Epizootic hemorrhagic disease virus in Montana: isolation and serologic survey

Epizootic hemorrhagic disease virus (EHDV) was isolated in Vero cell culture from the spleen and whole blood of a white-tailed deer (Odocoileus virginianus). A 10% spleen suspension caused acute hemorrhagic disease (HD) when inoculated into an experimental white-tailed deer and resulted in the recovery of EHDV from the blood of the experimental animal at 5 days after inoculation. The virus was identified as EHDV serotype 2 through indirect fluorescent antibody tests, electron microscopy, and reciprocal cross-neutralization tests. Approximately 73% (36/49) of the mule deer, 5% (2/42) of the white-tailed deer, and 79% (249/314) of the cattle samples tested from areas where HD had been reported were EHDV seropositive. Although none of the white-tailed deer was bluetongue virus seropositive, 29% of the mule deer and 3% of the cattle tested from "active" HD areas possessed bluetongue virus precipitating antibody.     

鹿流行性出血病毒分子生物学研究进展

鹿流行性出血病毒是一种在全世界野生及驯养的有蹄动物中广泛存在的重要病原体.该病毒属呼肠病毒科环状病毒属,有12个血清型,是一种有10个节段(L1-L3、M4-M6、S7-S10)的双链RNA病毒,编码10个蛋白(VP1-7和NS1、NS2、NS3/NS3A).VP7蛋白具有很强的抗原性且保守性最高.VP2与病毒型特异性有关,可诱导产生中和抗体.VP3为群特异性抗原,高度保守,具有亲水性保守区域.非结构蛋白NS1、NS2和NS3/NS3A及其编码序列均相当保守.该病毒的分子生物学诊断技术主要有PCR和核酸探针杂交技术.     

Isolation and characterization of epizootic hemorrhagic disease virus from sheep and cattle in Colorado

Epizootic hemorrhagic disease virus was isolated from cattle and sheep in northeastern Colorado during July and August 1984. The isolates were identified as serotype 2 by plaque-inhibition serotyping, genome electropherotyping, and protein analysis.     

鹿流行性出血病病毒单克隆抗体的制备与鉴定

为了更好的研究鹿流行性出血病病毒(EHDV)的生物学特性和建立EHDV免疫学检测方法,本研究用纯化的EHDV免疫Balb/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,经间接ELISA筛选,有限稀释法克隆,获得了2株(1A5和8H6)能够稳定分泌抗EHDV单抗的杂交瘤细胞株.其细胞培养上清效价分别为1∶256和1∶512,腹水效价分别为1∶1 024 000和1∶2 048 000;McAb 1A5和8H6 的相对亲和力分别为0.9 mg/L和0.7 mg/L.间接ELISA结果显示,2株单抗仅与EHDV反应,不与蓝舌病病毒、赤羽病病毒、水疱性口炎病毒、小反刍兽疫病毒和BHK细胞反应,表明2株单抗特异性良好.这2株单抗的获得为研究EHDV生物学特性和建立EHDV检测方法奠定了基础.     

Epizootic hemorrhagic disease virus infection of type I interferon receptor deficient mice

Type I interferon receptor deficient (IFNAR(-/-)) mice were infected with an Israeli isolate of serotype 7 of epizootic hemorrhagic disease virus (EHDV; Orbivirus, Reoviridae). Two out of two mice that received 5×10(5) 50% tissue culture infectious doses (TCID(50)) by intraperitoneal injection died or were euthanized in a moribund state on day 5 after infection. One mouse out of three that had been inoculated with 5×10(2) TCID(50) died on day 7 while the remaining mice did not show any clinical signs and survived until the end of the experiment. Spleens of all dead mice were highly positive in an EHDV real-time RT-PCR (quantification cycle values ≤15) and contained ≥10(5.8) TCID(50) of virus per ml of homogenate. The viral RNA content and virus titer in the spleens of the two surviving mice, on the other hand, were over 100-fold lower. Different from data reported for BTV, the outcome of EHDV infection of IFNAR(-/-) mice is dose-dependent and subclinical infections can occur.     

Diagnostic complementary DNA probes for genome segments 2 and 3 of epizootic hemorrhagic disease virus serotype 1

Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced. Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated. The polyadenylated RNA was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired. The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method. Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis. The cDNA clones of RNA segment 3 of EHDV-1 cross hybridized with the corresponding segment of EHDV serotype 2 by results of cDNA/RNA blot hybridization, but not with RNA of bluetongue virus serotypes isolated in the United States. After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.     

No evidence for involvement of sheep in the epidemiology of cattle virulent epizootic hemorrhagic disease virus

Epizootic hemorrhagic disease virus (EHDV) is an Orbivirus. While not previously considered as an important disease in cattle, several EHDV serotypes (EHDV-6 and 7) have recently been implicated in disease outbreaks. The involvement of sheep in the epidemiology of EHDV is still not understood. In this study we compared the prevalence of antibodies to EHDV and bluetongue virus (BTV) in sheep to their prevalence in cattle after an outbreak of EHDV that occurred in Israel during 2006. Sixty-six sheep and lambs scattered in seven herds were compared to 114 cows and calves scattered in 13 dairy cattle herds, matched to the sheep herds by location. While antibody prevalence to EHDV was high in cattle (35.2% within the outbreak zone) no evidence of exposure to EHDV was found in sheep (p<0.0001). Antibodies to BTV were apparent in both cattle and sheep though in the former it was significantly higher (63.2%, 16.7% respectively, p<0.0001), suggesting higher exposure of cattle to biting Culicoides midges. Taken together, these results imply that sheep have a negligible role in the epidemiology of EHDV.     

Replication of bluetongue virus and epizootic hemorrhagic disease virus in pulmonary artery endothelial cells obtained from cattle,sheep, and deer

OBJECTIVE: To compare replication of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in pulmonary artery endothelial cells (ECs) obtained from juvenile cattle, sheep, white-tailed deer (WTD; Odocoileus virginianus), and black-tailed deer (BTD; O hemionus columbianus). SAMPLE POPULATION: Cultures of pulmonary artery ECs obtained from 3 cattle, 3 sheep, 3 WTD, and 1 BTD. PROCEDURE: Purified cultures of pulmonary artery ECs were established. Replication, incidence of infection, and cytopathic effects of prototype strains of BTV serotype 17 (BTV-17) and 2 serotypes of EHDV (EHDV-1), and (EHDV-2) were compared in replicate cultures of ECs from each of the 4 ruminant species by use of virus titration and flow cytometric analysis. RESULTS: All 3 viruses replicated in ECs from the 4 ruminant species; however, BTV-17 replicated more rapidly than did either serotype of EHDV. Each virus replicated to a high titer in all ECs, although titers of EHDV-1 were significantly lower in sheep ECs than in ECs of other species. Furthermore, all viruses caused extensive cytopathic effects and a high incidence of cellular infection; however, incidence of cellular infection and cytopathic effects were significantly lower in EHDV-1-infected sheep ECs and EHDV-2-infected BTD ECs. CONCLUSIONS AND CLINICAL RELEVANCE: There were only minor differences in replication, incidence of infection, and cytopathic effects for BTV-17, EHDV-1, or EHDV-2 in ECs of cattle, sheep, BTD, and WTD. It is not likely that differences in expression of disease in BTV- and EHDV-infected ruminants are attributable only to species-specific differences in the susceptibility of ECs to infection with the 2 orbiviruses.     

A serologic investigation of blue tongue virus (BTV) in cattle,sheep and gazella subgutturosa subgutturosa in southeastern turkey

Bluetongue virus (BTV) is a vector-borne disease of ruminants disseminated in the tropic and sub-tropic zone of the world. It is also an important problem in the Middle East. Three serotypes (4–9–16) of the virus have been isolated so far in Turkey. Gazella subgutturosa subgutturosa (Goitred Gazella) is native species of Anatolia. Due to risk of extinction, captured Gazelles have been taken under protection in a restricted area at Ceylanpinar state farm. In this study, the presence of Bluetongue virus (BTV) was investigated serologically in 82 Goitred Gazella, 684 sheep and 100 cattle. Seropositivity rates in Gazella, sheep and cattle were detected as 40.2%, 295% and 88%, respectively.     

Experimental bluetongue virus infection of sheep; effect of previous vaccination: clinical and immunologic studies

Clinical and immunologic responses of sheep to vaccination and subsequent bluetongue virus (BTV) challenge exposure were studied and compared with those of non-vaccinated sheep. Sheep were vaccinated with inactivated BTV administered with aluminum hydroxide and cimetidine or levamisole. After sheep were vaccinated, precipitating group-specific antibodies to BTV were detected, but serotype-specific neutralizing antibodies were not detected. Cellular immune responses (lymphocyte blastogenesis) to BTV were not detected. After virulent BTV challenge exposure, vaccinated and nonvaccinated sheep developed acute clinical disease of similar severity. Clinical signs included hyperemia and petechiae of oral mucosa and coronary bands of the feet, excess salivation, nasal discharge with crusting, ulceration of the muzzle, and edema of lips and intermandibular space. Marked increases in serum creatine kinase activity were associated with stiff gait, reluctance to move, and vomiting. Fever and leukopenia were detected in most of the challenge-exposed sheep. Viremia and neutralizing antibodies were detected in vaccinated and nonvaccinated sheep after challenge exposure. Bluetongue virus-specific reaginic antibodies were not detected in sera from any of the sheep when the passive cutaneous anaphylaxis test was used.     

Neutralizing antibody responses to bluetongue and epizootic hemorrhagic disease virus serotypes in beef cattle

Blood samples were obtained from sentinel beef cattle at monthly intervals, and the sera were tested for antibodies, using a bluetongue virus (BTV) immunodiffusion test (IDT) and virus-neutralization test (VNT), for 5 BTV serotypes (2, 10, 11, 13, and 17) and 2 epizootic hemorrhagic disease virus (EHDV) serotypes (1 and 2). The cattle tested were transported from Tennessee to Texas in 1984 and 1985. All cattle were seronegative by the BTV IDT at the initial bleeding in Texas in 1984 and 1985. In 1984, 16 of 40 (40%) cattle seroconverted as assessed by results of the BTV IDT. In the 16 seropositive cattle in 1984, neutralizing antibodies were detected to BTV serotypes 10 (n = 7), 11 (n = 3), and 17 (n = 11), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1984, no cattle seroconverted to BTV-2 or BTV-13. In 1985, 10 of 36 (27.8%) cattle seroconverted as assessed by results of the IDT. Of the 10 seropositive cattle in 1985, neutralizing antibodies were detected to BTV serotypes 10 (n = 10), 11 (n = 10), 13 (n = 7), and 17 (n = 5), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1985, no cattle seroconverted to BTV-2. Clinical diseases attributable to BTV or EHDV was not detected in these cattle in 1984 or 1985.     

Foot-and-mouth disease in British deer: transmission of virus to cattle, sheep and deer.

After exposure for two hours to cattle with foot-and-mouth disease, each of the five species of deer found in the British countryside became infected. Clinical disease was typical and severe in the roe and muntjac deer, with some animals dying, less severe in the sika deer and usually subclinical in the fallow and red deer. Each species transmitted disease to its own species and to cattle and sheep. The amounts of virus present in the blood, and in oesophageal/pharyngeal samples and excreted as an aerosol during the course of the infection in the deer were similar to those recorded for the sheep and cattle in the same experiment. The fallow and sika deer commonly carried virus in the pharynx beyond 28 days after exposure; some red deer also became carriers. In epidemics of foot-and-mouth disease in the UK, it is likely that deer would have such intimate contact with farm animals as occurred in this study. The natural behavior of free-living deer in the UK suggests that, although the five species are susceptible to foot-and-mouth disease, they are unlikely to be an important factor in the maintenance and transmission of the virus during an epidemic of foot-and-mouth disease in domestic livestock.     

The airborne dispersal of foot-and-mouth disease virus from vaccinated and recovered pigs, cattle and sheep after exposure to infection.

Foot-and-mouth disease virus was detected during two periods in the air of looseboxes which housed susceptible, vaccinated or recovered pigs, cattle or sheep exposed to infection. The first was 30 min to 22 h after exposure and occurred in all animals. The second was two to seven days after exposure and occurred with those susceptible and vaccinated animals which developed clinical lesions, and with vaccinated and recovered pigs and sheep, which did not develop clinical lesions. Vaccination of animals before exposure resulted in less or no virus being detected. The virus during the first period was attributed to virus trapped on the animal during exposure, and the virus during the second period to limited multiplication in the respiratory tract. Control of movement for two weeks after contact with infection is suggested as a means of preventing spread of foot-and-mouth disease in areas that contain vaccinated animals.     

Bluetongue disease in Swiss sheep breeds: clinical signs after experimental infection with bluetongue virus serotype 8

Clinical disease of bluetongue (BT) in sheep may differ depending on breed, age and immunity of infected sheep and may also vary between serotype and strain of BT virus (BTV). Since there are no data available on the susceptibility of Swiss sheep breeds for BT, we performed experimental infection of the 4 most common Swiss sheep breeds and the highly susceptible Poll Dorset sheep with the BTV serotype 8 (BTV-8) circulating in Northern Europe since 2006. Clinical signs were assessed regarding severity, localisation, progression and time point of their appearance. The results clearly show that the Swiss sheep breeds investigated were susceptible to BTV-8 infection. They developed moderate, BT-characteristic symptoms, which were similar to those observed in Poll Dorset sheep. Regardless of breed, the majority of infected animals showed fever, swelling of the head as well as erosions of the mouth and subcutaneous haemorrhages.     

Bluetongue virus serotype 26: infection kinetics and pathogenesis in Dorset Poll sheep

Bluetongue virus serotype 26 (BTV-26) has recently been isolated from sheep in Kuwait. The aim of this study was to assess the pathogenicity and infection kinetics of BTV-26 in Dorset Poll sheep. Six sheep were experimentally infected with BTV-26 and samples taken throughout the study were used to determine the kinetics of infection using a pan specific BTV real time RT-PCR assay and two group specific ELISAs. Five of the six sheep showed mild clinical signs characteristic of bluetongue including conjunctivitis, reddening of the mouth mucosal membranes, slight oedema of the face and nasal discharge. Viral RNA was detected in 5 of the 6 sheep by real time RT-PCR, however the levels of viral RNA detected in the samples were lower and of shorter duration than seen with other field strains of BTV. Virus was isolated from the blood of infected animals at the peak of viraemia at around 9 dpi. Antibodies against BTV were first detected by 7 dpi using the early detection BTV ELISA and a little later (7-14 dpi) using a BTV specific competitive ELISA. Four of the five remaining sheep developed neutralising antibodies to BTV-26, measured by a serum neutralisation test (SNT), with titres (log(10)) ranging from 1.40 to 2.08.