中国淋巴囊肿病毒胸苷酸合酶基因结构特点及分析

胸苷酸合酶(Thymidylate synthase, TS)是进行 DNA 合成所必需的酶类,与细胞分化及肿瘤发生密切相关。淋巴囊肿病毒属于虹彩病毒科成员,是能引起百余种淡、海水鱼感染,并产生肿瘤的病毒病原。在已完成中国淋巴囊肿病毒株 (Lymphocystis disease virus-China, LCDV-C) 基因组序列测定的基础上,本文对位于 LCDV-C 基因组开放阅读框 ORF 011L 的 TS 基因结构、及其推定蛋白结构进行了分析。该基因全长 858bp,GC 含量为 28.2%,编

Isolation, characterization and genome sequence of a birnavirus strain from flounder Paralichthys olivaceus in China

A birnavirus strain, Paralichthys olivaceus birnavirus (POBV), was isolated and characterized from cultured flounder in China, and its complete genomic sequence was subsequently determined. The virus could induce cytopathic effects (CPE) in four of seven fish cell lines and was resistant to chloroform, 5-iodo-2'-deoxyuridine, acid and alkaline pH, and heat treatment. Purified virus particles had a typical icosahedral shape, with a diameter of approximately 55-60 nm. The genomic segments A and B of POBV were 3,091 and 2,780 bp in length and shared many of the features of the members of the family Birnaviridae. Segment A contained two partially overlapping ORFs encoding a polyprotein, pVP2-VP4-VP3, and a nonstructural protein, VP5, while segment B had only one ORF encoding for the VP1, a viral RNA-dependent RNA polymerase (RdRp). This is the first report about a birnavirus strain from a new non-salmonid host in China and its complete genome sequence.

Constitutive expression of thymidylate synthase from LCDV-C induces a transformed phenotype in fish cells

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumor formation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virus from China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4-5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity. (C) 2007 Elsevier Inc. All rights reserved.

Characterization and application of monoclonal antibodies against turbot (Scophthalmus maximus) rhabdovirus

Five monoclonal antibodies (mAbs), 1G8, 1H9, 2D2, 2D3, and 2F5, against Scophthalmus maximus rhabdovirus (SMRV) were prepared. Characterization of the mAbs included indirect enzyme-linked immunosorbent assay, isotyping, viral inhibition assay, immunofluorescence staining of virus-infected cell cultures, and Western blot analysis. Isotyping revealed that 1G8 and 1H9 were of the IgG2b subclass and that the other three were IgM. 2D2, 2D3, and 2F5 partially inhibited SMRV infection in epithelioma. papulosum cyprinid (EPC) cell culture. Western blotting showed that all five mAbs could react with two SMRV proteins with molecular masses of approximately 30 kDa (P) and 26 kDa (M). These two proteins were localized within the cytoplasm of SMRV-infected EPC cells by immunofluorescence assay. Also, progressive foci of viral replication in cell cultures were monitored from 6 to 24 h, using mAb 2D3 as the primary antibody. A flow cytometry procedure was used to detect and quantify SMRV-infected (0.01 PFU/cell) EPC cells with mAb 2D3, and 10.8% of cells could be distinguished as infected 36 h postinfection. Moreover, mAb 2D3 was successfully applied for the detection of viral antigen in cryosections from flounder tissues by immunohistochemistry tests.

Facteurs de risque associés à la prévalence d'aérosacculite à l'abattoir chez le poulet de chair

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy

Here, we analyze the complete coding sequences of all recognized tick-borne flavivirus species, including Gadgets Gully, Royal Farm and Karshi virus, seabird-associated flaviviruses, Kadam virus and previously uncharacterized isolates of Kyasanur Forest disease virus and Omsk hemorrhagic fever virus. Significant taxonomic improvements are proposed, e.g. the identification of three major groups (mammalian, seabird and Kadam tick-borne flavivirus groups), the creation of a new species (Karshi virus) and the assignment of Tick-borne encephalitis and Louping ill viruses to a unique species (Tick-borne encephalitis virus) including four viral types (i.e. Western Tick-borne encephalitis virus, Eastern Tick-borne encephalitis virus, Turkish sheep Tick-borne encephalitis virus and Louping ill Tick-borne encephalitis virus). The analyses also suggest a complex relationship between viruses infecting birds and those infecting mammals. Ticks that feed on both categories of vertebrates may constitute the evolutionary bridge between the three distinct identified lineages.

Implicações técnicas da vacinação na resposta imune contra o vírus da febre aftosa

Na bovinocultura brasileira, a vacinação contra o vírus da Febre Aftosa (FA) é fundamental para a fase inicial de erradicação da enfermidade. Mesmo a qualidade da vacina tendo controle rígido feito pelos órgãos oficiais, restam variáveis técnicas ainda não monitoradas como manipulação, transporte e conservação pelo consumidor, dose, local e forma de aplicação que interferem na resposta imune, preocupações essas, que direcionam o presente estudo. Assim, pela pesquisa de anticorpos neutralizantes do vírus da FA em placas de microtitulação com cultivo de células BHK-21, foram determinados os títulos, calculados em logaritmo decimal (SN), em soros sanguíneos de bovinos vacinados conforme o protocolo apresentado. No primeiro grupo com 25 animais, a média de SN foi igual a 2,37 e 2,19, respectivamente, 30 e 180 dias após a vacinação, cuja vacina foi manejada por especialista com todos os cuidados técnicos recomendados. Outro grupo com 140 bovinos, distribuídos em 5 fazendas distintas, apresentou média de SN igual a 1,66 e 1,5l depois de 30 e 180 dias após a vacinação, cuja vacina foi manejada sem acompanhamento técnico e por indivíduos não especializados. Finalmente um terceiro grupo com 10 animais, que ficaram sem vacinação, apresentou média de SN igual a 0,82 e 0,81, também 30 e 180 dias após a aplicação do placebo. Assim, só os cuidados com a qualidade da vacina são insuficientes para proporcionar títulos satisfatórios que determinam proteção dos rebanhos contra o vírus da FA, uma vez que a literatura pertinente considera rebanhos com 1,52 de média do SN como tendo 50% dos animais protegidos, e com 1,70 como tendo mais de 70% de proteção, no período de até 7 meses.

The use of scaning electron microscopy in postvaccinal evaluation of tracheal epithelium of Coturnix coturnix japonica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos

The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.

Estudo da eficácia de espuma para a depopulação de aves para situações de emergência sanitaria

There are several difficulties in the control of quick spreading and highly contagious poultry diseases such as avian influenza and Newcastle disease, which require large numbers of poultry to be rapidly killed inside the poultry houses in order to minimize human exposure and disease spreading. We have built and tested an equipment that uses a waterbased foam from high expansion fire detergent that induces poultry anoxia measured by sensorial signs, time to death, lethality and macro and microscopically lesions. Two hundred 78-weeks old hens were divided into ten groups and exposed to different times of death according to exposure to water-foam varying from 5min30s to 8min15s. All poultry died after six minutes of water-foam cover. Foam bubbles were observed in oral cavity and trachea of all poultry at each time of exposure to foam, revealing physical asphyxia. Macroscopically, congestion in oral cavity and in trachea mucosa was observed. Histopathology revealed diffuse and moderate hemorrhage in lungs and no lesion in trachea. The efficacy of water-foam procedure for poultry depopulation was demonstrated validating its recommendation in case of emergency.

Avaliação do perfil lipídico entre indivíduos com hepatite C

Metabolic profiles correlate with hepatitis C virus (HCV) infection and are prognostic for the viral response. However, little is known about the association between lipid profiles and viral load in chronic patients carrying HCV genotypes 1, 2 and 3. The aim of this study was to investigate the influence of the viremia and viral genotype on lipid metabolism by observing the variations in serum lipoprotein and apolipoprotein B, to assess whether HCV predisposes individuals to lipid imbalance and favors the appearance of vascular complications. A sample group of 150 chronic HCV patients with viral genotypes 1, 2 or 3 and a control group of 20 healthy adults (10 men and 10 women), all aged from 20 to 50 years were studied. The serum lipid profile of the chronic patients was analyzed and compared to that of the control group. The high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) and triglyceride levels of the sample group were lower than those of the control group, while the low-density lipoprotein (LDL) and apolipoprotein B levels of the patients were higher. These differences were more significant in patients carrying genotype 3a. There was a positive correlation between the viremia and the changes in apolipoprotein B levels in patients carrying genotype 1b. It was inferred that the risk of developing vascular complications raised in HCV patients. As 90% of LDL protein is composed of apolipoprotein B, the plasmatic concentration of the latter indicates the number of potentially atherogenic particles. Therefore, the lipid profile monitoring may aid in the diagnosis of hepatic infection severity and equally act as a good prognostic marker.

Fontes de óleo na dieta e sua importância no desempenho e na imunidade de frangos de corte

Pós-graduação em Ciência Animal - FMVA

Avaliação sorológica de Parainfluenzavirus Tipo 1, Salmonella spp., Mycoplasma spp. e Toxoplasma gondii em aves silvestres

Newcastle disease, salmonellosis and mycoplamosis are the most important infectious diseases in poultry. Toxoplamosis is a common disease in urban environment. The present study investigated serologic evidence of these diseases in captive and wildlife birds, with rapid plate agglutination test, haemagglutination inhibition test, and modified agglutination test. In a total of 117 blood serum samples, 20 showed the presence of Toxoplasma gondii, Mycoplasma gallisepticum, and Salmonella spp. antibodies. Amazona aestiva was the specie with the highest number of positive individuals (13/20). We also verified the first detection of T. gondii antibodies in birds of prey from Mivalgo chimachima and Rupornis magnirostris species.

Fontes de óleo na dieta e sua influência no desempenho e na imunidade de frangos de corte

A composição de ácidos graxos da dieta pode influenciar o desempenho produtivo e o sistema imune de frangos de corte. O objetivo deste estudo foi avaliar o efeito do consumo de óleos ricos em ácidos graxos poli-insaturados ômega-6 (PUFAs n-6) e ômega-3 (PUFAs n-3) sobre o desempenho e a resposta imunológica de frangos de corte frente a um desafio antigênico. Foram comparadas dietas formuladas com 7% de óleo de soja (OS), linhaça (OL) ou sardinha (OP), fornecidas a 240 frangos da linhagem Cobb, divididos em 24 grupos de 10 aves cada, num arranjo experimental 3x2 (3 tipos de óleo e aves vacinadas ou não vacinadas) e 4 repetições. O óleo de soja é rico em ácido linoleico, um PUFA n-6, o óleo de linhaça é fonte de ácido alfa-linolênico, um PUFA n-3, e o óleo de sardinha, de outros PUFAs n-3, como os ácidos eicosapentaenoico e docosahexaenoico. O consumo de ração, o ganho de peso e a conversão alimentar foram avaliados aos 21, 35 e 42 dias. Aos 7 e aos 21 dias de idade, metade das aves recebeu vacina contra doença de Newcastle. Quinze dias após a imunização, avaliou-se a produção de anticorpos pelo método de ELISA, expressa pela densidade óptica a 450 nm (D.O. 450nm). Apenas as aves alimentadas com ração contendo OS apresentaram maior imunidade humoral (P<0,05) após a vacinação. A resposta linfoproliferativa das aves, que expressa a imunidade celular, foi maior entre as aves vacinadas, em comparação às aves não vacinadas (P<0,05), independentemente do óleo utilizado. A fonte de óleo da ração ou a vacinação não influenciaram o ganho de peso das aves (P>0,05). Entre as aves que receberam dieta com OS, as aves vacinadas apresentaram pior conversão alimentar (P<0,05). Nos grupos que consumiram ração com OL ou OP, a vacinação não influenciou a conversão alimentar (P>0,05), considerando todo o período experimental. A utilização de óleo rico em PUFA n-6 na dieta de frangos de corte aumentou a resposta humoral, mas não influenciou a resposta celular frente a um desafio antigênico.