Growth of bluetongue and epizootic hemorrhagic disease of deer viruses in poikilothermic cell systems

Continuous cell lines from the ticks Dermacentor variabilis, D. parumapertus, D. nitens, Rhipicephalus sanguineus and R. appendiculatus, the mosquitoes Aedes albopictus and Culex quinquefasciatus and the African toad Xenopus laevis were tested for their ability to replicate bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses, and for their sensitivity as potential isolation systems. BT serotype 17 grew to peak titers of 10(4.5)-10(7.5) TCID50 ml-1 in all except one of the tick cell lines, EHD 2 virus attained titers similar to that of BT 17 in the mosquito and toads cells, but failed to replicate in tick cells. Only Aedes albopictus and Xenopus laevis cells were as sensitive to infection with low-passage BT 11 and EHD 2 viruses as control cultures of Vero and BHK cells. At 27 degrees C, persistent infection of Xenopus laevis cells occurred, producing low yields of BT 17 and EHD 2. When shifted to 32 degrees C, these cultures expressed virus in exponential increments. No cytopathic effect (CPE) was seen in any of the tick-virus systems, but infected mosquito and toad cells detached from the monolayer within 3-6 days after inoculation with either virus. In the toad cells, this CPE was presaged by the development of plaques within 48 h after infection. Potential applications of poikilotherm systems in orbivirus research are discussed.     

Plaque neutralization of bluetongue virus and epizootic hemorrhagic disease virus in BHK21 cells.

Plaque assay and plaque neutralization of blue-tongue virus and epizootic hemorrhagic disease virus were studied in baby hamster kidney (BHK21) cells grown under an overlay containing gum tragacanth. Tests were done in plastic panels, each with 24 wells, and variables were established for achieving reproducible results. Four serotypes of bluetongue virus were compared, and their antigenic differences were confirmed with this new plaque-neutralization test.     

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.     

云南省2株鹿流行性出血热病毒的分离鉴定

为了监测云南省反刍动物鹿流行性出血热病毒(epizootic hemorrhagic disease virus,EHDV)的感染情况,本试验在云南省师宗县设立了监控点,筛选出EHDV抗体阴性的10头牛和5只羊作为哨兵动物,每年的5～10月份每周采血1次,11月到次年4月份每月采血1次,通过酶联免疫吸附试验监测其抗体转阳情况,用转阳牛的红细胞接种BHK以分离病毒,用病毒RT-PCR和中和试验鉴定病毒。结果显示,从2头转阳牛的抗凝血中分离到2株EHDV,其TCID₅₀分别为10^-2.5/0.1 mL和10^-3.44/0.1 mL,血清型均为EHDV-5型。本试验在云南省分离到EHDV,明确了云南省反刍动物存在EHDV感染,为我国监控EHDV流行情况及疫病风险防范提供了重要借鉴意义。     

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.     

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.     

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.     

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.     

Survey for antibodies to viruses of bovine virus diarrhea, bluetongue, and epizootic hemorrhagic disease in hunter-killed mule deer in New Mexico

Sera from male mule deer (Odocoileus hemionus) collected in November 1977 in Otero County, New Mexico were tested fro antibodies to bovine virus diarrhea virus (BVDV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV). Neutralizing antibodies were detected in 26 of 76 (34%) sera tested for BVDV (titer greater than or equal to 1:16). Of 46 sera tested for antibodies to BTV and EHDV, 10 (22%) and 3 (7%), respectively, were positive. Three (7%) of 46 sera were suspect (titer < 1:20) for BTV, and 18 (38%) sera were suspect (titer < 1:20) for EHDV.     

Comparison of the epidemiology of epizootic haemorrhagic disease and bluetongue viruses in dairy cattle in Israel

An outbreak of epizootic haemorrhagic disease virus (EHDV) in cattle in Israel in 2006 enabled a comparison of the spatial distribution of epidemic exposure to EHDV with that of exposure to bluetongue virus (BTV), which is endemic in the country. The seroprevalence of both viruses was examined in 1650 serum samples collected from 139 farms representative of the spatial distribution of dairy cattle in Israel. A significant association between exposure to EHDV and BTV was demonstrated in both univariate and multivariate analyses. Recent exposure to BTV and EHDV (demonstrated by seroprevalence in calves) was clustered in different geographical locations, indicating that the two viruses had different patterns of spread, that of EHDV being influenced by winds and terrain barriers and that of BTV by herd immunity.     

Isolation and characterization of epizootic hemorrhagic disease virus from white-tailed deer (Odocoileus virginianus) in eastern Washington.

A virus was isolated from the spleen of a white-tailed deer (Odocoileus virginianus) that had died during an epizootic in Washington state in 1967. Inoculation of a 10% spleen suspension from the deer caused hemorrhagic disease in normal white-tailed deer. Studies were conducted on the biological, physicochemical, and serologic properties of the Washington isolate. An in vitro assay system, utilizing a cultured primary of white-tailed deer fetal cells from an entire fetus, was employed for isolation and propagation of the virus. Cytopathic effect was characterized by focal development of rounded and clumped cells. Propagation was unsuccessful in suckling mice, BHK-21, and Vero cell cultures. The virus was resistant to treatment with ether, sodium deoxycholate, trypsin, oxytetracycline hydrochloride, and was sensitive to chloroform. Virus yield was not affected when infected cultures were treated with 5-iodo-2'-deoxyuridine, but dactinomycin (actinomycin D) treatment of infected cultures reduced virus yield. The virus was inactivated when heated at 70 C for 5 minutes or when exposed to pH 5 for 18 hours at 4 C. The virus was completely excluded from the filtrate by a 0.10- micronm (APD) membrane filter. Staining of infected cells with acridine orange indicated the presence of double-standard nucleic acid in the cytoplasm. Serum-neutralization tests with antiserums against the homologous virus and the New Jersey and Alberta strains of epizootic hemorrhagic disease virus resulted in neutralization of the Washington isolate. The Washington virus was not neutralized by bluetongue virus antiserum. Cells infected with the Washington isolate exhibited intracytoplasmic fluorescence by the indirect fluorescent antibody method with New Jersey and Alberta epizootic hemorrhagic disease antiserums but not with bluetongue antiserum.     

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.     

Analysis of genetic variation of epizootic hemorrhagic disease virus and bluetongue virus field isolates by coelectrophoresis of their double-stranded RNA.

Thirty-two bovine field isolates of bluetongue virus (BTV), 6 field isolates of epizootic hemorrhagic disease virus (EHDV) from deer, 4 BTV prototype serotypes (10, 11, 13, and 17), and 2 EHDV prototype serotypes (1 and 2) were coelectrophoresed, using polyacrylamide gels. Field isolates were obtained from various regions of the United States. Analysis of polyacrylamide gels and scattered plots generated for comparison of migration patterns for different isolates within each serotype of BTV revealed wide variation among the individual segments. The BTV serotypes 10 and 11 had more variation, compared with BTV serotypes 13 and 17, especially for migration of genome segment 5. A definitive correlation was not seen between the double-stranded RNA migration profiles on polyacrylamide gel electrophoresis, geographic origin, herd of origin, or year of collection. One BTV field isolate contained more than 1 electropherotype, with 2 bands at the segment-7 position, and it was further characterized as BTV serotype 11. Segments 2 and 5 of EHDV isolates were more variable in their migration than were the other gene segments. Generally, migration profiles for EHDV double-stranded RNA were more variable, compared with those of BTV isolates. Although a correlation was found between migration profiles and serotype of 2 isolates of EHDV, a study of additional EHDV isolates is required before the diversity of electrophoretic patterns of EHDV can be determined.     

Antibodies against certain bluetongue and epizootic haemorrhagic disease viral serotypes in Indonesian ruminants.

The orbiviruses contain several important viruses of livestock including bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD) which share some group antigens. Preliminary screening of sera for antibodies to orbiviruses by the agar gel immunodiffusion (AGID) test has previously revealed widespread infections with the BT group in Indonesia. However serum neutralization (SN) tests give a more accurate estimate of exposure to each serotype in the BT and EHD groups, and in this study were applied to sera that had reacted previously in the AGID test. Five different serotypes of BT and one serotype of EHD virus were studied. Reactors to BT serotype 20 were the most prevalent, followed by EHD type 5 and BT types 21, 12, 1 and 17. Antibodies against BT serotype 20 were present in cattle, buffaloes, goats and sheep, but were most common in buffaloes. Buffaloes showed the highest exposure to the BT serotypes tested. Antibody to EHD type 5 occurred most frequently in cattle. Antibodies against all BT and EHD serotypes tested were found in buffaloes and cattle while goats had antibodies against BT types 20, 21 and EHD type 5 and sheep had antibodies only against BT type 20.