Molecular epidemiological tracing of a cattle rabies outbreak lasting less than a month in Rio Grande do Sul in southern Brazil.

BACKGROUND: Vampire bat-transmitted cattle rabies cases are typically encountered in areas where the disease is endemic. However, over the period of a month in 2009, an outbreak of cattle rabies occurred and then ended spontaneously in a small area of the Rio Grande do Sul State in southern Brazil. To investigate the epidemiological characteristics of this rabies outbreak in Rio Grande do Sul, 26 nucleotide sequences of rabies virus (RABV) genomes that were collected in this area were analyzed phylogenetically. RESULTS: Nucleotide sequence identities of the nucleoprotein gene and G-L intergenic region of the 26 RABVs were greater than 99.6 %. Phylogenetic analysis showed that all RABVs clustered with the vampire bat-related cattle RABV strains and that the RABVs were mainly distributed in southern Brazil. CONCLUSIONS: The findings of the present study suggested that a small population of rabid vampire bats carrying a single RABV strain produced a spatiotemporally restricted outbreak of cattle rabies in southern Brazil.

Isolation of a phylogenetically distinct rabies virus from a tufted capuchin monkey (Cebus apella) in Brazil.

A rabies virus isolate (BRmk1358 strain) was discovered from a rabid tufted capuchin monkey in Brazil. The present study determined the nucleotide sequence of the BRmk1358 strain and compared with the rabies viruses isolated from marmosets and other animals in the Americas. Phylogenetic analyses showed that the BRmk1358 strain formed a lineage distant from that of marmoset rabies virus within the Chiroptera-related rabies virus cluster. This result suggests that the source of rabies infection in the tufted capuchin monkey may have been bat, and that they have a risk to act as rabies reservoir in Brazil.

Molecular epidemiology of livestock rabies viruses isolated in the northeastern Brazilian states of Paraíba and Pernambuco from 2003 - 2009.

BACKGROUND: Limited or no epidemiological information has been reported for rabies viruses (RABVs) isolated from livestock in the northeastern Brazilian states of Paraíba (PB) and Pernambuco (PE). The aim of this study was to clarify the molecular epidemiology of RABVs circulating in livestock, especially cattle, in these areas between 2003 and 2009. FINDINGS: Phylogenetic analysis based on 890 nt of the nucleoprotein (N) gene revealed that the 52 livestock-derived RABV isolates characterized here belonged to a single lineage. These isolates clustered with a vampire bat-related RABV lineage previously identified in other states in Brazil; within PB and PE, this lineage was divided between the previously characterized main lineage and a novel sub-lineage. CONCLUSIONS: The occurrences of livestock rabies in PB and PE originated from vampire bat RABVs, and the causative RABV lineage has been circulating in this area of northeastern Brazil for at least 7 years. This distribution pattern may correlate to that of a vampire bat population isolated by geographic barriers.

Determination and molecular analysis of the complete genome sequence of two wild-type rabies viruses isolated from a haematophagous bat and a frugivorous bat in Brazil.

The complete genome sequences of two Brazilian wild-type rabies viruses (RABV), a BR-DR1 isolate from a haematophagous bat (Desmodus rotundus) and a BR-AL1 isolate from a frugivorous bat (Artibeus lituratus), were determined. The genomes of the BR-DR1 and BR-AL1 had 11,923 and 11,922 nt, respectively, and both encoded the five standard genes of rhabdoviruses. The complete nucleotide sequence identity between the BR-DR1 and BR-AL1 isolates was 97%. The BR-DR1 and BR-AL1 isolates had some conserved functional sites revealed by the fixed isolates, whereas both isolates had unique amino acid substitutions in the antigenic region IV of the nucleocapsid gene. Therefore, it is speculated that both isolates were nearly identical in virologic character. According to our phylogenetic analysis based on the complete genomes, both isolates belonged to genotype 1, and to the previously defined "vampire bat-related RABV lineage" which consisted of mainly D. rotundus- and A. lituratus-isolates; however, a branch pattern with high bootstrap values suggested that BR-DR1 was more closely related to the 9001FRA isolate, which was collected from a dog bitten by a bat in French Guiana, than to BR-AL1. This result suggests that the vampire bat-related RABV lineage includes Brazilian vampire bat and Brazilian frugivorous bat RABV and is further divided into Brazilian vampire bat and Brazilian frugivorous bat RABV sub-lineages. The phylogenetic analysis based on the complete genomes was valuable in discriminating among very closely related isolates.

Multiple substitutions in biologically active domains ofrabies virus glycoprotein can be related to pathogenicprofile / Múltiplas substituições em domínios biologicamente ativos da glicoproteína do vírusrábico podem estar relacionadas com o perfil patogênico

Pathogenic profile of a rabies virus isolated from an insectivorous bat Lasiurus ega was compared with a rabies fixedvirus strain (CVS/32) in hamster and mouse. Incubation and clinical periods, clinical manifestation and death rateswere compared. Challenge of hamsters with L. ega was performed using: 10 2,611-4,021 LD50 /0,05 mL;. For CVS were used10 3,7-4,7 LD50 /0,05 mL. Were tested intramuscular (IM), intradermal (ID), intranasal (IN), epidermal abrasion (EA)inoculation routes. Viral antigen in brains was confirmed by Direct Immunofluorescence Test. Mortality percentagesobserved with L. ega rabies virus isolate were the following in hamster: 3,5 % IM, 10,710% IN; in mice: 50.0% IM, 30.0%IN. Furious rabies was predominant. Mortality percentages observed with CVS/32 in hamster: 12.5% IM, 62.5% ID,12.5% IN; in mice 100.0% IM, 70.0% ID, 10.0% IN. Paralytic rabies was found with this strain in both animal models.Epidermic abrasion was not a suitable challenge route. Incubation period was 5-7 days for CVS and 11-16 days for L. egaisolate, meanwhile clinical periods were comprehended between 4–7 days for both viruses. Several substitutions weredetected at antigenic domains of glycoprotein: AI (position 231), AII (34–42 and 198-200), domain of fusion dependenton low pH (102–179), transmembrane domain (440–461) and residue 242. These viruses showed contrasting biologicalbehaviors that can be linked to those substitutions at antigenic domains previously described.

O perfil patogênico de um vírus da raiva isolado de um morcego insetívoro Lasiurus ega foi comparado com o de vírusfixo de raiva (CVS/32) em hamster e camundongo, determinando os períodos de incubação e clínico, manifestaçãoclínica e mortalidade. Os animais foram desafiados com 10 2,611 - 4,021 DL50 /0,05 mL do isolado de L. ega e 10 3,7- 4,7 LD50 /0,05 mL do CVS/32, usando as vias: intramuscular (IM), intradermica (ID), intranasal (IN) e abrasão epidermica (AE).A presença do antígeno viral foi confirmada pela prova de imunofluorescência direta. As porcentagens de mortalidadeobservadas com o isolado de L. ega foram as seguintes em hamster: 3,5% IM, 10,71% IN; em camundongo: 50.0%IM, 30.0% IN. A forma furiosa da doença foi predominante. As porcentagens de mortalidade observadas com o vírusCVS/32 em hamster foram as seguintes: 12.5% IM, 62.5% ID, 12.5% IN; em camundongo 100.0% IM, 70.0% ID,10.0% IN. Com este vírus foi observada raiva paralitica. A via AE mostrou-se inadequada para induzir doença. Operíodo de incubação foi de 5–7 dias para o CVS/32 e 11-16 dias para o isolado de L. ega, entre tanto os períodosclínicos oscilaram entre 4–7 dias para ambos os vírus. Varias substituições foram achadas em domínios antigênicos daglicoproteína: AI (posição 231), AII (34–42 e 198-200), domínio de fusão dependente de baixo pH (102–179), domínioda transmembrana (440–461) e resíduo 242. Esses vírus mostraram comportamentos biológicos distintos o que poderiaestar ligado às substituições nos domínios antigênicos anteriormente descritos.

Multiple substitutions in biologically active domains of rabies virus glycoprotein can be related to pathogenic profile / Múltiplas substituições em domínios biologicamente ativos da glicoproteína do vírus rábico podem estar relacionadas com o perfil patogênico

Pathogenic profile of a rabies virus isolated from an insectivorous bat Lasiurus ega was compared with a rabies fixedvirus strain (CVS/32) in hamster and mouse. Incubation and clinical periods, clinical manifestation and death rateswere compared. Challenge of hamsters with L. ega was performed using: 10 2,611-4,021 LD50 /0,05 mL;. For CVS were used10 3,7-4,7 LD50 /0,05 mL. Were tested intramuscular (IM), intradermal (ID), intranasal (IN), epidermal abrasion (EA)inoculation routes. Viral antigen in brains was confirmed by Direct Immunofluorescence Test. Mortality percentagesobserved with L. ega rabies virus isolate were the following in hamster: 3,5 % IM, 10,710% IN; in mice: 50.0% IM, 30.0%IN. Furious rabies was predominant. Mortality percentages observed with CVS/32 in hamster: 12.5% IM, 62.5% ID,12.5% IN; in mice 100.0% IM, 70.0% ID, 10.0% IN. Paralytic rabies was found with this strain in both animal models.Epidermic abrasion was not a suitable challenge route. Incubation period was 5-7 days for CVS and 11-16 days for L. egaisolate, meanwhile clinical periods were comprehended between 4–7 days for both viruses. Several substitutions weredetected at antigenic domains of glycoprotein: AI (position 231), AII (34–42 and 198-200), domain of fusion dependenton low pH (102–179), transmembrane domain (440–461) and residue 242. These viruses showed contrasting biologicalbehaviors that can be linked to those substitutions at antigenic domains previously described.(AU)

O perfil patogênico de um vírus da raiva isolado de um morcego insetívoro Lasiurus ega foi comparado com o de vírusfixo de raiva (CVS/32) em hamster e camundongo, determinando os períodos de incubação e clínico, manifestaçãoclínica e mortalidade. Os animais foram desafiados com 10 2,611 - 4,021 DL50 /0,05 mL do isolado de L. ega e 10 3,7- 4,7 LD50 /0,05 mL do CVS/32, usando as vias: intramuscular (IM), intradermica (ID), intranasal (IN) e abrasão epidermica (AE).A presença do antígeno viral foi confirmada pela prova de imunofluorescência direta. As porcentagens de mortalidadeobservadas com o isolado de L. ega foram as seguintes em hamster: 3,5% IM, 10,71% IN; em camundongo: 50.0%IM, 30.0% IN. A forma furiosa da doença foi predominante. As porcentagens de mortalidade observadas com o vírusCVS/32 em hamster foram as seguintes: 12.5% IM, 62.5% ID, 12.5% IN; em camundongo 100.0% IM, 70.0% ID,10.0% IN. Com este vírus foi observada raiva paralitica. A via AE mostrou-se inadequada para induzir doença. Operíodo de incubação foi de 5–7 dias para o CVS/32 e 11-16 dias para o isolado de L. ega, entre tanto os períodosclínicos oscilaram entre 4–7 dias para ambos os vírus. Varias substituições foram achadas em domínios antigênicos daglicoproteína: AI (posição 231), AII (34–42 e 198-200), domínio de fusão dependente de baixo pH (102–179), domínioda transmembrana (440–461) e resíduo 242. Esses vírus mostraram comportamentos biológicos distintos o que poderiaestar ligado às substituições nos domínios antigênicos anteriormente descritos.(AU)

A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field.

Complete genome analysis of a rabies virus isolate from Brazilian wild fox.

The complete genome sequence of wild-type rabies virus (RABV) isolated from a wild Brazilian hoary fox (Dusicyon sp.), the BR-Pfx1 isolate, was determined and compared with fixed RABV strains. The genome structure and organization of the BR-Pfx1 isolate were composed of 11,924 nt and included the five standard genes of rhabdoviruses. Sequences of mRNA start and stop signals for transcription were highly conserved among all structural protein genes of the BR-Pfx1 isolate. All amino acid residues in the glycoprotein (G) gene associated with pathogenicity were retained in the BR-Pfx1 isolate, while unique amino acid substitutions were found in antigenic region I of the nucleoprotein gene and III of G. These results suggest that although the standard genome structure and organization of the RABV isolate are common between the BR-Pfx1 isolate and fixed RABV strains, the unique amino acid substitutions in functional sites of the BR-Pfx1 isolate may result in different biological characteristics from fixed RABV strains.

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.