Isolation and characterization of a novel strain (YH01) of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the baculovirus expression system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300-500 nm in length and 100-200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1-458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.

Áreas propícias para o ataque de morcegos hematófagos Desmodus rotundus em bovinos na região de São João da Boa Vista, Estado de São Paulo / Areas prone for vampire bat (Desmodus rotundus) attack on cattle in the São João da Boa Vista region, State of São Paulo

Baseada em estudos anteriores, a Coordenadoria de Defesa Agropecuária da Secretaria da Agricultura e Abastecimento do Estado de São Paulo (CDA) vinha realizando nas regiões administrativas onde é feita a vigilância da raiva em herbívoros o controle de populações de Desmodus rotundus em áreas próximas aos principais rios. A descoberta de abrigos e de animais mordidos pelos morcegos vampiros em outras áreas, além das áreas estabelecidas de risco levaram a questionamentos dos métodos em vigência. Isso estimulou a elaboração de um modelo descritivo para analisar áreas de risco para o ataque espoliativo de morcegos hematófagos em bovinos na região de São João da Boa Vista, estado de São Paulo. Para tanto, foram utilizadas imagens de satélite, parâmetros de estudos anteriores sobre o comportamento dos morcegos vampiros, dados da CDA e da Fundação Instituto Brasileiro de Geografia e Estatística (IBGE). Todas as informações foram agrupadas em um banco de dados e integradas em um Sistema de Informação Geográfica. Contraditoriamente ao que se pensava, observou-se que áreas próximas aos principais rios da região das cidades de Aguaí, Casa Branca, Santa Cruz das Palmeiras e Tambaú não deveriam ser consideradas de risco para o ataque dos morcegos hematófagos em bovinos. Isso sugere que é necessária uma reavaliação dos métodos adotados para o controle das populações da espécie e enfermidade em questão.

Supported by previous studies that selected areas prone for the development of Desmodus rotundus roosts and bovine rabies transmitted by vampire bats in São Paulo state, the Department of Agriculture of the State of São Paulo (CDA) has conducted interventions for bovine rabies control in areas mostly close to the main rivers of surveillance regions. Inquiries of the actual concepts led to the development of a descriptive model to analyze the vampire bat Desmodus rotundus attacks on cattle far beyond main rivers and water streams in the São João da Boa Vista region, São Paulo state. The model was elaborated using satellite images classification, parameters from previous studies about vampire bats behaviour, data from the CDA and the Brazilian Institute of Geography and Statistics (IBGE). All this information was used to build a geographical database and integrate in a Geographical Information System then analyzed through spatial statistical and analytical methods. The results showed that areas close to the main rivers of the towns Aguaí, Casa Branca, Santa Cruz das Palmeiras and Tambaú were found not to be under risk of bat attacks, suggesting that the hypothesis behind the CDA control areas definition does not hold when contracted with the obtained spatial data. The results indicate that the technicians from CDA need to reformulate their strategy for defining the surveillance of vampire bat populations and rabies control.

[Rabies situation in Thailand]

Rabies statistics have been officially recorded since 1929 and rabies became a notifiable disease in Thailand in 1980. The direct fluorescent antibody test is the test most frequently used to diagnose rabies. Although rabies incidence has been declining, animal specimens submitted for testing have been inadequate and it is not justifiable to conclude that the rabies situation is controlled. Laboratory confirmation of rabies in man relies on demonstration of rabies antigen on brain impression smear or RNA in secretions and hair follicles. Nevertheless, almost all specimens belonged to patients who manifested as furious rabies only. Reliability of the reporting system needs improvement in public understanding and close cooperation between public and government sectors.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

The Expression of a Novel 90 kDa Stress Protein in Human Malignant Neoplasms / 대한암학회지

PURPOSE: When cells are subjected to stressful stimuli such as, heat shock, toxic metal, nutrient deprivation, and metabolic disruption, they increase production of specific stress proteins that buffer them from harm. We reported that the expression of a navel 90 kDa cellular protein was increased by the infection of a fish rhabdovirus and heat shock in a fish cell. This new 90 kDa protein is not expressed in normal animal tissues but is highly induced in progressively transforming tissues or cells. That gives us some ideas tl at it is possible for this stress protein to be expressed in specific human cancer tissues. MATERIALS AND METHODS: Commercialized checkerboard multi-tumor block (DAKO Co. Carpinteria, CA) was used for immunohistochemical analysis. The samples of human gastric cancer, colon cancer and breast cancer tissues were evaluated by Western blot and Northern blot for overexpression of the novel 90 kDa stress protein. Sera of those patients were analyzed by ELISA for the presence of antibody against the novel 90 kDa stress protein. RESULTS: Immunohistochemical staining of human tumor tissue blocks showed significant immunostaining of novel 90 kDa stress protein in carcinomas such as colon cancer, breast cancer and stomach cancer but no apparent immunostaining in sarcomas. Coinciding with the immunohistochemical result, Western blotting and Northern blotting analyses indicate that the expression of the novel 90 kDa stress protein was increased in carcinomas. In addition, the antibody titer against the novel 90 kDa stress protein was found to be elevated in the sera of cancer patients. CONCLUSIONS: The novel 90 kDa stress protein gene expression was elevated in carcinomas such as gastric cancer, breast cancer and colon cancer. These findings suggest that this new stress protein can be used as a tumor marker and may function as a chaperone in tumor growth.

Historical, Epidemiological and Ecological Aspects of Arboviruses in Argentina: Flaviviridae, Bunyaviridae and Rhabdoviridae

This is a review of the arboviruses in Argentina belonging to families Flaviviridae, Bunyaviridae and Rhabdoviridae. Of the many viruses beloging to these families, the flavivirus St. Louis encephalitis (SLE), has been most intensively studies. SLE virus strains have been recovered from three sources: 2 strains from humans with an undifferentiated, febrile disease; 6 from mosquitoes; and 2 from rodents. The viruses recovered from rodents are attenuated and those from mosquitoes are virulent based on a neuroinvasiveness test in mice; the degree of virulence of the mosquito strains remain to be analyzed. Serological surveys indicate a wide distribution and endemicity of SLE virus in the temperate and subtropical areas (central and northern Argentina), but no data are available from the Andean region or from the South. The virulent SLE virus strains appear to be transmitted between Culex (Cx.) spp., from which they were isolated, and wild birds, based on antibody prevalence. A urban cycle may involve Cx. quinquefasciatus (source of a viral isolate and a competent experimental vector) and abundant birds (house sparrows, doves, and/or chickens), chickens are experimentally competent host species. Despite similarities in the ecology of SLE between Argentina and North America, urban outbreaks of SLE have not recognized. Possible explanations for this discrepancy include virus strain differences in virulence, ecologic factors determining the rate os virus transmission, and the lack of disease recognition and specific laboratory diagnosisof human meningoencephalitis. The transmission cycle of attenuated SLE virus strains isolated from rodents has not been studied. Ilheus virus has been isolated only once from a human being. The available serological data are difficult to interpret due to cross-reactivity with other flaviviruses, and the ecology and medical importance of this agent remain uncertain. Dengue has not been recognized in Argentina since 1916, although its vector, Aedes aegypti, was not eradicated until 1963. Dengue was previously present in coastal localities of Chaco, Corrientes and Misiones Provinces. Within the last few years, Argentina was reinfested by Ae. aegypti. Although no human cases have yet been reported, outbreaks of dengue in bordering countries (Brazil, Paraguay, Bolovia) since 1986, clearly signal that the country in once again at risk of importation and spread of the viruse

Analysis of Brazilian rabies virus isolates with monoclonal antibodies to lyssavirus antigens

Brazilian rabies virus isolates from different host species obtained from different geographical areas in the country were analysed in their reactivity profile with a panel of monoclonal antibodies (Mabs) prepared against members of the Lyssavirus genus of the family Rhabdoviridae. The panel included fourteen Mabs prepared against rabies-related viruses and two Mabs prepared to the CVS-31 rabies vaccine strain. Isolates were multiplied in mice and brain impressions examined by indirect immunoflurescence. Epitopes common to lyssaviruses were identified in all rabies viruses examined. differential patterns of reactivity were obtained with ten of the sixteen Mabs. Two Mabs reacted only with viruses from cattle and horses with the same reactivity profile, suggesting a common origin for the viruses, most likely vampire bats.

Comparaçäo de dois testes de neutralizaçäo, utilizados no diagnóstico sorológico do vesiculovírus Piry / Comparison of two neutralization test for the use in the Piry serologic vesiculovirus diagnose

Realizou-se inquérito sorológico para pesquisar anticorpos neutralizantes contra o vesiculovírus Piry, na cidade de Catolândia, Bahia, Brasil. Duas técnicas de vírus-neutralizaçäo foram comparadas em cultura das células C6/36, com revelaçäo pelo método imunoenzimático (TN-C6/36) e em camundongos recém-nascidos (TN-camundongos), que é considerada a prova maior. Em 204 soros, dos 1.274 colhidos, a concordância das duas técnicas foi de 98,7 por cento (K=0,9853). Com este resultado do TN-C6/36, que também é mais exeqüível, decidiu-se desenvolver o estudo soro-epidemiológico do vesiculovírus Piry, em Catolândia, baseado nesta nova técnica

Preparaçåo de soro anti-rábico e desenvolvimento de ensaio imunoenzimático para quantificaçåo de seus anticorpos / Purification of antirabic seurm and establischment of immune assay o quantify entobodies agaist rabic virus

O método imunoenzimático de ELISA foi padronizado, utilizando como fonte de antígeno extrato de cérebro de camundongo infectado com vírus rábico, para detectar e quantificar anticorpos antivírus rábico e adaptá-lo ao acompanhamento da produçåo de soros anti-rábicos em cavalos, e para titular anticorpos antivírus rábico presentes em soros humanos. Em paralelo, foram utilizados como fonte de antígeno para este ensaio imunoenzimático, sobrenadantes de culturas celulares infectadas com vírus rábico, tanto para soros humanos quanto para plasma imune eqüino. Os resultados obtidos no ensaio imunoenzimático tendo como fonte de antígeno extrato de cérebro de camundongo infectado com vírus rábico foram comparados com os resultados obtidos por imunofluorescência indireta. Comparando os resultados obtidos, estatisticamente, verificou-se que existe concordância entre os dois métodos. Métodos alternativos de purificaçåo e concentraçåo do plasma hiperimune eqüino foram sugeridos para obtençåo do IgG e seus fragmentos F(ab')². No acompanhamento da produçåo de anticorpos antivírus rábico em cavalos e nos processos de obtençåo de IgG e dos fragmentos F(ab')², feito em paralelo pelos métodos de CIE, SN e pelo métodos de ELISA, verificou-se concordância nos resultados obtidos através dos três métodos. Dentre os métodos alternativos sugeridos para purificaçåo e concentraçåo do soro hiperimune anti-rábico, o método onde se purificou IgG utilizando ácido caprílico, mostrou-se eficaz tanto na obtençåo da pureza desejada como na atividade específica e porcentagem de rendimentos. Utilizando também um ensaio imunoenzimático demonstrou-se que a IgG(T) de cavalo apresenta atividade específica para o vírus rábico

Susceptibility of Aedes krombeini cell line to some arboviruses.

The susceptibility of the newly established Ae. krombeini cell line (NIVI-AK-453) to six arboviruses, belonging to four different families, was studied. Sindbis (SIND), Vesicular stomatitis (VSV) Chandipura (CHP) and African horse sickness (AHS) viruses multiplied in these cultures. A four-to-five-fold increase in the virus titres was observed. The maximum titre of SIND, VSV, CHP and AHS viruses were observed on 1st, 4th, 3rd and 10th post infection days, respectively. A steady and significant increase in the titre of AHS was observed over a period of ten days. The sandfly fever virus (SFV) and the tick-borne, Kaisodi virus did not multiply in the cultures.

Frequência de anticorpos neutralizantes contra o vesiculovirus piry, em doadores de sangue de Uberaba, Minas Gerais, Brasil / Frequency of neutralizing antibodies to the vesiculovirus piry, in blood donors of Uberaba, Minas Gerais, Brazil

Na cidade de Uberaba, Estado de Minas Gerais, dos 162 doadores de sangue, 13 (8.0%) apresentaram anticorpos neutralizantes contra o vesiculovirus Piry. Nos indivíduos positivos foi estaticamente mais freqüente o relato de residência anterior em area rural (p<0,0001) em cidades do Estado de Säo Paulo (p<0,05)

Uso de células de Aedes albopictus C6/36 na propagaçäo e classificaçäo de arbovirus das famílias Togaviridae, Flaviviridae, Bunyaviridae e Rhabdoviridae / The use of Aedes albopictus C6/36 cells in the propagation and classification of arbovirus of the Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae families

Colônias de células de mosquito Aedes albopicus C6/36 foram infectadas com 23 arbovirus, sendo 19 destes existentes no Brasil, pertencentes às famílias Togavitidae, Flaviviridae, Bunyaviridae e Rhabdoviridae. A Replicaçäo viral foi detectada por imunofluorescência indireta com todos os vírus estudados enquanto que o efeito citopático foi observado durante a infecçäo por alguns deste. No teste de imunofluorescência indireta utilizou-se fluidos ascíticos imunes de camundongos, especificos para os vírus estudados. A replicaçäo viral caracterizada por grande produçäo de antígeno recomenda a utilizaçäo de células C6/36 na propagaçäo e em tentativas de isolamento desses arbovírus. A técnica de imunofluorescência ofereceu subsídios na classificaçäo e identificaçäo de vírus que replicam nestas células

Estudo epizootiológico da estomatite vesicular no Vale do Paraíba, SP / Epizootiological study of the vesicular estomatitis in Paraiba Valley, SP

Realizou-se estudo epizootiologico da estomatite vesicular ocorrida em bovinos e equídeos da regiäo do Vale do Paraíba, SP. Coletaram-se amostras de sangue de 2.673 animais, pertencentes a 367 rebanhos. A pesquisa de anticorpos foi feita pela técnica de dupla difusäo em gel de ágar e revelou 41 rebanhos positivos (11,17 por cento) sendo que a prevalência nos equídeos (4,36 por cento) foi maior que nos bovinos (1,64 por cento). Dos rebanhos positivos, apenas dois apresentaram histórico de ocorrência de enfermidade vesicular. Foram aplicados testes estatísticos com a finalidade de se estabelecerem relaçöes entre rebanhos infectados pelo vírus Indiana 3 e fatores ambientais. Os testes de associaçäo revelaram-se positivos para a presença de rio, como local onde os animais bebiam água, e a presença de cultura de milho e feijäo. O teste de duas proporçöes mostrou que, quando comparadas as faixas etárias, a espécie eqüina foi mais susceptível ao vírus da estomatite vesicular e os animais que mostraram maior susceptibilidade situaram-se entre as faixas etárias de 3-5 e 5-7 anos. Os resultados obtidos demonstraram a necessidade de maiores estudos relacionados à epidemiologia da doença, em nosso meio