Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus.

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.     

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.     

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.     

Antigenic relationship of Ibaraki,bluetongue, and epizootic Hemorrhagic disease viruses

Ibaraki virus, which causes a bluetongue-like disease of cattle in Japan, was compared antigenically with the four serotypes of bluetongue virus (BTV) found in the U.S. and with the two serotypes of epizootic hemorrhagic disease virus (EHDV). No antigenic relationship was found between Ibaraki virus and BTV serotypes 10, 11, 13, and 17 in tests for group or serotype-specific antigens. However, Ibaraki virus and EHDV were related antigenically. The agar gel precipitin and indirect fluorescent antibody tests for group antigens showed two-way cross relationships between Ibaraki virus and EHDV serotypes 1 and 2. The more restrictive serotype-specific neutralization test revealed that antigenic relatedness was stronger between Ibaraki virus and the serotype 2 (Alberta strain) of EHDV than between Ibaraki virus and the serotype 1 (New Jersey strain) of EHDV.     

A survey of cattle for antibodies against bluetongue and epizootic hemorrhagic disease of deer viruses in British Columbia and southwestern Alberta in 1987.

In 1987 a serological survey of cattle for antibodies (Ab) to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) was undertaken in British Columbia and southwestern Alberta after infection with the viruses was diagnosed in wild and domestic ruminants in the Okanagan Valley. Of 4610 cattle tested, five had Ab only to BTV, 125 had antibodies only to EHDV and 16 had Ab to both viruses. The Ab were identified as specific for BTV type 11 (BT-11) or EHDV type 2 (EHDV-2). All but one of the seropositive cattle originated in the Okanagan Valley of British Columbia. The remaining one seropositive animal which had Ab to EHDV-2 was pastured with a bull purchased from the Okanagan Valley.     

Epizootic hemorrhagic disease virus serotype 7 in European cattle and sheep: Diagnostic considerations and effect of previous BTV exposure

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants that is closely related to bluetongue virus (BTV). The present study examines the outcome of an experimental EHDV-7 infection of Holstein cattle and East Frisian sheep. Apart from na?ve animals that had not been exposed to BTV, it included animals that had been experimentally infected with either BTV-6 or BTV-8 two months earlier. In addition, EHDV-infected cattle were subsequently challenged with BTV-8. Samples were tested with commercially available ELISA and real-time RT-PCR kits and a custom NS3-specific real-time RT-PCR assay. Virus isolation was attempted in Vero, C6/36 and KC cells (from Culicoides variipennis), embryonated chicken eggs and type I interferon receptor-deficient IFNAR(-/-) mice. EHDV-7 productively infected Holstein cattle, but caused no clinical signs. The inoculation of East Frisian sheep, on the other hand, apparently did not lead to a productive infection. The commercial diagnostic kits performed adequately. KC cells proved to be the most sensitive means of virus isolation, but viremia was shorter than 2 weeks in most animals. No interference between EHDV and BTV infection was observed; therefore the pre-existing immunity to some BTV serotypes in Europe is not expected to protect against a possible introduction of EHDV, in spite of the close relation between the viruses.     

Cloning and expression of the M5 RNA segment encoding outer capsid VP5 of epizootic hemorrhagic disease virus Japan serotype 2, Ibaraki virus

The complete nucleotide sequence of a cDNA clone representing the M5 RNA segment of epizootic hemorrhagic disease virus Japan serotype 2 (EHDV-2), Ibaraki virus, was determined. The M5 segment is 1641 base pairs long with the single open reading frame which predicts a polypeptide of 527 amino acids. The comparison of the amino acid sequence of the VP5 with those of EHDV-1, bluetongue virus serotype 10, and African horse sickness virus serotype 4 revealed that the protein shared 67%, 57% and 42% homologies, respectively. In addition, the VP5 protein was expressed in insect cells by recombinant baculovirus, which could be recognized by the mouse anti-EHDV-2 sera at a position of the expected 59 kDa on immunoblot analysis.     

云南边境地区流行性出血热病毒血清学监测及血清型鉴定

为了解近几年云南边境地区牛、羊流行性出血热病毒（EHDV）的感染和流行情况,本研究从2014年起连续3年在与老挝、越南接壤的江城县设置EHDV监测点,每年选择投放EHDV抗体阴性的10头牛和5只山羊作为哨兵动物进行跟踪监测。每年5~10月份对哨兵动物采血,每周1次,11、12月每月采集一次,进行EHDV抗体、抗原监测和病毒分离。针对致细胞病变的样品,采用EHDV群特异性S7基因片段引物进行RT-PCR方法检测,同时利用EHDV-1、-5、-6、-7、-10标准阳性血清对分离到的病毒进行中和试验鉴定。结果显示,2014－2016年江城县牛EHDV抗体阳性率分别为41.9%、58.6%和75.4%;3年期间共监测到15头EHDV抗体阳性黄牛,并从中分离到20个可致细胞病变样品,经RT-PCR确认为EHDV,遗传进化分析发现有11个毒株与1997和2003年日本分离的EHDV毒株亲缘关系较近,9个毒株与1977和1981年澳大利亚分离的EHDV毒株亲缘关系较近,5个毒株与2015年广西分离株的亲缘关系较近;3年期间在山羊体内未检测出抗体,未发现抗原阳性动物;经中和试验血清型鉴定,确定20株毒株包括EHDV-5、-6、-7、-10型4种血清型,感染时间均在5~9月之间。本研究发现,江城县长期存在多种血清型EHDV同时流行,2014－2016年EHDV抗体阳性率逐年增加,亟需加强对EHDV感染情况及活动规律的持续研究,提高流行性出血热的防控效率。     

Bluetongue and epizootic hemorrhagic disease in ruminants in Georgia: survey by serotest and virologic isolation

The frequencies of precipitating antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in domestic ruminants and white-tailed deer (WTD) in Georgia were 36% and 32%, respectively (n = 2,200). The frequencies of seropositivity to BTV and EHDV were high among cattle (47% and 42%, respectively [n = 1,068]) and less so in WTD (36% and 34% [n = 414]). The frequencies among sheep were 34% for BTV and 29% for EHDV (n = 286), whereas among goats, seropositivity was 8% for BTV and 7% for EHDV (n = 433). Serum samples from northeastern Georgia (1 of the 4 regions in the survey) had the highest frequency of precipitating antibodies for BTV (45%) and EHDV (38%). The lowest frequency was in southeastern Georgia, with 29% seropositivity for BTV and 24% seropositivity for EHDV. Of the 175 farms or herds in the serosurvey, 70% included animals that had BTV-precipitating antibodies, and 67% included animals which had EHDV-precipitating antibodies. Seventeen viral isolates were obtained from individual animals on 9 different farms. Fifteen of the isolates were BTV--8 from cattle, 4 from sheep, and 3 from WTD; 8 of them were serotype 11, and 7 were serotype 17. Viral isolates from each of 2 WTD were identified as EHDV serotype 1 and serotype 2. Of the total 17 isolates, 11 were from clinically healthy ruminants, and 6 were from animals with clinical signs of BT or EHD. Five of the viral isolates originated from northeastern Georgia, 7 from the northwestern region, and 5 from the southwestern region; none was obtained from specimens from the southeastern region.     

Molecular and genetic comparisons of two serotypes of epizootic hemorrhagic disease of deer virus

The virus-specific double-stranded genome RNA of 2 serotypes of epizootic hemorrhagic disease of deer virus (EHDV) was evaluated by use of coelectrophoresis in polyacrylamide and agarose gel systems. The molecular weights of virion RNA segments were 0.32 to 2.57 X 10(6) for EHDV-1 and 0.33 to 2.54 X 10(6) for EHDV-2. Seven of 10 double-stranded RNA segments of the 2 serotypes had different electrophoretic mobilities in the polyacrylamide-gel electrophoresis system. Although the individual RNA segments of each serotype contained unique RNA sequences determined on the basis of 2-dimensional polyacrylamide-gel electrophoresis analysis of oligonucleotides, the corresponding segments of the 2 serotypes were found to be comparable and at least 1 pair of RNA segment was almost identical. Virus-specific polypeptides for the 2 serotypes were compared by use of gel electrophoresis. Eleven polypeptides were detected for EHDV-1 and 10 for EHDV-2. Six corresponding polypeptides of these 2 serotypes had different electrophoretic mobilities, indicating that these corresponding polypeptides differ in their molecular weights. A genetic relationship was not determined between the 2 EHDV serogroups and the blue-tongue serogroup viruses, using oligonucleotides mapping.     

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.     

Development of PCR-based tests for the identification of North American isolates of epizootic haemorrhagic disease virus.

A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) and nucleotide sequencing) were developed for the detection of North American isolates of epizootic haemorrhagic disease virus (EHDV). PCR primers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1. Total nucleic acid was extracted from preparations of infected tissue culture and PCR was performed using a cDNA-PCR kit, according to the manufacturer's specifications. The PCR assay generated a 459 base pair product from North American isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) serotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products. Slight modifications allowed for the PCR detection of EHDV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR fragments were not amplified from uninfected deer blood. A number of restriction endonucleases and sequencing primers were evaluated for their utility in isolate identification experiments. Specifically, REA employing HincII and cycle sequencing with an internal primer (EHDV-1-pr3) proved most successful for identifying isolate-specific genome markers. The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species.     

Simultaneous screening of bovine sera for antibodies to bluetongue and epizootic hemorrhagic disease of deer viruses by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (I-ELISA) is described for simultaneous screening of bovine sera for detection of antibodies to bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses (V). Optimal dilutions of BTV and EHDV antigens were combined and allowed to absorb on to the wells of microtiter plates. Appropriately diluted (1:100) bovine sera were allowed to incubate and the bound antibodies were detected by a murine monoclonal antibody (MAb) to bovine immunoglobulin (H-Chain) conjugated with horseradish peroxidase. The performance of the combined (C) I-ELISA in detecting antibodies to BTV and EHDV in sequential serum samples from calves experimentally inoculated with BTV, serotype 10, EHDV, serotype 1 (New Jersey) or EHDV serotype 2 (Alberta) was evaluated. Comparable antibody profiles were demonstrable by the CI-ELISA and separate I-ELISAs using either BTV or EHDV antigens. The results suggest that the CI-ELISA offers many advantages over the standard agar gel immunodiffusion (AGID) test and has potential application as a rapid, sensitive, inter-group-specific and inexpensive test for simultaneous screening of bovine sera for antibodies to BTV and/or EHDV.     

A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses

In the present study, a multiplex RT-PCR-based assay for simultaneous detection and differentiation of North American serotypes of bluetongue (BT) virus (BTV) and epizootic hemorrhagic disease (EHD) virus (EHDV) in cell culture and clinical samples was developed. Two pairs of primers (B1 and B4) and (E1 and E4) were designed to hybridize to non-structural protein 1 (NS1) genomes of (BTV-11) and (EHDV-1), respectively. The multiplex PCR-based assay utilized a single tube-PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The BTV primers generated a 790 base pair (bp) specific PCR product from RNA samples of North American BTV serotypes 2, 10, 11, 13 and 17; whereas EHDV serotypes 1 and 2 or total nucleic acid extract from non-infected baby hamster kidney (BHK) cells failed to demonstrate the 790bp specific BTV PCR product. Likewise, the EHDV primers produced a 387bp specific PCR product from RNA samples of EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17 or from total nucleic acid extract of BHK cell controls.Two pairs of nested primers (B2 and B3) and (E2 and E3), internal to the annealing sites of primers (B1and B4) and primers (E1 and E4), produced a 520bp specific BTV and a 224bp specific EHDV PCR product from BTV and EHDV first amplification products, respectively. These nested amplifications increased the sensitivity of the PCR assay and confirmed the specificity of the first amplified EHDV or BTV PCR products. The described multiplex RT-PCR-based assay could be used to facilitate rapid detection and differentiation of North American BTV and EHDV serotypes and to provide a valuable tool to study the epidemiology of these orbivirus infections in susceptible animal populations.     

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.     

Experimental infection of calves with epizootic hemorrhagic disease virus.

OBJECTIVE: To determine whether experimental inoculation with a field strain of epizootic hemorrhagic disease virus serotype-2 (EHDV-2) suspected of causing clinical disease in naturally infected cattle would cause clinical disease in calves. ANIMALS: 8 calves. PROCEDURE: A strain of EHDV-2 isolated from a white-tailed deer that died of hemorrhagic disease was passaged twice in deer and used to inoculate 6 calves SC and ID; the other 2 calves were used as controls. Physical examinations, CBC, lymphocyte blastogenesis assays, and coagulation assays were performed; rectal temperature, interferon production, and serum neutralizing antibody responses were measured; and virus isolation was attempted every other day for 21 days after inoculation and then every fourth day for another 30 days. Calves were euthanatized on postinoculation day 51, and necropsy was performed. RESULTS: Calves inoculated with EHDV-2 became infected, as evidenced by development of viremia and seroconversion. However, the virus did not cause detectable clinical disease, clinicopathologic abnormalities, or gross lesions. Viremia was prolonged despite development of a serum neutralizing antibody response. A white-tailed deer inoculated with the same EHDV-2 strain developed clinical signs of epizootic hemorrhagic disease, demonstrating that the inoculum was virulent. CONCLUSION: Calves experimentally infected with EHDV-2 developed viremia and seroconverted but did not develop detectable clinical disease.     

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.     

Bluetongue and epizootic hemorrhagic disease virus isolation from vertebrate and invertebrate hosts at a common geographic site

One serotype of bluetongue virus (BTV) and two serotypes of epizootic hemorrhagic disease virus (EHDV) were isolated from vertebrate and invertebrate hosts on a farm in Colorado. The isolations were from blood samples collected a week apart from a dairy heifer with stomatitis and laminitis; EHDV serotypes 1 and 2 were isolated from the first blood sample, and BTV serotype 13 and EHDV serotype 1 were isolated from the second. Antibodies to EHDV and BTV were detected in the serum from this heifer. Both EHDV serotypes and BTV serotype 13 were isolated from pools of female biting gnats (Culicoides variipennis) that had not had a recent blood meal. The BTV insect isolate was biologically transmitted by female gnats from an infected donor sheep to a recipient host sheep. Culicoides variipennis was the predominant insect collected during three nights of light trap captures at the farm.