Dispersion and diversification of Lyssavirus rabies transmitted from haematophagous bats Desmodus rotundus: a phylogeographical study.

Rabies is worldwide zoonosis caused by Lyssavirus rabies (RABV) a RNA negative sense virus with low level of fidelity during replication cycle. Nucleoprotein of RABV is the most conserved between all five proteins of the virus and is the most used gene for phylogenetic and phylogeographic studies. Despite of rabies been very important in Public Health concern, it demands continuous prophylactic care for herbivores with economic interest, such as cattle and horses. The main transmitter of RABV for these animals in Brazil is the hematophagous bats Desmodus rotundus. The aim of this study was to determine the dispersion over time and space of RABV transmitted by D. rotundus. Samples of RABV from the State of São Paulo (SP), Southeast Brazil isolated from the central nervous system (CNS) of cattle, were submitted to RNA extraction, RT-PCR, sequencing and phylogeographic analyzes with BEAST (Bayesian Evolutionary Analysis Sampling Trees) v 2.5 software. Was possible to identify high rate of diversification in starts sublineages of RABV what are correlated with a behavior of D. rotundus, the main transmitter of rabies to cattle. This study also highlights the importance of continuous monitoring of genetic lineages of RABV in Brazil.

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

Epidemiologia molecular de surto de raiva bovina na região central do Rio Grande do Sul, 2012 / Molecular epidemiology of an outbreak of bovine rabies in central Rio Grande do Sul, Brazil, 2012

A raiva é uma doença infecciosa do sistema nervoso central de mamíferos causada pelo vírus da raiva (RabV), geralmente, transmitido pela mordedura de animais infectados. No Brasil, os morcegos hematófagos Desmodus rotundus são as principais fontes de infecção do RabV para bovinos e equinos. Este artigo descreve uma investigação epidemiológica e molecular de surtos de raiva ocorridos na região central do Rio Grande do Sul, entre maio e agosto de 2012. Nesse período, 45 casos suspeitos de raiva foram relatados em 22 pequenos rebanhos, localizados dentro de um raio de 4,7km, no município de Pinhal Grande. Desses, 32 amostras foram submetidas para diagnóstico da raiva, sendo que o RabV e/ou antígenos virais foram identificados em 27 amostras. Em um segundo momento, 11 amostras foram submetidas à transcrição reversa/reação em cadeia da polimerase (RT-PCR) para o gene da nucleoproteína (N) do RabV, seguido de sequenciamento nucleotídico e análise filogenética. Sete das 11 amostras apresentaram sequências nucleotídicas idênticas e uma apresentou mutação sinônima, não-codificante, indicando uma provável origem comum dos vírus. Por outro lado, três amostras apresentaram mutações que resultaram em alterações de aminoácidos, sugerindo uma origem diferente do vírus. Esses resultados sugerem que RabV de diferentes origens/linhagens co-circulam na região e foram envolvidos nos surtos descritos. Investigações sobre a circulação de ambos os genótipos em morcegos na região estão em andamento.

Rabies is an infectious disease of the central nervous system of mammals caused by rabies virus (RabV), generally transmitted by the bite of rabid animals. In Brazil, vampire bats Desmodus rotundus are the main reservoirs of RabV for livestock. The present study describes a molecular and epidemiological investigation of outbreaks of bovine rabies occurring in the central region of Rio Grande do Sul state, Brazil, between May and August 2012. In this period, 45 cases suspected of rabies were reported in 22 small herds, located within a 4.7km range, in the county of Pinhal Grande. From these, 32 samples were submitted to rabies diagnosis and RabV and/or viral antigens were identified in 27 samples. Subsequently, 11 brain samples were submitted to reverse transcription/polymerase chain reaction (RT-PCR) for the nucleoprotein gene (N) followed by nucleotide sequencing and phylogenetic analysis. Seven out of 11 samples yielded identical sequences; one presented a synonymous, non-coding mutation, indicating a likely common origin of the virus. However, three other samples presented nucleotide mutations which resulted in amino acid changes, suggesting a different origin of the virus. In summary, these results suggest that RabV strains of different origin/lineages co-circulate in the region and were involved in the outbreaks. Investigations on the circulation of both genotypes in bats in the region are currently underway.

First detection of adenovirus in the vampire bat (Desmodus rotundus) in Brazil.

This paper describes the first detection of adenovirus in a Brazilian Desmodus rotundus bat, the common vampire bat. As part of a continuous rabies surveillance program, three bat specimens were captured in Southern Brazil. Total DNA was extracted from pooled organs and submitted to a nested PCR designed to amplify a 280 bp long portion of the DNA polymerase gene of adenoviruses. One positive sample was subjected to nucleotide sequencing, confirming that this DNA fragment belongs to a member of the genus Mastadenovirus. This sequence is approximately 25 % divergent at the nucleotide level from equine adenovirus 1 and two other recently characterized bat adenoviruses.

Genetic characterization of rabies virus isolated from bovines and equines between 2007 and 2008, in the States of São Paulo and Minas Gerais.

INTRODUCTION: Rabies is an acute disease of the central nervous system and is responsible for the deaths of thousands of humans, wild animals and livestock, particularly cattle, as well as causing major economic losses. This study describes the genetic characterization of rabies virus variants that circulate in Desmodus rotundus populations and are transmitted to herbivores. METHODS: Fifty rabies virus isolates from bovines and equines in the States of São Paulo and Minas Gerais, Brazil, were genetically characterized and compared with sequences retrieved from GenBank. RESULTS: Two clusters (I and II) with mean nucleotide identities of 99.1 and 97.6% were found. The first of these contained nearly all the samples analyzed. Lineages from other Brazilian states grouped in cluster II. CONCLUSIONS: Analysis of the amino acid sequences of the N proteins revealed the existence of genetic markers that may indicate possible variations between geographic regions, although the biologically active regions are conserved within the species over space and time.

Genetic characterization of rabies virus isolated from bovines and equines between 2007 and 2008, in the States of São Paulo and Minas Gerais / Caracterização genética de vírus da raiva isolado de bovinos e equinos entre 2007 e 2008, nos Estados de São Paulo e Minas Gerais

INTRODUCTION: Rabies is an acute disease of the central nervous system and is responsible for the deaths of thousands of humans, wild animals and livestock, particularly cattle, as well as causing major economic losses. This study describes the genetic characterization of rabies virus variants that circulate in Desmodus rotundus populations and are transmitted to herbivores. METHODS: Fifty rabies virus isolates from bovines and equines in the States of São Paulo and Minas Gerais, Brazil, were genetically characterized and compared with sequences retrieved from GenBank. RESULTS: Two clusters (I and II) with mean nucleotide identities of 99.1 and 97.6 percent were found. The first of these contained nearly all the samples analyzed. Lineages from other Brazilian states grouped in cluster II. CONCLUSIONS: Analysis of the amino acid sequences of the N proteins revealed the existence of genetic markers that may indicate possible variations between geographic regions, although the biologically active regions are conserved within the species over space and time.

INTRODUÇÃO: A raiva é uma doença aguda do sistema nervoso central e é responsável por mortes de milhares de humanos, animais silvestres e animais de criação - especialmente bovinos - além de causar elevadas perdas econômicas. Este trabalho descreve a caracterização genética das variantes do vírus da raiva que circulam em populações de Desmodus rotundus e são transmitidas aos herbívoros. MÉTODOS: Cinquenta isolados de vírus da raiva de bovinos e equinos provenientes dos Estados de São Paulo e Minas Gerais, Brasil, foram caracterizadas geneticamente e comparadas com sequências recuperadas do GenBank. RESULTADOS: Dois clusters, I e II, apresentando identidades médias de nucleotídeos de 99,1 e 97,6 por cento, foram obtidos, sendo o primeiro composto de quase a totalidade das amostras analisadas. Linhagens de outros estados do Brasil "clustered" no II. CONCLUSÕES: A análise das sequências de aminoácidos da proteína N revelou que existem marcadores genéticos que podem determinar uma possível regionalidade embora as regiões biologicamente ativas apresentem-se conservadas dentro das espécies ao longo do tempo e espaço.

Caracterização genética de vírus da raiva isolados de bovinos e eqüínos no período de 1997-2002 em área epidêmica do Estado de São Paulo, Brasil

A história biogeográfica da raiva pode ser reconstruída utilizando dados moleculares. Este trabalho descreve a caracterização do Vírus da Raiva(RABV) que circula na população do morcego hematófago Desmodus rotundus em uma área epidêmica do Estado de São Paulo e que é transmitido para herbívoros de interesse econômico como, por exemplo, os bovinos e eqüínos. Os genes N e G dos vírus foram seqüenciados e as árvores filogenéticas geradas são topologicamente concordantes. Três agrupamentos filogenéticos (clusters) foram identificados na área epidêmica e foram designados como RD1, RD2 e RD3. Os resultados mostram que a origem dos clusters RD1 e RD2 são diferentes e que a epidemia da área RD3 é o resultado da expansão da área RD2. As seqüências genéticas dos dois genes analisados neste estudo foram comparadas entre si e conseqüências obtidas no GenBank apresentando alta identidade (> 98%), mantidas no tempo e espaço. Os resultados sugerem que as linhagens do RABV que circulam em D. rotundus na costa atlântica da América do Sul são altamente conservadas.

Caracterização genética de vírus da raiva isolados de bovinos e eqüínos no período de 1997-2002 em área epidêmica do Estado de São Paulo, Brasil / Genetic characterization of rabies virus isolated from cattle and equine between 1997 and 2001 in an epidemic area in the State of São Paulo, Brazil

A história biogeográfica da raiva pode ser reconstruída utilizando dados moleculares. Este trabalho descreve a caracterização do Vírus da Raiva(RABV) que circula na população do morcego hematófago Desmodus rotundus em uma área epidêmica do Estado de São Paulo e que é transmitido para herbívoros de interesse econômico como, por exemplo, os bovinos e eqüínos. Os genes N e G dos vírus foram seqüenciados e as árvores filogenéticas geradas são topologicamente concordantes. Três agrupamentos filogenéticos (clusters) foram identificados na área epidêmica e foram designados como RD1, RD2 e RD3. Os resultados mostram que a origem dos clusters RD1 e RD2 são diferentes e que a epidemia da área RD3 é o resultado da expansão da área RD2. As seqüências genéticas dos dois genes analisados neste estudo foram comparadas entre si e conseqüências obtidas no GenBank apresentando alta identidade (> 98%), mantidas no tempo e espaço. Os resultados sugerem que as linhagens do RABV que circulam em D. rotundus na costa atlântica da América do Sul são altamente conservadas.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA.

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

Padronização e aplicação da técnica de isolamento do vírus da raiva em células de neuroblastoma de camundongo (N2A) / Patterning and appliance of virus isolation techniques in mice neuroblastoma cells (N2A)

O diagnóstico laboratorial da raiva é de suma importância para o controle e prevenção da doença, uma vez que os diagnósticos clínicos não são precisos. A imunofluorescência direta (IFD) é o teste mais utilizado e mesmo sendo altamente sensível, acurado e relativamente rápido, pode gerar resultados falsos negativos. Desta forma, recomenda-se o isolamento do vírus da raiva em camundongos (IVC) em amostras de Sistema Nervoso Central (SNC) de animais suspeitos de estarem infectados, teste que atualmente vem sendo substituído em vários laboratórios pelo isolamento viral em cultura de células (IVCC). O objetivo do presente estudo foi comparar a sensibilidade do teste de isolamento do vírus em cultura de células de neuroblastoma de camundongos (N2A), com o teste de IVC e com a IFD, bem como avaliar os resultados obtidos na rotina de diagnóstico do Instituto Pasteur, em relação à redução de custo, tempo e trabalho. Foram analisadas 105 amostras de SNC de diferentes espécies de animais pela IFD, pelo IVC e pelo IVCC: 50 de morcegos, 32 de cães, 13 de raposas e 10 de bovinos. Todas as amostras de morcegos e de bovinos apresentaram resultados concordantes para os três testes, enquanto que as de cães e raposas apresentaram concordância em somente 24 amostras (69%). Com base nestes resultados, a partir de 2004 estabeleceu-se que todas as amostras de morcegos enviadas ao Laboratório do Instituto Pasteur, após o diagnóstico por IFD, seriam submetidas ao IVCC, substituindo o uso de camundongos. No período de janeiro de 2004 a setembro de 2007, foram analisadas 11.298 amostras de morcegos. Um total de 67 amostras positivas por IFD e/ou IVCC foram também submetidas ao IVC e 61 amostras apresentaram resultados concordantes nos três testes, mostrando que o uso de células N2A é mais sensível para o isolamento de “vírus de rua” em uma rotina laboratorial para amostras de morcegos, sendo rápido e de menor custo do que o IVC.

Short-interfering RNAs as antivirals against rabies

This study aimed to test in vitro a RNA-interference based antiviral approach for rabies with short-interfering RNAs (siRNAs) against rabies virus nucleoprotein mRNA. BHK-21 cells were infected with serial dilutions of PV rabies virus strain and transfected with a pool of three siRNAs. Direct immunofluorescence staining showed a 5-time decrease in virus titer when compared to a non-treated plate, showing a promising new approach to the development of antivirals for rabies treatment.

Reservatórios silvestres do vírus da raiva: um desafio para a saúde pública / Wildlife reservoirs of rabies virus: a new challenge to a public health

Estima-se que 55.000 óbitos humanos sejam causados pela raiva transmitida pelo cão, anualmente, em especial na Ásia e África. Na América Latina, onde a raiva canina era endêmica até 1980, houve, recentemente, uma redução do número de casos em cães e, conseqüentemente, em humanos. Com o desenvolvimento do Plano de Ação para Eliminação da Raiva Urbana, a raiva em animais silvestres assumiu maior importância, especialmente porque nos anos 2004-2005 o morcego hematófago (Desmodus rotundus) passou a ser o principal transmissor da raiva humana no continente (68%). Além das três espécies de morcegos hematófagas nas quais há relatos de isolamento do vírus da raiva, 33 outras espécies de morcegos também já foram infectadas e identificadas com o mesmo vírus. Juntamente com os morcegos (Ordem Chiroptera), os canídeos (Ordem Carnivora) são considerados os principais reservatórios silvestres do vírus da raiva. No Nordeste do Brasil a doença tem sido cada vez mais freqüente em Cerdocyon thous (cachorro do mato) e há um outro ciclo epidemiológico da raiva em Callithrix jacchus (sagüi do tufo branco), espécie em que a distribuição da doença é desconhecida. Os autores descrevem características da doença em quirópteros e carnívoros, estratégias de controle e ressaltam a importância dos estudos antigênicos e genéticos como instrumento da vigilância epidemiológica.

Diagnosis of human rabies cases by polymerase chain reaction of neck-skin samples

Rapid diagnosis of rabies in suspected human cases influences post-exposure prophylaxis for potential contacts of the patient and ensures appropriate patient management. Apart from the central nervous system (CNS), rabies virus (RABV) is usually present in small sensory nerves adjacent to hair follicles of infected humans. We used an RT-PCR, with primers targeted to the 3' terminal portion of the nucleoprotein gene (N), to test neck-skin samples of nine patients who had rabies in order to validate a diagnostic method that could serve as an additional tool for rabies diagnosis, particularly in antemortem samples. Six of eight postmortem samples were found to be positive for rabies by RT-PCR, and one of two samples collected antemortem was positive with this same technique. Results were confirmed by DNA sequencing; this validates RT-PCR and neck-skin as a suitable technique and type of sample, respectively, for use in the diagnosis of human rabies. RT-PCR applied to neck-skin biopsies could allow early diagnosis and lead to more effective rabies treatment.

Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection.

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100% agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.

Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100 percent agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.