Rabies virus related to vampire bats (Desmodus rotundus) isolated from a crab-eating fox (Cerdocyon thous) in Southeast Brazil

In this study, rabies virus was isolated from a naturally infected crab-eating fox [Cerdocyon thous] in a rural area of São Paulo State, Brazil. The sample was positive for the standard techniques used for rabies diagnosis. The isolate was antigenically characterized using anti-nucleoprotein monoclonal antibodies. Studies on molecular epidemiology by patterns of nucleotide substitution in the nucleoprotein gene showed that the isolate was genetically homologous to the rabies variant maintained and transmitted by the common vampire bat Desmodus rotundus. This is the first report of detection of this variant from a crab-eating fox in Brazil and represents evidence of cross-species transmission. This was the unique report where this variant was isolated from a crab eating fox in Brazil.

[Hematophagous bats as reservoirs of rabies]. / Murciélagos hematófagos como reservorios de la rabia.

Rabies continues to be a challenge for public health authorities and a constraint to the livestock industry in Latin America. Wild and domestic canines and vampire bats are the main transmitter species and reservoirs of the disease. Currently, variations observed in the epidemiological profile of rabies, where the species of hematophagous bat Desmodus rotundus constitutes the main transmitting species. Over the years, knowledge has accumulated about the ecology, biology and behavior of this species and the natural history of rabies, which should lead to continuous development of methods of population control of d. Rotundus as well as prevention and diagnostic tools for rabies. Ecological relationships of this species with other hematophagous and non-hematophagous bats is unknown, and there is much room for improvement in reporting systems and surveillance, as well as creating greater awareness among the farming community. Understanding the impact of human-induced environmental changes on the rabies virus in bats should be cause for further investigation. This will require a combination of field studies with mathematical models and new diagnostic tools. This review aims to present the most relevant issues on the role of hematophagous bats as reservoirs and transmitters of the rabies virus.

Murciélagos hematófagos como reservorios de la rabia / Hematophagous bats as reservoirs of rabies

La rabia continua siendo un desafío para las autoridades de salud pública y una limitante para la industria ganadera en América Latina. Caninos silvestres y domésticos, así como murciélagos hematófagos son las principales especies transmisoras y reservorios de la enfermedad. Actualmente, se observa variaciones en el perfil epidemiológico de la rabia, donde la especie de murciélago hematófago Desmodus rotundus se constituye en la principal especie transmisora. A lo largo del tiempo se ha acumulado conocimiento sobre la ecología, biología y comportamiento de esta especie y sobre la historia natural de la rabia, lo cual debe conducir a una continua evolución de los métodos de control poblacional de D. rotundus, prevención y técnicas de diagnóstico. Aún se desconoce la relación ecológica de esta especie con otras hematófagas y no hematófagas, y queda mucho por mejorar en los sistemas de notificación y vigilancia epidemiológica, así como crear una mayor conciencia entre los ganaderos ante el tema. La comprensión del impacto que las modificaciones ambientales inducidas por el hombre ejercen sobre la dinámica de infección del virus de la rabia en los murciélagos debe ser motivo de investigaciones posteriores. Esto requerirá la combinación de estudios de campo con modelos matemáticos y nuevas herramientas diagnósticas. La presente revisión pretende presentar los aspectos más relevantes sobre el rol de los murciélagos hematófagos como reservorios y transmisores del virus de la rabia.

Rabies continues to be a challenge for public health authorities and a constraint to the livestock industry in Latin America. Wild and domestic canines and vampire bats are the main transmitter species and reservoirs of the disease. Currently, variations observed in the epidemiological profile of rabies, where the species of hematophagous bat Desmodus rotundus constitutes the main transmitting species. Over the years, knowledge has accumulated about the ecology, biology and behavior of this species and the natural history of rabies, which should lead to continuous development of methods of population control of d. Rotundus as well as prevention and diagnostic tools for rabies. Ecological relationships of this species with other hematophagous and non-hematophagous bats is unknown, and there is much room for improvement in reporting systems and surveillance, as well as creating greater awareness among the farming community. Understanding the impact of human-induced environmental changes on the rabies virus in bats should be cause for further investigation. This will require a combination of field studies with mathematical models and new diagnostic tools. This review aims to present the most relevant issues on the role of hematophagous bats as reservoirs and transmitters of the rabies virus.

In vitro and in vivo inhibition of rabies virus replication by RNA interference

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

In vitro and in vivo inhibition of rabies virus replication by RNA interference

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.(AU)

In vitro and in vivo inhibition of rabies virus replication by RNA interference.

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

Outbreaks of Eastern equine encephalitis in northeastern Brazil.

Outbreaks of eastern equine encephalitis observed from May 2008 to August 2009 in the Brazilian states of Pernambuco, Ceará, and Paraíba are reported. The disease occurred in 93 farms affecting 229 equids with a case fatality rate of 72.92%. Main clinical signs were circling, depression or hyperexcitability, ataxia, and progressive paralysis with a clinical manifestation period of 3-15 days. Main histologic lesions were a diffuse lymphocytic encephalomyelitis with neuronal death, satellitosis, neuronophagia, and hemorrhages being more severe in the cerebral gray matter of the telencephalon, diencephalon, and mesencephalon. Some animals also had areas of malacia in the telencephalon, thalamus, and basal nuclei. From 1 case, the virus was isolated by mice inoculation, and in other 13 cases was identified as Eastern equine encephalitis virus by semi-nested reverse transcription polymerase chain reaction. After DNA sequencing, all samples were identified as eastern equine encephalitis through the BLASTn analysis, but samples from the Ceará and Paraíba states corresponded to the same cluster, while the sample from the state of Pernambuco corresponded to a different cluster.

Sensibilidade da técnica de imuno-histoquímica em fragmentos de sistema nervoso central de bovinos e equinos naturalmente infectados pelo vírus da raiva / Sensitivity of the Immunohistochemistry technique in central nervous system fragments of cattle and horses naturally infected by rabies virus

A raiva é uma zoonose viral que acomete o sistema nervoso central (SNC) de mamíferos, considerada um grave problema de saúde pública. Herbívoros (bovinos e equinos) são frequentemente acometidos pela in-fecção após serem atacados por morcegos hematófagos (Desmodus rotundus). A técnica de imunofluorescência direta (IFD) realizada em tecidos frescos, recomendada pela Organização Mundial de Saúde (OMS), é utilizada para o diagnóstico da raiva. A técnica de imuno-histoquímica (IHQ) é utilizada para detectar antígenos em tecidos fixados, pelo uso de anticorpos monoclonais/policlonais. O objetivo deste trabalho foi avaliar a sensibilidade da IHQ na detecção de antígenos do vírus da raiva em amostras de SNC de herbívoros fixadas em formol, analisando a distribuição antigênica em diferentes fragmentos do SNC. Os resultados demonstraram concordância das técnicas de IFD e IHQ. A IHQ mostrou maior sensibilidade em amostras de bovinos em relação às de equinos, especialmente quando realizada em fragmentos de cerebelo e tronco encefálico. A detecção de antígeno nestes fragmentos foi mais consistente para ambas as técnicas, nas duas espécies. Estes resultados demonstram que a IHQ pode ser empregada para a vigilância epidemiológica da raiva, entretanto, recomenda-se cautela ao se empregar a IHQ para diagnóstico de doença em herbívoros, especialmente quando o fragmento encaminhado ao laboratório for apenas o hipocampo.

Rabies is a viral zoonosis that causes disease in the central nervous system (CNS) of mammals and it is considered a serious problem of public health. Herbivorous (bovines and equines) are often infected after being attacked by vampire bats (Desmodus rotundus). The direct fluorescent antibody technique is used as a diagnostic test to detect viral antigens in fresh tissues and is recommended by the World Health Organization. The immunohistochemistry technique (IHC) is used to detect the viral antigen through the use of monoclonal/policlonal antibodies in formalin-fixed tissues. The aim of this work was to evaluate the sensitivity of the IHC in samples of CNS of herbivorous fixed in formol, analyzing the antigenic distribution in different fragments of the CNS. The results demonstrated good agreement between the two techniques for the rabies diagnosis. The IHC presented higher sensitivity in samples of cattle comparing to horse samples, especially in fragments of cerebellum and brain stem. These fragments demonstrated to be more suitable for antigen detection by both techniques in the two species. These data demonstrate that the IHC is suitable for rabies vigilance yet cautions should be taken in examining cattle and horses samples, when the submitted specimen is only the hippocampus.

Sensibilidade da técnica de imuno-histoquímica em fragmentos de sistema nervoso central de bovinos e equinos naturalmente infectados pelo vírus da raiva / Sensitivity of the Immunohistochemistry technique in central nervous system fragments of cattle and horses naturally infected by rabies virus

A raiva é uma zoonose viral que acomete o sistema nervoso central (SNC) de mamíferos, considerada um grave problema de saúde pública. Herbívoros (bovinos e equinos) são frequentemente acometidos pela in-fecção após serem atacados por morcegos hematófagos (Desmodus rotundus). A técnica de imunofluorescência direta (IFD) realizada em tecidos frescos, recomendada pela Organização Mundial de Saúde (OMS), é utilizada para o diagnóstico da raiva. A técnica de imuno-histoquímica (IHQ) é utilizada para detectar antígenos em tecidos fixados, pelo uso de anticorpos monoclonais/policlonais. O objetivo deste trabalho foi avaliar a sensibilidade da IHQ na detecção de antígenos do vírus da raiva em amostras de SNC de herbívoros fixadas em formol, analisando a distribuição antigênica em diferentes fragmentos do SNC. Os resultados demonstraram concordância das técnicas de IFD e IHQ. A IHQ mostrou maior sensibilidade em amostras de bovinos em relação às de equinos, especialmente quando realizada em fragmentos de cerebelo e tronco encefálico. A detecção de antígeno nestes fragmentos foi mais consistente para ambas as técnicas, nas duas espécies. Estes resultados demonstram que a IHQ pode ser empregada para a vigilância epidemiológica da raiva, entretanto, recomenda-se cautela ao se empregar a IHQ para diagnóstico de doença em herbívoros, especialmente quando o fragmento encaminhado ao laboratório for apenas o hipocampo.(AU)

Rabies is a viral zoonosis that causes disease in the central nervous system (CNS) of mammals and it is considered a serious problem of public health. Herbivorous (bovines and equines) are often infected after being attacked by vampire bats (Desmodus rotundus). The direct fluorescent antibody technique is used as a diagnostic test to detect viral antigens in fresh tissues and is recommended by the World Health Organization. The immunohistochemistry technique (IHC) is used to detect the viral antigen through the use of monoclonal/policlonal antibodies in formalin-fixed tissues. The aim of this work was to evaluate the sensitivity of the IHC in samples of CNS of herbivorous fixed in formol, analyzing the antigenic distribution in different fragments of the CNS. The results demonstrated good agreement between the two techniques for the rabies diagnosis. The IHC presented higher sensitivity in samples of cattle comparing to horse samples, especially in fragments of cerebellum and brain stem. These fragments demonstrated to be more suitable for antigen detection by both techniques in the two species. These data demonstrate that the IHC is suitable for rabies vigilance yet cautions should be taken in examining cattle and horses samples, when the submitted specimen is only the hippocampus.(AU)

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.