Caracterização antigênica e genética do primeiro isolado de vírus da raiva em morcego Eumops perotis no Brasil

Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other).No Brasil, embora os principais transmissores da raiva sejam cães e morcegos hematófagos, o papel de outras espécies, tais como morcegos insetívoros e frugívoros, merece atenção especial, uma vez que o vírus da raiva já foi isolado em 36 espécies de morcegos. Este estudo descreve o primeiro isolamento do vírus da raiva em um morcego insetívoro Eumops perotis. O animal infectado foi encontrado na cidade de Ribeirão Preto, São Paulo. O vírus foi identificado pelo teste de imunofluorescência direta (IFD) em amostras de sistema nervoso central (SNC), e o isolamento foi realizado em cultura de células N2A e em camundongos adultos. A amostra foi submetida à tipificação antigênica, utilizando um painel de oito anticorpos monoclonais (CDC/Atlanta/USA). A seqüência de DNA do gene da nucleoproteína, localizada entre os nucleotídeos 102 a 1385, foi alinhada com seqüências homólogas presentes no GenBank, usando o método CLUSTAL/W e o alinhamento foi utilizado para a construção da árvore filogenética de distância "neighbor-joining" com o modelo K-2-P. O SNC testado foi negativo por IFD, e somente um camundongo morreu após inoculação com a suspensão do SNC do morcego. A tipificação antigênica apresentou resultado não-compatível com os padrões definidos pelo painel. A análise filogenética mostrou que o vírus isolado segregou no mesmo grupo relacionado com outros vírus isolados de morcegos insetívoros, gênero Nyctinomops ssp. (98,8% de identidade de nucleotídeos entre elas)

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

Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog), 3 (Desmodus rotundus), 4 (Tadarida brasiliensis), 5 (vampire bat from Venezuela), 6 (Lasiurus cinereus) and Lab (reacted to all used antibodies). Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus). These findings may be related to the existence of multiple independent transmission cycles, involving different bat species.Amostras de vírus rábico isoladas de animais e humanos no período de 1989 a 2000 foram tipificadas antigenicamente com a utilização de um painel de anticorpos monoclonais contra a nucleoproteína viral, pré-estabelecido para o estudo da epidemiologia molecular do vírus rábico isolado nas Américas. As amostras testadas foram isoladas no laboratório de diagnóstico do Instituto Pasteur e outros centros de diagnóstico de raiva no Brasil. Além das cepas de vírus rábico fixo CVS-31/96-IP, mantida em cérebro de camundongos e a PV-BHK/97, mantida em cultura de células, cepas de vírus rábico isoladas de cães, gatos, bovinos, eqüinos, morcegos, ovinos, caprino, suínos, raposa, sagüí, coatí, guaxinim e humanos, totalizaram 330 amostras. Seis variantes antigênicas foram definidas, compatíveis com perfís observados no painel de anticorpos monoclonais pré-estabelecido utilizado, as de número 2 (cão), 3 (Desmodus rotundus), 4 (Tadarida brasiliensis), 5 (Vampiro da Venezuela), 6 (Lasiurus cinereus) e Lab (reagente a todos os anticorpos utilizados), além de outros seis perfís desconhecidos, não compatíveis com aqueles observados no painel utilizado. A maior variabilidade foi observada entre as amostras isoladas de morcegos insetívoros e a variante mais comum isolada entre as espécies foi a variante 3 (Desmodus rotundus). Estes fatos podem representar a existência de múltiplos ciclos de transmissão independentes, envolvendo diferentes espécies de morcegos

Humoral immune response in capuchin monkeys (Cebus apella) after vaccination with inactivated suckling mouse brain rabies vaccine: comparison of two schedules of immunization

Foram vacinados contra a raiva, dois grupos de macacos-pregos adultos, com a vacina inativada preparada em cérebros de camundongos lactentes, administrada pela via intramuscular, na Fundação Parque Zoológico de São Paulo. Os animais em momento algum haviam sido imunizados contra a raiva. O grupo I consistia de nove animais, que receberam três doses de 1,0 mL nos dias 0, 30 e uma dose de reforço aos 210 dias, e o grupo II continha 10 animais que receberam duas doses de 1,0 mL no dia 0 e uma dose de reforço aos 210 dias. As amostras de sangue foram colhidas aos 0, 30º, 60º, 90º, 150º, 210º, 240º, 300º e 365º dias, e os anticorpos neutralizantes titulados pela técnica simplificada da inibição de focos fluorescentes. A vacina induziu uma resposta imune de curta duração com títulos de anticorpos neutralizantes acima de 0.5 UI/mL em ambos os grupos; entretanto a resposta imune persistiu por apenas 54,9 + 57,0 e 36,1 + 60,2 dias nos Grupos I e II respectivamente após a primo vacinação, e, por apenas 62,6 + 74,0 e 86,4 + 61,5 dias nos Grupos I e II respectivamente após o reforço. Não houve diferença estatística significante entre os grupos estudados (p >; 0,05).Two groups of adult capuchin monkeys (Cebus apella) were vaccinated i.m. against rabies with inactivated suckling mouse brain rabies vaccine (SMBV), at the Zoo Park Foundation in São Paulo, SP, Brazil. The animals had not been immunized against rabies at any time before. Group I consisted of nine animals, to which were given three 1.0mL doses on days 0 and 30, plus a booster on day 210. Group II comprehended ten animals, to which were given two 1.0mL doses: one, on day 0 and, another, a booster, on day 210. The blood samples were collected on days 0, 30, 60, 90, 150, 210, 240, 300 and 365, being the titrated neutralizing antibodies determined by the simplified fluorescent focus inhibition technique. The vaccine induced immune responses with neutralizing antibody titers above 0.5 IU/m in both groups of capuchin monkeys; however, they were short, lasting 54.9 ± 57.0 days and 36.1 ± 60.2 days in Groups I and II, respectively, after primo-vaccination, and 62.6 ± 74.0 and 86.4 ± 61.5 days in Groups I and II, respectively, after a booster. No statistically significant differences were observed out between the two groups studied (p >; 0.05)

Vírus da raiva em quirópteros naturalmente infectados no Estado de São Paulo, Brasil

OBJECTIVE: To identify the species of bats involved in maintaining the rabies cycle; to investigate the distribution of the rabies virus in the tissues and organs of bats and the time taken for mortality among inoculated mice. METHODS: From April 2002 to November 2003, bats from municipalities in the State of São Paulo were screened for the presence of the rabies virus, by means of direct immunofluorescence. The virus distribution in the bats was evaluated by inoculating mice and N2A cells with 20% suspensions prepared from fragments of different organs and tissues, plus the brain and salivary glands. The time taken for mortality among the mice was monitored daily, following intracerebral inoculation. RESULTS: Out of the 4,395 bats received, 1.9% were found positive for the rabies virus. They belonged to ten genera, with predominance of insectivores. The maximum mean times taken for mortality among the mice following inoculation with brain and salivary gland material were 15.33±2.08 days and 11.33±2.30 days for vampire bats, 16.45±4.48 days and 18.91±6.12 days for insectivorous bats, and 12.60±2.13 days and 15.67±4.82 days for frugivorous bats, respectively. CONCLUSIONS: The species infected with the rabies virus were: Artibeus lituratus, Artibeus sp., Myotis nigricans, Myotis sp., Eptesicus sp., Lasiurus ega, Lasiurus cinereus, Nyctinomops laticaudatus, Tadarida brasiliensis, Histiotus velatus, Molossus rufus, Eumops sp. and Desmodus rotundus. Virus investigation in the different tissues and organs showed that the brain and salivary glands were the most suitable sites for virus isolation.OBJETIVO: Identificar as espécies de morcegos envolvidas na manutenção do ciclo da raiva, verificar a distribuição do vírus da raiva em tecidos e órgãos de morcegos e os períodos de mortalidade dos camundongos inoculados. MÉTODOS: A positividade para o vírus da raiva foi avaliada por imunofluorescência direta em morcegos de municípios do Estado de São Paulo, de abril de 2002 a novembro de 2003. A distribuição do vírus nos morcegos foi avaliada pela inoculação de camundongos e infecção de células N2A, com suspensões a 20% preparadas a partir de fragmentos de diversos órgãos e tecidos, além de cérebro e glândula salivar. A mortalidade dos camundongos foi observada diariamente, após inoculação intracerebral. RESULTADOS: Dos 4.393 morcegos pesquisados, 1,9% foram positivos para o vírus da raiva, pertencentes a dez gêneros, com predomínio de insetívoros. A média do período máximo de mortalidade dos camundongos pós-inoculação a partir de cérebros e glândulas salivares de morcegos hematófagos foi de 15,33±2,08 dias e 11,33±2,30 dias; insetívoros, 16,45±4,48 dias e 18,91±6,12 dias; e frugívoros, 12,60±2,13 dias e 15,67±4,82 dias, respectivamente. CONCLUSÕES: As espécies infectadas com o vírus da raiva foram: Artibeus lituratus, Artibeus sp., Myotis nigricans, Myotis sp., Eptesicus sp., Lasiurus ega, Lasiurus cinereus, Nyctinomops laticaudatus, Tadarida brasiliensis, Histiotus velatus, Molossus rufus, Eumops sp. e Desmodus rotundus. A pesquisa de vírus em diferentes tecidos e órgãos mostrou-se que os mais apropriados para o isolamento foram cérebro e glândulas salivares

Diagnóstico ante-mortem de raiva humana em médico veterinário infectado por manipulação de herbívoro, Minas Gerais, Brasil

The Ministry of Health's National Human Rabies Control Program advocates pre-exposure prophylaxis (PEP) for professionals involved with animals that are at risk of contracting rabies. We report an antemortem and postmortem diagnosis of rabies in a veterinarian who became infected when handling herbivores with rabies. The antemortem diagnosis was carried out with a saliva sample and a biopsy of hair follicles using molecular biology techniques, while the postmortem diagnosis used a brain sample and conventional techniques. The veterinarian had collected samples to diagnose rabies in suspect herbivores (bovines and caprines) that were subsequently confirmed to be positive in laboratory tests. After onset of classic rabies symptoms, saliva and hair follicles were collected and used for antemortem diagnostic tests and found to be positive by RT-PCR. Genetic sequencing showed that the infection was caused by variant 3 (Desmodus rotundus), a finding confirmed by tests on the brain sample. It is essential that professionals who are at risk of infection by the rabies virus undergo pre-exposure prophylaxis. This study also confirms that molecular biology techniques were used successfully for antemortem diagnosis and therefore not only allow therapeutic methods to be developed, but also enable the source of infection in human rabies cases to be identified accurately and quickly.O Programa Nacional de Controle da Raiva Humana do Ministério da Saúde preconiza o esquema profilático pré-exposição (PEP) para profissionais envolvidos com animais expostos ao risco de contraírem raiva. O presente trabalho relata o diagnóstico de raiva (ante e post-mortem) em veterinário infectado por manipulação de herbívoros raivosos. O diagnóstico laboratorial ante-mortem foi efetuado a partir da saliva e biópsia de folículo piloso, utilizando técnicas de biologia molecular e o post-mortem a partir do tecido cerebral e de técnicas convencionais. O médico veterinário coletou amostras para diagnóstico de raiva em herbívoros (bovinos e caprinos) suspeitos que, posteriormente, foram confirmados positivos em laboratório. Após a apresentação dos sintomas clássicos de raiva e realizadas as provas de diagnóstico ante-mortem com saliva e folículo piloso, ambas as amostras apresentaram resultados positivos pelo nested-RT-PCR. O sequenciamento genético revelou que a infecção se deu pela variante 3 do Desmodus rotundus, resultados estes confirmados com a amostra do cérebro. É indispensável que profissionais expostos ao risco de infecção pelo vírus da raiva realizem a profilaxia pré-exposição. Ressalta-se, também, que as técnicas de biologia molecular apresentaram bons resultados para a realização de diagnóstico ante-mortem, propiciando o desenvolvimento de métodos terapêuticos, e determinando com precisão e rapidez a fonte de infecção dos casos de raiva humana

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. (C) 2012 Elsevier Editora Ltda. All rights reserved.CAPESCAPESCNPqCNP

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

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. (C) 2012 Elsevier Editora Ltda. All rights reserved.CAPESCAPESCNPqCNP