV lineage that circulates in bats. As there has been a change in the rabies transmission cycle in cats and dogs, we decided to analyze the tests used to diagnose rabies in these animals in the 15-year period from 2002 to 2016 in the state of São Paulo. During this period, 85,508 central nervous system (CNS) samples from dogs and cats were submitted to the Rabies Diagnosis Section at the Pasteur Institute of São Paulo for testing. All of the samples were tested by the fluorescent antibody test (FAT) and at least one of the following three tests: mouse inoculation test (MIT), rabies tissue culture infection test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). Of all the samples tested, twenty were positive in at least one of these assays. Four other positive samples were identified at other institutions in the state of São Paulo. Of the twenty samples that tested positive at the Pasteur Institute of São Paulo, nine were tested by FAT, and the results were subsequently confirmed by other techniques; five gave inconclusive results, and therefore, other techniques had to be used as soon as possible in case the samples were positive; and six were negative by FAT and positive by one or more of the following tests:...

Analysis of rabies diagnosis in dogs and cats in the state of São Paulo, Brazil.

The genetic lineage of rabies virus (RABV) associated with dogs has not been found in the state of São Paulo since 1998, and all cases of rabies in domestic animals reported since then have involved the RABV lineage that circulates in bats. As there has been a change in the rabies transmission cycle in cats and dogs, we decided to analyze the tests used to diagnose rabies in these animals in the 15-year period from 2002 to 2016 in the state of São Paulo. During this period, 85,508 central nervous system (CNS) samples from dogs and cats were submitted to the Rabies Diagnosis Section at the Pasteur Institute of São Paulo for testing. All of the samples were tested by the fluorescent antibody test (FAT) and at least one of the following three tests: mouse inoculation test (MIT), rabies tissue culture infection test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). Of all the samples tested, twenty were positive in at least one of these assays. Four other positive samples were identified at other institutions in the state of São Paulo. Of the twenty samples that tested positive at the Pasteur Institute of São Paulo, nine were tested by FAT, and the results were subsequently confirmed by other techniques; five gave inconclusive results, and therefore, other techniques had to be used as soon as possible in case the samples were positive; and six were negative by FAT and positive by one or more of the following tests: RTCIT, MIT and RT-PCR. Genetic typing of isolates from eighteen samples identified them as the lineage circulating in bats. In light of this finding, which indicates that genetic lineages associated with bats are circulating in domestic animals in the state of São Paulo, when the results of FAT carried out with samples from aggressive cats and dogs are inconclusive, complementary tests should be used. Decomposing samples and samples for which FAT was inconclusive should be tested using molecular techniques so that a definitive result can be obtained quickly and timely post-exposure prophylaxis can be administered to exposed individuals.

Phylogeography of rabies isolated from cattle and horses transmitted by haematophagous bat Desmodus Rotundus in Brazil

Rabies is Public Health problem and is very important in Animal Health too. The illness demands continuous prophylactic care for herbivores with economic interest, such as cattle and horses. The main vector of rabies virus (RABV) for these animals are the hematophagous bats Desmodus rotundus. RABV is a RNA genome with low level of fidelity during replication cycle due to lack of repair of its polymerase. This causes the incorporation of mutations that increase the genotypic variation of the viral population. In the project, the nucleoprotein (N) gene of the RABV isolated mainly from cattle in different cities of State of São Paulo (SP), will be sequenced. In addition, genetic sequences deposited in GenBank will be also used. N is the most conserved gene of RABV for these reason is the most appropriated for phylogeographic studies. Because the RABV display evolutionary and ecological dynamics on the same time scale reliable phylogeographic inferences can be obtained from molecular data. As phylogeography expresses the contemporary pattern of geographic distribution of an organism according to gene genealogies the objective of this project is to determine the dispersion over time and space of the RABV transmitted by D. rotundus in SP. The phylogeography of RABV will be studied by phylogenetic analysis using Bayesian statistics using Monte Carlo methods via Markov Chains (MCMC), available on the Bayesian Evolutionary Analysis Sampling Trees (BEAST) plataform. In this way and after the test of different evolutionary models the data of the phylogenetic trees of substitution and more probable time will be converted into a KML file that allows the visualization of the spatial projection of the diffusion of the genetic lineages in the time and space using Google Earth. In this way, the final results can aid epidemiological surveillance and also strategic planning for the control of rabies.

The epidemiological importance of bats in the transmission of rabies to dogs and cats in the State of São Paulo, Brazil, between 2005 and 2014

In Brazil, rabies control in dogs and cats was pioneered by the state of São Paulo with the adoption of the Pan American Health Organization recommendations for prophylaxis and control, which led to a reduction in rabies cases from 1994 onwards. As a result of these measures, the rabies virus (RABV) genetic lineage associated with dogs has not been found in the state since 1998, and all the cases in domestic animals reported since then have been caused by bat­associated lineages of RABV. In the light of this, this study sought to investigate rabies cases in dogs and cats in the state of São Paulo between 2005 and 2014 and identify the associated transmission cycles by characterizing the RABV lineages responsible for these cases. Nine samples from dogs (n = 5) and from cats (n = 4) were collected between 2005 and 2014. The tenth animal, a rabid cat, was analysed by a different laboratory. The N gene nucleotide sequences obtained were analysed with the neighbor­joining algorithm and Kimura 2­parameter model using the MEGA 6 program. Phylogenetic analysis revealed that the genetic lineages identified in all the samples were those circulating in Brazilian bats. The findings of this study demonstrate that bats play an important role in the transmission of rabies to domestic animals in São Paulo state and that emphasis should be placed on the implementation of public policies to support surveillance of chiropterans for rabies.(AU) i

Diagnóstico de cinomose canina por RT-PCR em amostras de cães do Estado de São Paulo enviadas para a vigilância laboratorial da raiva

A cinomose canina (CD) é uma das doenças infecciosas mais importantes dos cães domésticos. No Brasil é ainda a principal causa de mortalidade de cães em algumas populações urbanas. Embora sequências de diferentes genes do vírus sejam utilizadas como alvo para detecção do vírus da cinomose canina (CDV), o gene N parece ser o melhor para a amplificação de todas as suas linhagens. Utilizando-se a técnica de RT-PCR direcionada ao gene N do CDV, foram analisadas 190 amostras de sistema nervoso central (SNC) de cães do estado de São Paulo com quadros sugestivos de encefalite e que foram encaminhadas ao Instituto Pasteur para o diagnóstico da raiva, durante o ano de 2014. A positividade foi superior a 50% indicando que a cinomose continua a ser uma importante causa de mortalidade canina.

Genetic identification of species of bats that act as reservoirs or hosts for viral diseases

Introduction: Viruses have been identified as the main etiologic agents of both zoonoses and emerging infectious diseases (EIDs) and various species of wild fauna can be involved in the maintenance of these diseases. The very wide variety of bats, together with their ability to adapt to different environments and fly long distances, means that these animals are currently one of the main reservoirs for zoonoses and EIDs. For these reasons the correct identification of different bat species is essential.Aims: This paper describes the genetic identification of 56 samples isolated from different bat species.Methodology: Sequencing and phylogenetic analysis of the mitochondrial DNA cytochrome b (mtDNA cyt-b) gene. Results: Four families (Molossidae, Vespertilionidae, Noctilionidae and Phyllostomidae), twelve genera and nineteen different species of bats were identified, and the Basic Local Alignment Search Tool (BLAST) was used to confirm species identity. The phylogenetic tree constructed revealed two main clusters (1 and 2), both consist in two subclusters.Conclusions: Our results were concordant with those obtained by morphometric identification and genetic identification carried out by other authors, showing that the method described here can be used as an effective alternative to, or in combination with, morphometric identification of bats

Eastern equine encephalitis cases among horses in Brazil between 2005 and 2009.

Eastern equine encephalitis is a viral zoonosis that exhibits complex distribution and epidemiology, and greater importance should be given to this disease by the public-health authorities. In Brazil, although eastern equine encephalitis virus (EEEV) has been identified in vectors and antibodies are sometimes detected in horses and humans, there have been no records of equine encephalitis in horses caused by this virus during the last 24 years. This study describes eighteen cases of eastern equine encephalomyelitis that occurred in six Brazilian states between 2005 and 2009. Viral RNA was identified using semi-nested RT-PCR to detect members of the genus Alphavirus, and by genetic sequencing. The gene encoding NSP1 was partially amplified, and after genetic sequencing, eighteen sequences were generated. All eighteen strains were classified as belonging to lineage III of American EEEV. These findings could be an indication of the importance of this virus in animal and human public health.

Phylogeographic dispersion and diversification of rabies virus lineages associated with dogs and crab-eating foxes (Cerdocyon thous) in Brazil

Genetic lineages of dog-associated RABV still circulate in some areas of the North and Northeast of Brazil. In parallel, another RABV lineage circulates among wild canids in the Northeast, particularly the crab-eating fox (Cerdocyon thous). Although previous studies and phylogenetic analyses have been carried out, the way in which these lineages are dispersed temporally and spatially remained to be elucidated. In this study, RABV N gene sequences isolated from canids in North and Northeast Brazil were analyzed by the Bayesian Markov Chain Monte Carlo Method, and the results were then used in a phylogeographic study. It was inferred from the findings that the most recent common ancestor became established at the end of the nineteenth century on the border of the Brazilian states of Paraíba and Pernambuco and diversified into the lineages associated with dogs and C. thous. Around 1910, the original C. thous lineage diversified into two main sublineages in the same area, one of which migrated to the south and the other to the north. The dog-associated lineage diversified around 1945 and moved toward the north and south. From the phylogeographic analysis it was possible to infer not only the movement of the virus lineages but also the probable location where dispersion and diversification occurred. The methodology used here enabled the phylogeographic history of RABV in the region to be reconstructed, and the dispersion pattern of the virus can be used to predict its movements, making it easier to stop the advance of a rabies epidemic.

Phylogeographic dispersion and diversification of rabies virus lineages associated with dogs and crab-eating foxes (Cerdocyon thous) in Brazil.

Genetic lineages of dog-associated RABV still circulate in some areas of the North and Northeast of Brazil. In parallel, another RABV lineage circulates among wild canids in the Northeast, particularly the crab-eating fox (Cerdocyon thous). Although previous studies and phylogenetic analyses have been carried out, the way in which these lineages are dispersed temporally and spatially remained to be elucidated. In this study, RABV N gene sequences isolated from canids in North and Northeast Brazil were analyzed by the Bayesian Markov Chain Monte Carlo Method, and the results were then used in a phylogeographic study. It was inferred from the findings that the most recent common ancestor became established at the end of the nineteenth century on the border of the Brazilian states of Paraíba and Pernambuco and diversified into the lineages associated with dogs and C. thous. Around 1910, the original C. thous lineage diversified into two main sublineages in the same area, one of which migrated to the south and the other to the north. The dog-associated lineage diversified around 1945 and moved toward the north and south. From the phylogeographic analysis it was possible to infer not only the movement of the virus lineages but also the probable location where dispersion and diversification occurred. The methodology used here enabled the phylogeographic history of RABV in the region to be reconstructed, and the dispersion pattern of the virus can be used to predict its movements, making it easier to stop the advance of a rabies epidemic.

Antigenic and genetic characterization of rabies virus isolates from Uruguay

After 25 years without any reported cases of rabies in Uruguay, the northern region of the country experienced an epizootic of bovine paralytic rabies in October 2007. The outbreak affected bovines and equines, and the main source of infection was the bat Desmodus rotundus, the only hematophagous species in the country. From October 2007 to July 2008, 42 bovine, 3 equine and 120 chiropteran samples were submitted to the National Veterinary Diagnostic Laboratory for rabies testing. A total of 12 samples (7 bovine, 2 equine and 3 from D. rotundus) were positive by the fluorescent antibody test, and viruses were isolated by the mouse inoculation test. The objective of this study was to compare the antigenic and genetic characteristics of these isolates and three isolates from insectivorous bats from other regions. Antigenic typing using a panel of eight monoclonal antibodies identified all 12 viruses as variant 3 (AgV3), a variant associated with D. rotundus. Two isolates from insectivorous bats (Tadarida brasiliensis and Molossus sp.) were characterized as antigenic variant 4 (AgV4) while the third, from Myotis sp., could not be characterized using this panel as its reactivity pattern did not match that of any of the known antigenic variants. Partial N-gene sequences (nt 149–1420) of these isolates were aligned with homologous sequences derived from GenBank by the CLUSTAL/W method and used to build a neighbor-joining distance tree with the Kimura 2-parameter model. All 12 isolates were genetically grouped into the D. rotundus cluster as they shared 100% identity. In the phylogenetic analysis, the three isolates from insectivorous bats segregated into three clusters: one related to T. brasiliensis, one to Myotis sp. and the other to Lasiurus sp., although the isolate associated with the latter came from a Molossus sp. specimen. These results indicate that AgV3 was associated with the outbreak of bovine paralytic rabies in Uruguay. This is the first report...

Método de diagnóstico antemortem da raiva humana por meio de técnicas de biologia molecular, utilizando saliva e biópsia de pele da região da nuca / An antemortem method for diagnosis of human rabies by molecular techniques using saliva and neck-skin biopsy

Por meio das técnicas de RT-PCR com primers direcionados para o gene da

glicoproteína e RT-PCR e hemi-nested RT-PCR com primers direcionados para o gene da nucleoproteína, o RNA do vírus da raiva foi identificado em 95,2% de 21 amostras, 18 de saliva e três de biópsia de pele da região da nuca, coletadas entre a hospitalização e a morte de um paciente com sinais clínicos da raiva. O tratamento administrado ao paciente incluiu a indução de coma e terapia antiviral. Cada técnica, isoladamente, detectou RNA viral em 90,5%; 57,1% e 85,7% das amostras, respectivamente. Nossos resultados sugerem que a amplificação em paralelo de diferentes regiões do genoma do vírus da raiva pode fornecer maior confiabilidade ao diagnóstico antemortem da doença, auxiliando a decisão médica quanto à aplicação do protocolo de tratamento com antivirais.

Antigenic and genetic characterization of rabies virus isolates from Uruguay.

After 25 years without any reported cases of rabies in Uruguay, the northern region of the country experienced an epizootic of bovine paralytic rabies in October 2007. The outbreak affected bovines and equines, and the main source of infection was the bat Desmodus rotundus, the only hematophagous species in the country. From October 2007 to July 2008, 42 bovine, 3 equine and 120 chiropteran samples were submitted to the National Veterinary Diagnostic Laboratory for rabies testing. A total of 12 samples (7 bovine, 2 equine and 3 from D. rotundus) were positive by the fluorescent antibody test, and viruses were isolated by the mouse inoculation test. The objective of this study was to compare the antigenic and genetic characteristics of these isolates and three isolates from insectivorous bats from other regions. Antigenic typing using a panel of eight monoclonal antibodies identified all 12 viruses as variant 3 (AgV3), a variant associated with D. rotundus. Two isolates from insectivorous bats (Tadarida brasiliensis and Molossus sp.) were characterized as antigenic variant 4 (AgV4) while the third, from Myotis sp., could not be characterized using this panel as its reactivity pattern did not match that of any of the known antigenic variants. Partial N-gene sequences (nt 149-1420) of these isolates were aligned with homologous sequences derived from GenBank by the CLUSTAL/W method and used to build a neighbor-joining distance tree with the Kimura 2-parameter model. All 12 isolates were genetically grouped into the D. rotundus cluster as they shared 100% identity. In the phylogenetic analysis, the three isolates from insectivorous bats segregated into three clusters: one related to T. brasiliensis, one to Myotis sp. and the other to Lasiurus sp., although the isolate associated with the latter came from a Molossus sp. specimen. These results indicate that AgV3 was associated with the outbreak of bovine paralytic rabies in Uruguay. This is the first report of rabies virus having been detected in non-hematophagous bats in this country.

Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages

In Brazil, bats have been assigned an increasing importance in public health as they are important rabies reservoirs. Phylogenetic studies have shown that rabies virus (RABV) strains from frugivorous bats Artibeus spp. are closely associated to those from the vampire bat Desmodus rotundus, but little is known about the molecular diversity of RABV in Artibeus spp. The N and G genes of RABV isolated from Artibeus spp. and cattle infected by D. rotundus were sequenced, and phylogenetic trees were constructed. The N gene nucleotides tree showed three clusters: one for D. rotundus and two for Artibeus spp. Regarding putative N amino acid-trees, two clusters were formed, one for D. rotundus and another for Artibeus spp. RABV G gene phylogeny supported the distinction between D. rotundus and Artibeus spp. strains. These results show the intricate host relationship of RABV's evolutionary history, and are invaluable for the determination of RABV infection sources.