Culicoides: biological vectors of Akabane virus

Akabane virus replicated in Culicoides nubeculosus and Culicoides variipennis after intrathoracic inoculation and was maintained in both species of midge for at least 9 days post-infection. The virus also replicated to high concentration in C. variipennis after oral infection and was transmitted through a membrane by this species of midge 7-10 days after infection. The experiments described in this paper provided the first definitive evidence that Culicoides spp. are able to act as fully competent vectors of Akabane virus.     

赤羽病毒G1基因的真核表达及抗原性检测

通过RT-PCR获得赤羽病毒（AKAV）OBE-1株的Gl基因,克隆至pMD18-T载体。经测序鉴定正确后,将该片段作为目的基因亚克隆至Bac-to-Bac杆状病毒表达系统中的pFastBacHTA转移载体质粒中,利用Tn7转座子与穿梭我体Bacmid DNA同源重组,获得了重组杆状病毒DNA,用CELLFECTIN介导其转染Sf9昆虫细胞,获得重组杆状病毒BAC-G1P1。SDS-PAGE结果表明,感染BAC-GIP1后的Sf9昆虫细胞表达可溶形式的120ku目的蛋白,其大小与预期相符。Western blot和间接免疫荧光试验显示表达产物具有良好的免疫学活性。     

赤羽病病毒RT-PCR检测方法的建立

利用BHK 2 1细胞繁殖赤羽病病毒 (AKV)美国株 ,以 30 %蔗糖垫底超速离心浓缩病毒。根据病毒SRNA序列设计 3对引物 ,初步建立了检测AKV的RT PCR方法 ,850bp扩增产物的序列与已知序列同源性 99%以上。人工攻毒鼠检测特异灵敏。     

广西牛羊赤羽病和蓝舌病流行病学调查

为了解赤羽病病毒(AKAV)和蓝舌病病毒(BTV)2种虫媒病毒(Arbovirus)在广西的流行与分布情况,本研究对采集自2010年～2011年广西11市的706份牛、羊血清样品进行检测,共检测出AKAV抗体阳性样品382份,总阳性率为54.11％.其中山羊血清82份,阳性率为28.37％(82/289);牛血清300份,阳性率为71.94％(300/417).BTV抗体阳性样品279份,总阳性率为39.52％.其中山羊血清95份,阳性率为32.87％(95/289),牛血清样品184份,阳性率44.12％(184/417).同时检测出2种虫媒病毒抗体185份,占样品总数的26.20％.本研究结果表明2种虫媒病毒在广西广泛流行,并首次证明广西存在AKAV的感染.     

Experimental placental transfer of Akabane virus in the hamster

Transplacental infection of hamster fetuses was produced by inoculation of pregnant hamsters with 10(6.3) plaque-forming units (PFU) of Akabane virus by either the intraperitoneal or the subcutaneous route. Virus with titers as high as 10(7.5) PFU/g of tissue was detected first in the placenta and later in the fetus. Virus could also be readily isolated from blood, lung, spleen, and liver of both pregnant and nonpregnant hamsters, but it reached higher titers and persisted longer in the placenta and fetus. Young dying at birth had Akabane virus titers as high as 10(7.3) PFU/g of brain tissue. Litter size was reduced by inoculation of the pregnant hamster at gestational day 11 or earlier, and survival of the newborn to 1 week of age was decreased by inoculation at gestational day 9 or later.     

Akabane and bovine ephemeral fever virus infections.

Akabane and bovine ephemeral fever viruses are exotic to the American continent. Both viruses are spread by insect vectors, and each causes disease of varying severity in food-producing animals. However, there are few other similarities between the agents and the diseases that they cause. They do not share the same insect vectors, the mammalian host range is different, and the clinical manifestations of virus infection vary markedly. Akabane virus is a cause of severe congenital defects, but adult animals show no signs of infection. In contrast, bovine ephemeral fever virus causes a febrile illness affecting mainly mature animals. If introduced to North America, it is probable that there would be significant economic losses, at least until endemic virus transmission patterns were established. Subsequently, it is likely that there would be patterns of alternate disease outbreaks followed by interepidemic periods in which there is a minor clinical effect.     

Neutralising antibodies to Akabane virus in ruminants in Cyprus

Summary Neutralising antibodies to Akabane virus, a cause of arthrogryposis and hydranencephaly, were demonstrated in serum samples from 33 sheep, 3 goats and 1 bovine among 285 serum samples collected in south-eastern Cyprus from December 1970 onwards. Twenty-four of the 29 sheep having positive antibodies came from one farm in Liopetri. No positive sera came from animals born after 1969, no association with abortions or stillbirths was noted and no arthrogryposis or hydranencephaly was observed in Cypriot animals in 1969 or before. It is suggested that Akabane virus was carried to Cyprus from the eastern Mediterranean mainland by infected midges on the wind in 1969 and possibly also in 1968, but that no disease was observed since infection took place after 50 days of gestation when damage to the foetus was unlikely.

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Anticuerpos Neutralizantes Del Virus Akabane En Rumiantes En Chipre

Resumen Se encontraron anticuerpos neutralizantes del virus Akabane, la causa de artrogriposis e hidranencefália, en muestras de suero de 33 ovejas, 3 cabras y 1 bovino, entre 285 muestras en total colectadas en la región suroriental de Chipre. El muestreo empezó en Diciembre de 1970. Veinticuatro de las 29 ovejas positivas se sangraron en una granja de Liopetri.No se encontraron anticuerpos específicos en animales nacidos después de 1969; tampoco se encontró asociación alguna con abortos o daños fetales. No se observó artrogriposis o hidranencefália en animales chipriotas en 1969 o anterior a esa fecha. Esto sugiere, que el virus Akabane llegó a Chipre de la región oriental mediterránea, en insectos del ordenDiptera, en 1969 y posiblemente también en 1968. La enfermedad en esa época no se vió, posiblemente debido a que la infección ocurrió después de los 50 días de gestación, cuando el feto es poco susceptible al virus.

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Anticorps Neutralisants, Specifiques Du Virus Akabane, Chez Des Ruminants A Chypre

Résumé Des anticorps neutralisants, spécifiques du virus Akabane, agent de l'arthrogrypose et de l'hydrocéphalie, ont été mis en évidence dans des échantillons de sérum, provenant de 33 moutons, 3 chevres et 1 vache, sur 285 échantillons sérologiques prélevés dans la partie orientale de Chypre depuis décembre 1970.24 des 29 moutons positifs venaient d'une ferme de Liapetri. Aucun sérum ne provenait d'animaux nés en 1969 ou avant; de même aucun syndrome d'avortement ou de mortinatalité associé à des cas d'arthrogrypose ou d'hydrocéphalie n'avait été décelé chez les animaux de Chypre en 1969 on avant. On pense que le virus Akabane a pu être importé à Chypre depuis les rivages de la Méditerranée orientale par des moucherons infectés transportés par le vent en 1969 peut être aussi en 1968 mais qu'aucun cas n'avait été observé puisque l'infection s'était déclarée après 50 jours de gestation alors que l'atteinte du foetus était devenue peu probable.

     

赤羽病病毒核衣壳蛋白基因的表达纯化及活性鉴定

为获得赤羽病病毒（Akabane virus, AKAV）核衣壳蛋白重组抗原,经RT-PCR扩增了OBE-1株的S节段,将其插入到pMD 18-T载体,然后用带有酶切位点的引物从该重组载体亚克隆出表达核衣壳蛋白的N基因,用BamHⅠ和XhoⅠ双酶切后与同样处理的表达载体pET-28a（＋）进行连接,转化感受态细胞BL21（DE3）.将筛选出的阳性克隆进行测序、IPTG诱导表达和融合蛋白纯化及活性鉴定.结果表明,N基因含有1个全长702 bp的ORF,编码233个氨基酸,通过SDS-PAGE和Western-blotting分析,证实重组菌株可高效表达有免疫活性的分子质量约27 ku的可溶性融合蛋白,用薄层扫描仪分析,其最高表达量占可溶性菌体蛋白总量的58.5%.工程菌经超声波裂解高速离心后,上清液在ProBondTM Purification System（含Ni2＋）天然状态下纯化,可获得高纯度的融合蛋白.     

Genetic analysis of Akabane virus isolates from cattle in Korea

Bayesian Inference (BI) and Neighbor Joining (NJ) analyses of the phylogenetic relationships between the nucleotide sequences of the N gene of Akabane virus revealed an unclear topology among genogroups I–III, which was probably caused by genetic reassortment or recombination between these genogroups. In contrast, nucleotide and amino acid phylogenetic tree analyses of the M RNA segment agreed with the topologies obtained by using the BI and NJ methods. Therefore, distinct genogrouping of Akabane virus isolates should be performed using the M RNA segment. Four Korea isolates were classified into genogroup II together with Akabane virus strains isolated from all areas of Japan, including Okinawa Island. However, more nationwide isolates and more clinical data from Korean cattle farms will be required in the future to confirm the precise relationships between genotypes and pathogenicity.     

Prevalence of neutralising antibodies to Akabane virus in the Arabian peninsula

Serum-neutralising antibodies to Akabane virus were found in a wide range of domestic animals in all countries of the Arabian Peninsula but the virus does not seem to be endemic there. Sentinel herds in Oman and N. Yemen did not detect any Akabane activity between August 1984-November 1986 and May 1983-November 1984, respectively. However, there is strong evidence to suggest that Akabane virus incursions have recently taken place in Kuwait, Saudi Arabia and Bahrain as neutralising antibodies were detected in 1-year-old cattle bled during 1986 in each of these countries. The possibility of windborne infected vectors, from virus-endemic areas, initiating these incursions into the Arabian Peninsula is discussed.     

Elisa test for the serodiagnosis of Akabane virus infection in cattle

Summary A simple ELISA test has been developed for the detection of IgG and IgM antibodies to Akabane virus in bovine serum. The test is specific and its sensitivity higher than the serum neutralisation assay. Detection of IgM antibodies can serve as a rapid method of diagnosing primary infection with Akabane virus. The superiority of ELISA resides mainly in the rapidity of performance and that it can be performed with inactivated reagents at high dilutions of serum samples. Thus it might enable the surveillance of spread of infection in zones prone to be affected by insect-borne viral diseases.

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La Prueba Elisa Para El Serodiagnostico De La Infeccion En Bovinos Con El Virus De Akabane

Resumen Se desarrolló una prueba ELISA simple, para la detección en sueor bovino de anticuerpos IgG e IgM del virus de Okabane. La prueba es específica y la sensibilidad superior a aquella de la seroneutralización. La detección de anticuerpos IgM puede servir como un método diagnóstico rápido para detectar infecciones primarias causadas por el virus. La superioridad de la prueba ELISA reside principalmente, en la rapidez de ejecución de la misma y que ésta puede realizarse con reactivos inactivados, a altas diluciones de la muestra de suero. Esta prueba ELISA podría facilitar la vigilancia epidemioloógica de la extensión de la enfermedad, en zonas en donde las condiciones ecológicas facilitarían el establecimiento de infecciones transmitidas por insectos.

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Un Test Elisa Pour Le Serodiagnostic De L'Infection A Virus Akabane Chez Le Betail

Résumé Un test ELISA simple a été développé pour la détection dans le sérum bovin des anticorps IgG et IgM dirigés contre le virus Akabane. Le test est spécifique et sa sensibilité est supérieure à celle du test de séro-neutralisation. La détection des anticorps IgM peut servir de méthode rapide pour diagnostiquer une infection primaire par le virus Akabane. La supériorité de l'ELISA réside essentiellement dans la rapidité de son exécution et en ce qu'il peut être réalisé avec des réactifs inactivés, et a de hautes dilutions des échantillons de sérums. De la sorte, il permet la surveillance de la propagation de l'infection dans les zones aptes à être affectées par les maladies transmises par arthropodes.

     

Isolation of Akabane virus from the biting midge Culicoides oxystoma in Japan

Akabane virus was isolated from the biting midge, Culicoides oxystoma, collected in a cowshed in Kagoshima on Kyushu Island of Japan. This is the first report on the isolation of Akabane virus from biting midges of the genus Culicoides in Japan. Two calves kept as bait in the cowshed seroconverted to Akabane virus. These results strongly suggest that C. oxystoma may be a vector of Akabane virus.     

Demonstration of Akabane virus antigen using immunohistochemistry in naturally infected newborn calves

Eight newborn calves showing ataxia were necropsied and examined histologically. Six of seven cerebrospinal fluid samples collected from these animals had neutralizing antibody for Akabane virus (AKV). All examined calves had nonsuppurative encephalomyelitis, localized mainly in the midbrain and spinal cord. Corresponding to the encephalitic lesion, AKV antigen was demonstrated in neuroglial cells in the brain stem and neuronal cells in the ventral horn of the spinal cord. This is the first study to demonstrate AKV antigen by immunohistochemistry in naturally infected newborn calves.     

赤羽病病毒间接荧光抗体快速检测方法的初步研究

以兔抗赤羽病毒重组核蛋白血清作一抗,利用间接荧光抗体法在AKAV感染的BHK-21培养细胞和攻毒的乳鼠脑组织中特异性地检测到了AKAV抗原,为赤羽病的病原学诊断提供了简便的检测方法。     

Myopathy and encephalopathy in chick embryos experimentally infected with Akabane virus

Chick embryos infected with Akabane virus by the yolk sac route at 6 days of incubation developed polymyositis and encephalitis. At 3 to 7 days after inoculation, skeletal muscles had myotubule degeneration, clumping of muscle cell nuclei, and infiltration of heterophils; dysplasia and aplasia were evident at 9 to 15 days after inoculation. Changes in the cerebral neostriatum and optic lobes at 2 to 11 days after inoculation included necrosis of primordial nervous tissue, hemorrhages, and hyperplasia of the vascular endothelial cells. Cavities were in nervous tissue subsequent to encephalitis. Hydranencephaly and vascular wall thickening were found 13 and 15 days after inoculation. Embryos infected intravenously at 15 days incubation had foci of encephalitis 3 to 6 days after inoculation, including neuronal degeneration, neuroglial hyperplasia, vascular endothelial proliferation, and heterophil infiltration.     

Neutralising antibodies to Akabane virus in free-living wild animals in Africa

Summary A total of 2,895 sera collected between 1963 and 1983 from 41 different species of free-living wildlife in 11 African countries South of the Sahara was examined for serum neutralising antibodies to Akabane virus. Antibodies were demonstrated in 25 species including 16 species not previously reported to be susceptible to this virus. Results indicate that infection is widespread in Africa and has been present for a considerable time. The prevalence of antibodies suggests that some free-living species may act as reservoir hosts of infection in the wild.

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Anticuerpos Neutralizantes Contra El Virus De Akabane En Animales Salvajes En Africa

Resumen Se examinaron 2,895 sueros para detectar anticuerpos contra el virus de Akabane, los cuales habían sido colectados entre 1963 y 1983 en 41 especies diferentes de animales salvajes. El muestreo incluyó 11 países africanos al Sur del Sahara. Se demostraron anticuerpos en 25 especies, incluyendo 16 especies las cuales no habían sido reportadas como susceptibles al virus. Los resultados indican que la infección está ampliamente difundida en Africa y que ha estado presente por tiempo considerable. La prevalencia de anticuerpos sugiere, que algunas especies de animales salvajes posiblemente actúan como reservorios del virus.

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Anticorps Neutralisant Le Virus Akabane Chez La Faune Sauvage Libre En Afrique

Résumé On a recherché les anticorps sériques neutralisant le virus Akabane sur 2.895 sérums récoltés entre 1963 et 1983 à partir de 41 espèces de la faune sauvage libre dans 11 pays africains au sud du Sahara. On a retrouvé des anticorps dans 25 espèces, dont 16 n'avaient pas encore été signalées comme sensibles à ce virus. Les résultats indiquent que l'infection est répandue en Afrique et qu'elle y est présente depuis un temps considérable. La prévalence des anticorps suggère que quelques espèces sauvages peuvent être des hôtes de l'infection dans la nature.

     

Evaluating the protective efficacy of a trivalent vaccine containing Akabane virus,Aino virus and Chuzan virus against Schmallenberg virus infection

Schmallenberg virus (SBV), an arthropod borne pathogen, spread rapidly throughout the majority of Europe since 2011. It can cause a febrile disease, milk drop, diarrhea, and fetal malformation in ruminants. SBV, a member of the Simbu serogroup within the genus Orthobunyavirus, is closely related to Akabane virus (AKAV) and Aino virus (AINOV) among others. In the present study, 4 Holstein-Friesian calves were immunized twice four weeks apart with a multivalent, inactivated vaccine against AKAV and AINOV. Another 4 calves were kept as unvaccinated controls. All animals were clinically, serologically and virologically examined before and after challenge infection with SBV. AKAV- and AINOV-specific neutralizing antibodies were detected one week before challenge infection, while SBV-specific antibodies were detectable only thereafter. SBV genome was detected in all vaccinated animals and 3 out of 4 controls in serum samples taken after challenge infection. In conclusion, the investigated vaccine was not able to prevent an SBV-infection. Thus, vaccines for other related Simbu serogroup viruses can not substitute SBV-specific vaccines as an instrument for disease control.