First record of antibodies to the bluetongue virus in ewe (Ovis aries) in the state of Amazonas, Brazil / Primeiro registro de anticorpos para o vírus da língua azul em ovino (Ovis aries) no estado do Amazonas, Brasil

The objective of this work was to describe the first record of antibodies to the Bluetongue Virus (BTV) in ewe, in the state of Amazonas. The ewe, which was in twin pregnancy, gave birth on May 9, 2015, but a lamb died hours after delivery. Veterinary service was then requested by the owner, where emaciation, loss of wool, pyrexia, apathy, dyspnea, mucoid nasal secretion, facial, lingual and submandibular edema were observed. There was a visit by the Agricultural Defense Agency of the State of Amazonas to the property and blood samples were collected from the animal. The whole blood and serum were sent to the National Agricultural Laboratory, where it was possible to detect the presence of specific antibodies to BTV, through the Agar Gel Double Immunodiffusion. The ewe was submitted to a new blood collection, following the same protocols and the samples were sent to the Biological Institute of São Paulo, confirmed diagnosis. The animal in a serious clinical condition, could not resist and died in July 2015. The occurrence of an allochthonous case, in an area where vector insects occur, can trigger an endemic process in the Amazon region. With this, the epidemiological control of these occurrences is necessary, in order to avoid the spread of the disease in the country.

O objetivo do trabalho foi descrever o primeiro registro de anticorpos para o Vírus da Língua Azul (VLA) em ovino, no estado do Amazonas. A ovelha, que se encontrava em gestação gemelar, pariu no dia 9 de maio de 2015, porém um cordeiro faleceu horas após o parto. Foi então solicitado serviço veterinário por parte do proprietário, onde foi observado emaciação, perda de lã, pirexia, apatia, dispneia, secreção nasal mucoide, edema facial, lingual e submandibular. Houve visita da Agência de Defesa Agropecuária do Estado do Amazonas na propriedade e coletadas amostras de sangue do animal. O sangue total e soro foram enviados ao Laboratório Nacional Agropecuário, no qual foi possível detectar a presença de anticorpos específicos para VLA, através do teste de Imunodifusão Dupla em Gel de Ágar. A ovelha foi submetida a uma nova coleta de sangue, seguindo os mesmos protocolos e as amostras foram enviadas ao Instituto Biológico de São Paulo, confirmando diagnóstico. O animal em estado clínico grave, não resistiu e veio a óbito em julho de 2015. A ocorrência de um caso alóctone, em uma área de ocorrência de insetos vetores, pode desencadear um processo de endemia na região amazônica. Com isso, o controle epidemiológico destas ocorrências, se fazem necessários, afim de se evitar a disseminação da doença no país.

Advances in the release mechanisms of bluetongue virus / 生物工程学报

Bluetongue virus (BTV) causes Bluetongue (BT) of ruminants vectored by culicoides midges. It is also a classic model for studying the release mechanism of non-enveloped virus. This review begins with the infection and assembly of BTV, then summarizes the advances of different ways of releasing BTV. This includes BTV-induced autophagy and the release as extracellular vesicles via multivesicular bodies, BTV-induced apoptosis and the lytic release, as well as different pathways of release through budding via plasma membrane. The regulatory mechanisms of NS3 which is a key non-structural protein during the release of BTV are also discussed, providing a basis for further understanding the molecular mechanisms underpinning the infection, proliferation and release of BTV.

Bluetongue disease in sheep: a review / O vírus da língua azul em ovinos: uma revisão

The present review aims to show the main aspects related to bluetongue virus (BTV) infection in sheep. The bluetongue (BT) is a viral, infectious, and non-contagious disease caused by a virus (BTV) of the Orbivirus genus, transmited by a hematophagous vector of the Culicoides genus, to domestic and wild ruminants, mainly to sheep, the most susceptible species. It is caused by the association of endemic with climate conditions, with high temperatures and humidity. Economic loss is directly linked to death, abortion, weight loss, loss of milk, and meat production, and, indirectly, to the restriction on the export of animals and their by-products. The study concludes that the BTV is worldwidely spread, and probably persists due to the warm and humid climate that leads to the proliferation of Culicoides sp., being necessary to adopt measures that reduce the risk factors associated to the BTV infection.(AU)

A presente revisão objetivou apresentar os principais aspectos relacionados à infecção causada pelo vírus da língua azul em ovinos. A língua azul é uma doença viral, infecciosa e não contagiosa, causada por um vírus (BTV) do gênero Orbivírus, transmitida por meio de vetores hematófagos do gênero Culicoides a ruminantes domésticos e selvagens, principalmente aos ovinos, a espécie mais susceptível. A infecção ocorre de forma endêmica, associada a condições climáticas com elevada temperatura e umidade. As perdas econômicas estão ligadas diretamente à morte, ao abortamento, à perda de peso, à perda na produção de leite e carne, e, indiretamente, devido à restrição na exportação de animais e seus subprodutos. O estudo conclui que a língua azul está disseminada mundialmente e persiste, provavelmente, devido ao clima quente e úmido que propicia a proliferação de Culicoides sp., sendo necessário adotar medidas que diminuam os fatores de risco associados à infecção pelo vírus.(AU)

Ocorrência e fatores de risco da infecção pelo vírus da língua azul em bovinos no Estado de Pernambuco / Occurrence and risk factors of the Bluetongue Virus infeccion in cattle in Pernambuco state, Brazil

Objetivou-se com este estudo determinar os aspectos epidemiológicos da infecção pelo Vírus da Língua Azul (VLA) em bovinos leiteiros na microrregião de Garanhuns, Estado de Pernambuco, Brasil. Foram coletadas 384 amostras de soro de bovinos fêmeas em idade reprodutiva, procedentes de 20 propriedades dos 19 municípios que compõem a região. As amostras foram testadas com a prova de imunodifusão em gel de agarose (IDGA) para pesquisa de anticorpos anti-VLA. Observou-se ocorrência de 71,3% (274/384; IC 95% - 66,5% - 75,7%) de animais positivos. Em 100% das propriedades houve ao menos um animal soropositivo. Os fatores de risco identificados foram: presença de áreas alagadas (OR=11,8; p=0,001), não realizar controle de insetos (OR=2,1; p=0,033), rebanho aberto (OR=2,1; p=0,001) e utilização de inseminação artificial (OR=8,8; p=0,003). Este é o primeiro registro de detecção de anticorpos anti-VLA em bovinos no Estado de Pernambuco. Conclui-se que a infecção pelo VLA ocorre em bovinos na área estudada e sugere-se que medidas de controle baseadas no manejo higiênico-sanitário e biosseguridade sejam implantadas para evitar a propagação do vírus, tais como: eliminação de áreas alagadiças; controle de insetos; utilizar sêmen na inseminação artificial com atestado sanitário; realizar exames sorológicos ao adquirir animais.(AU)

The objective of this study was to determine epidemiological aspects of Bluetongue Virus (BTV) infection on dairy cattle in the Garanhuns microregion, Pernambuco state, Brazil. Three hundred eighty-four (384) serum samples of female bovines of reproductive age were collected from 20 farms of the 19 municipalities that make up the region. Samples were tested with the agarose gel immunodiffusion test (AGID) for anti-VLA antibody screening. There were 71.3% (274/384, 95% CI - 66.5% - 75.7%) positive animals. In 100% of the farms there was at least one seropositive animal. The risk factors identified were: presence of flooded areas (OR=11.8, p=0.001), absence of insect control (OR=2.1, p=0.033), open herd (OR=2.1; p=0.001) and use of artificial insemination (OR=8.8, p=0.003). This is the first record of detection of anti-BTV antibodies in cattle in Pernambuco state. It is concluded that BTV infection occurs in cattle in the studied area, and it is suggested that control measures based on hygienic-sanitary management and biosecurity are in place to prevent the spread of the virus, such as elimination of wetlands; Insect control; semen used in artificial insemination with health certificate; Serological tests when acquiring animals.(AU)

Clinical, pathological and epidemiological aspects of outbreaks of bluetongue disease in sheep in the central region of Rio Grande do Sul / Aspectos epidemiológicos, clínicos e patológicos de surtos de língua azul em ovinos na Região Central do Rio Grande do Sul

This article describes the clinical, pathological and epidemiological aspects of 17 outbreaks of bluetongue (BT) disease in sheep occurring between December 2014 and July 2015 in the central region of Rio Grande do Sul state (RS), southern Brazil. Affected farms were visited for clinical examination, necropsy, sample collection and epidemiological investigation. The outbreaks were seasonal and occurred during the summer and autumn. A total of 180 sheep (20.4%) out of 884 in 17 small herds were affected. All ages of Texel and mixed breed sheep were affected. However, lambs (younger than one year) had higher morbidity than adult sheep. The most frequent clinical signs were anorexia, lethargy, loss of body condition, facial swelling mainly involving the lips, and greenish seromucous or mucous nasal discharge. Pulmonary lesions characterized by edema were the most prevalent findings; however, erosive and ulcerative lesions in the upper gastrointestinal tract, as well as cardiac, skeletal muscle and esophageal striated muscle necrosis, and hemorrhage in the pulmonary artery were also frequent. The bluetongue virus (BTV) genome was detected by RT-PCR in blood and tissue samples (spleen and lungs) of 21 animals from 17 outbreaks. The virus involved in the outbreak 3 was subsequently isolated and shown to belong to serotype 17, for the first time reported in Brazil. In summary, our data support the BTV genotype 17 as the etiological agent of the outbreaks and indicate that the central region of RS is an area at risk for BT in sheep, a disease previously not recognized in the region.(AU)

O objetivo deste artigo é descrever os aspectos epidemiológicos, clínicos e anatomopatológicos de 17 surtos de língua azul (BT) em ovinos, que ocorreram entre dezembro de 2014 a julho de 2015, na Região Central do Rio Grande do Sul, Brasil. Para isso, foram realizadas visitas as propriedades nas quais ocorreram surtos da doença para investigação epidemiológica e clínica, realização de necropsias e coleta de amostras. Os surtos foram sazonais e ocorreram durante o verão e outono. Em 17 pequenos rebanhos, de um total de 884 ovinos, 180 adoeceram (20,4%). Ovinos de todas as faixas etárias, da raça Texel e sem raça definida, foram acometidos. Entretanto, ovinos com menos de um ano de idade tiveram taxa de morbidade maior do que ovinos com um ano ou mais. Os sinais clínicos mais frequentes caracterizaram-se por anorexia, apatia, acentuada perda de peso, edema facial, envolvendo principalmente os lábios, e secreção nasal seromucosa ou muco-esverdeada. Lesões pulmonares, caracterizadas por edema, foram as mais prevalentes. Porém, lesões erosivas e ulcerativas no trato gastrointestinal superior, assim como necrose da musculatura cardíaca e esquelética e do músculo estriado do esôfago e hemorragia na artéria pulmonar foram frequentes. O genoma do BTV foi detectado por RT-PCR em amostras de sangue e tecidos (baço e pulmão) de 21 animais de 17 surtos. O vírus envolvido no surto 3 foi subsequentemente isolado e pertence ao sorotipo 17, que pela primeira vez é descrito no Brasil. Em síntese, nossos dados permitem concluir que o BTV é o agente causador dos surtos e indicam que a Região Central do RS é uma área de risco para a ocorrência de BT em ovinos, uma doença, até então, não reconhecida nessa região.(AU)

Full genome sequencing of the bluetongue virus-1 isolate MKD20/08/Ind from goat in India

ABSTRACT This communication reports full genome sequencing of the bluetongue virus-1 (BTV-1) isolate MKD20/08/Ind from goat in northern India. The total BTV-1 genome size was found to be 19,190 bp. A comparison study between the Indian isolate and other global isolates revealed that it belongs to the 'Eastern' BTV topotype. The full genome sequence of BTV-1 will provide vital information on its geographical origin and it will also be proved useful for comparing the Indian isolate with global isolates from other host species.

Seasonal effects on the prevalence of bluetongue in small ruminants in west Azarbaijan, Iran

Bluetongue virus [BTV] is recognized to infect ruminants all around the world, and its prevalence mainly depends on the BTV situation, vectors distribution, weather patterns and susceptibility of the host. The purpose of this study was to draw a correlation between the prevalence of BTV antibodies and climate changes in Iranian Azerbaijan Province. Sera samples were collected from sheep, in a period of a year between 2008- 2009. The seroprevalance of Bluetongue antibodies were evaluated using an Enzyme-linked Immunosorbent Assay, considering monthly weather changes in the Province, recorded by the Iran Meteorological Organization. The infection rate was found to be 55.9% and there was a peak BTV prevalence of at 81% [p<0.001]. The Pearson correlation coefficient ranges were found to be between 0.36 and 0.005. The vector distribution is based on climatic changes and environmental conditions. It was concluded that the phenotypic expression of the BTV gene may be influenced by the weather

Anticorpos contra o vírus da língua azul em rebanhos ovinos da microrregião de Juazeiro, Bahia / Antibodies against the bluetongue virus in sheep flocks of Bahia state, Brazil

O objetivo desta pesquisa foi verificar a frequência de ovinos soropositivos para o vírus da língua azul na microrregião de Juazeiro, Bahia. O teste de imunodifusão em gel de ágar (IDGA) foi utilizado para pesquisar 469 amostras de soro oriundas de 58 rebanhos. Durante as colheitas, um questionário foi aplicado a cada criador a fim de se obter dados sobre o sistema de criação e correlacioná-los com a sorologia. Os resultados demonstraram que 0,43% (2/469) das amostras analisadas apresentaram anticorpos contra o agente. Esta região é caracterizada pelo clima semiárido e pela predominância do tipo de exploração extensiva, com presença de animais nativos, mestiços e sem raça definida para produção de carne e pele, com baixa produtividade e tecnificação.

The objective of this work was to verify the frequency of sheep with positive serology for Bluetongue virus in the micro-region of Juazeiro, Bahia State, Brazil. The agar gel immunodifusion test (AGID) was used to examine 469 serum samples of 58 herds. During collection, an epidemiological questionnaire was applied to each farmer. The results demonstrated that 0.43% (2/469) of the analyzed samples presented antibodies for the agent. This region is characterized by a semi-arid climate, and the predominant livestock management system is the extensive one, with a presence of native and crossbred animals, aiming at the production of meat and fleece, with low productivity and technification.

Sorologia para o vírus da língua azul em bovinos de corte, ovinos e veados campeiros no Pantanal sul-mato-grossense / Bluetongue virus serosurvey in beef cattle, sheep, and pampas deer from Brazilian Pantanal

This investigation was carried out in beef cattle (n=219), sheep (n=55), and pampas deer (Ozotoceros bezoarticus) (n=49) from Nhecolândia, sub region of Brazilian Pantanal in Mato Grosso do Sul State, Brazil. It was aimed to assess the seropositivity of these species to bluetongue virus (BTV) by agar gel immunodiffusion test. Seropositivity rates were 42.0% for cattle and 10.9% for sheep. The pampas deer showed to be all seronegative. In cattle, seropositivity to BTV significantly increased with age (P<0.001). These data, the favorable environmental conditions to development of BTV vectors, and the bovine reproductive disorders reported by farmers may indicate that BTV infection occurrs in herds of Brazilian Pantanal, and probably induces to economical losses.

Infección por el virus de la Lengua azul: activación de señales celulares que inducen apoptosis / Bluetongue virus infection: signaling pathway activated during apoptosis

El virus de la Lengua azul (VLA) es un ARN virus de doble cadena que induce apoptosis tanto en cultivos celulares como en tejidos blanco. Con el fin de dilucidar el mecanismo de apoptosis en la infección por el VLA, en el presente trabajo examinamos en detalle, por la técnica de Western blot, las señales celulares de caspasas, Bax, citocromo c, Smac/DIABLO y factor nuclear kappa B (NF-kB) que se activan en la infección viral. Hemos comprobado que luego de la infección in vitro con el VLA, se detectó la activación de la caspasa 8 y con ello el mecanismo extrínseco de la apoptosis. También detectamos por primera vez no sólo la activación de miembros de la familia Bcl-2 (Bax), sino también la liberación del citocromo c y la proteína Smac/DIABLO, confirmando que en la infección por el VLA está involucrado el mecanismo secuencial intrínseco de la apoptosis. Asimismo, demostramos que la infección por el VLA activa el NF-kB y que la apoptosis es sustancialmente reducida mediante la inhibición del mismo. La activación de las señales celulares tales como Bax, citocromo c, Smac/DIABLO y NF-kB presentados en este trabajo, esclarecen los mecanismos apoptóticos durante la infección por el VLA para una mayor comprensión del papel primario que juega la apoptosis en la patogénesis del virus.

Bluetongue (BTV) is a double-stranded RNA virus that induces apoptosis both in mammalian cell cultures and in target tissues. To elucidate the apoptosis pathways in BTV infection, we have examined in detail the apoptosis mechanism by examination of caspases, Bax, cytochrome c, Smac/DIABLO and NF-kB signalling pathways. In this report, after cell infection with BTV, the activation of caspase 8 was detected, proving the extrinsic receptor binding apoptotic pathway. Apoptosis followed a sequential pathway involving the detection of activated Bcl-2 family members. Furthermore, its translocation to the mitochondria, as well as the release of cytochrome c and Smac/Diablo confirmed that BTV apoptosis involves the sequential intrinsic pathway. In addition, we demonstrated that NF-kB was activated following BTV infection and cell treatment with an inhibitor peptide before BTV infection, prevented NF-kB activation and substantially reduced cellular apoptosis. Our accumulating data concerning the activation of Bax, cytochrome c, Smac/DIABLO and NF-kB clarify the mechanism of apoptosis during BTV infection, and confer a better understanding of the primary role of apoptosis in BTV pathogenesis.

Restriction enzyme analysis of VP7 gene of Indian isolates of bluetongue virus.

The genome segment 7 of two Indian isolates of bluetongue virus (BTV) from Avikanagar (BTV-1-western India) and Hyderabad (BTV-Untyped Hyderabad-southern India) was amplified by RT-PCR using two sets of VP7 specific primers. A sequence of 1137 bp comprising the complete coding sequence of the VP7 gene from Avikanagar isolate and a 1154 bp full-length sequence from BTV-UT Hyderabad isolate were amplified. Further, restriction enzyme digestion of these full-length amplicons, using EcoRI, PstI and TaqI revealed that genome segment 7 from both isolates were different from each other by absence of any EcoRI site in the BTV-UT Hyderabad isolate. There were also variations in the number and position of restriction sites for TaqI enzyme in these two isolates. Taql has two sites in the Avikanagar isolate whereas four sites in BTV-UT Hyderabad. The restriction digestion fragments obtained after PstI digestion were differentiated on the basis of their distinct sizes in both isolates. Comparison of their in silico restriction profiles with that of other isolates from different countries revealed that the two Indian isolates belonging to different parts of India had variations in their VP7 gene which was also distinguishable from at least some isolates from Australia and South Africa. Hence the restriction enzyme (RE) based analysis might be a useful tool in determining the genetic diversity in genome segment 7 and also for tracing their evolutionary relationships.

Isolation and primary identification of viruses in mosquitoes in the south of Xinjiang / 中华预防医学杂志

<p><b>OBJECTIVE</b>To isolate viruses from mosquitoes in the south of Xinjiang and identify these viruses primarily.</p><p><b>METHODS</b>A total of 13 491 mosquitoes were collected in the south of Xinjiang from Jul to Aug, 2005. These mosquitoes were divided into 130 groups and grinded respectively. The supernates were inoculated in C6/36 and Vero cells. Viruses isolated were detected, the genomic nucleic types by electrophoresis of viral genomes and the morphologies observed under electronmicroscope.</p><p><b>RESULTS</b>All 42 viruses were isolated, which caused CPEs on C6/36 but not on Vero cells. 27 viruses showed similar genomic profiles with 12 dsRNA segments. 1 virus displayed genomic profile with 10 dsRNA segments. 5 viruses took on similar genomic profiles with about 4 kbp DNA band. 9 viruses did not get any taxonomy information. Electromicroscopic pictures of these viruses revealed that above four types of viruses had distinguished morphologies indicating different virus species.</p><p><b>CONCLUSION</b>There should be several virus species in the mosquitoes in the south of Xinjiang. dsRNA virus with 12 genomic segments should play analysis a predominant role in the south of Xinjiang.</p>

Death mode of Hep-3B and A549 tumor cells induced by bluetongue virus strain HbC3 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the death mode of human hepatocellular carcinoma Hep-3B cells and human lung adenocarcinoma A549 cells induced by bluetongue virus strain HbC3 (BTV-HbC3) and the mechanism of its action.</p><p><b>METHODS</b>BTV-HbC3 was used to infect the tumor cells, and the cytopathic effects (CPE) was observed. TUNEL staining was used to detect the apoptosis of tumor cells induced by BTV-HbC3. The changes of endoplasmic reticulum and nuclei treated with BTV-HbC3 were further examined by laser scanning confocal microscopy. The activities of caspase-3/7, caspase-8 and caspase-9 were determined by fluorescence analysis.</p><p><b>RESULTS</b>Hep-3B cells were sensitive to BTV-HbC3. Lots of early apoptotic cells were found by TUNEL staining. The laser scanning confocal microscopic examination showed characteristics of apoptosis, such as pyknotic nuclei, margination of nuclear chromatin and vacuolization of endoplasmic reticulumin in Hep-3B cells exposed to BTV-HbC3. The activity of caspase-3/7 was increased, but the activity changes of caspase-8 and caspase-9 were not found. A549 cells were sensitive to BTV-HbC3 too. But no apoptotic cells were observed by TUNEL staining. The results of laser scanning confocal microscopy showed marked vacuolization of endoplasmic reticulum, but chromatin margination was not found after A549 cells was exposed to BTV-HbC3. The activity of caspase-3/7 and caspase-9 was increased, but the activity of caspase-8 was not changed.</p><p><b>CONCLUSION</b>BTV-HbC3 induces apoptosis of Hep-3B tumor cells mainly through endoplasmic reticulum signal transduction pathway, and the features of cell death in A549 cells could be described as paraptosis.</p>

Prevalência de anticorpos contra o vírus da língua azul em bovinos e ovinos do Sudoeste e Sudeste do Rio Grande do Sul / Bluetongue virus antibodies in cattle and sheep in Southwest and Southeast regions of Rio Grande do Sul, Brazil

It was studied bluetongue virus antibodies prevalence for sheep and cattle in Southwest and Southeast regions of Rio Grande do Sul State. A total of 2613 serum samples (1272 bovine and 1341 ovine) were tested by agar gel immunodiffusion. Eight bovine and two ovine samples were positive meaning a prevalence of 0.63 percent and 0.15 percent, respectively. These results show that most of animals in these regions are negative to bluetongue.

Restriction analysis of conserved and variable regions of VP2 gene of Indian isolates of bluetongue virus serotype 1.

Bluetongue virus (BTV) is a member of Orbivirus genus in family Reoviridae. The virus genome is composed of 10 double-stranded RNA segments. The RNA segment L2 encodes an outer capsid viral protein VP2, which is the main determinant of neutralization and serotype-specific immune response. BTV serotype 1 (BTV-1) specific novel primer pair was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1 from different geographical regions of the country. The 604 bp PCR product of VP2 gene of all six BTV-1 yielded two fragments of 273 and 331 bp when digested with Taq1 restriction enzyme. This indicated that there is only one TaqI site at 1513 bp (within 1240-1844 bp region) of VP2 gene of BTV-1 Indian isolates. The in silico restriction analysis revealed that in BTV-1 South African isolate (BTV-1SA) there is no TaqI site while in BTV-1 Australian isolates (BTV-1AUS), there are two TaqI sites (at 1513 and 1567 bp) within 1240-1844 bp region of VP2 gene. The earlier reported VP2 gene based primer pair for BTV-1 was used in the present study to amplify 2242-2933 bp region of six BTV-1 Indian isolates as three conserved regions have been reported within these 691 nucleotides. The digestion of 691 bp PCR products with XmnI yielded three fragments of 364, 173 and 154 bp with all the six Indian isolates of BTV-1 suggesting that there are two XmnI sites within 2242-2933 bp region of VP2 gene. A single XmnI site was observed in silico in BTV-1AUS and BTV-1SA isolates at different positions within this region. The in vitro and in silico restriction profile analyses of partial VP2 gene sequences using TaqI and XmnI restriction enzymes indicated a close relationship of Indian isolates of BTV-1 with BTV-1AUS isolates but not with BTV-1SA isolate.

Lengua Azul: Aislamiento y caracterización del virus e identificación de vectores en el noreste argentino / Bluetongue: isolation and characterization of the virus and identification of vectors in northeastern Argentina

To establish if BTV was circulating in Argentina, 94 bovines from the Santo TomÚ and Ituzaingó Departments of Corrientes Province were sampled every 30-60 days during 14 months. Red blood cells from those animals that showed seroconvertion with a c-ELISA were processed for virus isolation by inoculation in embryonated chicken eggs and cell cultures. Cells with CPE were positive by direct and indirect immunofluorescence with BTV specific reagents. These samples examined by electron microscopy showed virus particles with BTV morphological characteristics. Blood samples and tissue culture supernantants were positive by RT-PCR technique with primers corresponding to the segment 3 of the BTV genome. Haematophagous insects were captured in one farm using light traps and Culicoides insignis Lutz was the predominant species detected. This is the first isolation of BTV in Argentina from northeastern bovines without any disease symptom.

Lengua Azul. Actualización sobre el agente y la enfermedad / Blue tongue. Update on the agent and the disease

Bluetongue (BT) is a viral disease of domestic and wild ruminants. It is particularly damaging in sheep, where up to half of infected animals may die, showing inflammation and hemorrhages of the mucous membranes of the mouth, nose, and intestines. In cattle and goats, BT rarely causes disease, however it can affect the animal's reproductive ability, so that losses are not easily estimated. Bluetongue virus spreads from animal to animal by biting insects of the genus Culicoides; and this is the reason why the disease is more prevalent in geographic areas where climate conditions are favourable for their development. The disease was first recognized in South Africa in the late 1700's, but it was not until the early 1900's that it was described in detail, and at present, epizootiology and pathogenesis studies are still being carried on.

RT-PCR and its detection limit for cell culture grown bluetongue virus 1 using NS1 gene group specific primers.

RT-PCR was standardised for the detection of bluetongue viral RNA using highly expressed non structural protein 1 gene as the target gene with specific primers targeted to 274 bp of 5' end of NS1 gene. PCR product was consistently obtained in 30 PCR cycles. Further, detection limit of RT-PCR was estimated using serial 10 fold dilutions of BHK 21 cells grown BTV 1. The study suggested that RT-PCR can be used for detection of BTV in Indian conditions with the sensitivity limit of 10 infectious particles of the virus. The study suggested that this technique may be used as a tool for sensitive detection of BTV in carrier/reservoir animals, insect vectors and certification of animals and their germ- plasm for export and import purposes.

Comparative evaluation of sensitivity of RNA-polyacrylamide gel electrophoresis and dot immunobinding assay for detection of bluetongue virus in cell culture.

Bluetongue virus serotype 1 (Avikanagar isolate) was grown in BHK-21 cell line and titrated. The titre of the virus in BHK-21 cell line was 10(6) TCID50/ml. RNA-polyacrylamide gel electrophoresis (RNA-PAGE) and dot immunobinding assay (DIA) were performed on 10-fold serial dilutions of the sonicated cell culture material. The results indicated that the minimum limit of detection of the virus by RNA-PAGE and DIA was 10(5) TCID50/ml.

Detection of bluetongue virus genome segment 6 sequences by RT-PCR.

Reverse transcription-PCR (RT-PCR) technique was adopted to amplify a 101 basepair nucleotide sequence of bluetongue virus (BTV) genome segment 6. The specificity of the amplicon was determined by its approximate size in 3% agarose gel electrophoresis, digestion with restriction enzyme MspI, dot-blot hybridization and cycle sequencing. The technique was found to be suitable for detection of bluetongue virus in infected cell culture and clinical samples.