Bluetongue virus infection in pregnant ewes.

Inoculation of 53 ewes after 35, 45, 60, or 80 days of gestation with bluetongue virus serotypes 10, 11, 13, or 17, or with epizootic hemorrhagic disease virus serotypes 1 or 2, resulted in overt clinical disease in the 47 ewes inoculated with bluetongue virus but not in the 6 ewes inoculated with epizootic hemorrhagic disease virus. None of the lambs produced by these ewes had developmental defects or any evidence of persistence of viremia.

Cross-protective immunity between equine encephalomyelitis viruses in equids.

Eighteen equids were inoculated with eastern equine encephalomyelitis (EEE) and 18 equids with western equine encephalomyelitis (WEE) viruses to produce EEE virus- and WEE virus-immunized equids. Twelve surviving EEE virus-seropositive equids, 15 surviving WEE virus-seropositive equids, and 10 nonimmunized, seronegative equids (controls) were subsequently inoculated with an equine pathogenic (epizootic) strain of Venezuelan equine encephalomyelitis (VEE) virus to determine cross-protective immunity. Challenge infection produced 90% mortality in control (nonimmunized) equids, and 40% mortality in WEE virus-seropositive equids; all EEE virus-seropositive equids survived. Postchallenge exposure VEE viremia levels in EEE virus- or WEE virus-seropositive equids were lower than those in the 10 nonimmunized VEE virus-inoculated control equids. Plaque-neutralizing antibody responses to VEE virus in the EEE virus- and WEE virus-seropositive equids were similar in time of onset and titer to the antibody responses of nonimmunized equids. Neutralizing antibody to the third equine encephalomyelitis virus (either EEE virus or WEE virus) was detectable in 19 of 27 equids after inoculation with the challenge virus, VEE. Demonstration of cross-protective immunity between EEE or WEE virus and VEE virus in equids confirmed field observations made during the VEE epizootic in Texas in 1971.

Genotypic transitions among bluetongue viral isolates from domestic ruminants in Colorado during 1981-1984.

Two predominant electropherotypes of bluetongue virus (BTV) serotype 11 isolates from cattle during a 1981-1984 field study in eastern Colorado were characterized. The genomes of strains isolated from the first 2 years of the study had 1 predominant electropherotype (CO81), with the exception of 1 isolate that differed only in the migration of segment 3. A second electropherotype (CO83), with differences in the migration of 4 segments, coexisted in the same region during 1983 and 1984 with strains having the CO81 RNA profile. The genomes of CO81 and CO83 were also distinguishable from those of the US prototype of BTV 11. Analysis of the polypeptides of representative strains of each electropherotype by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the proteins were very similar. The occurrence of the CO81 electropherotype was apparently the result of multiple viral infections since the positions of 7 segments had faint second bands and single-banded variants were isolated after serial plaque purifications. In addition, protein 7 of 1 of the CO81 isolates and protein 7 of the single-banded variant differed as shown by reverse phase-high performance liquid chromatography of 35S-methionine-labeled tryptic peptides.

Serologic survey of selected pathogens in white-tailed and mule deer in western Nebraska.

Exposure of free-ranging white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus) in western Nebraska to selected livestock pathogens was determined by serology and attempted virus isolation. Antibodies to bluetongue virus, epizootic hemorrhagic disease virus, and bovine respiratory syncytial virus were present in both species of deer. No serologic reactors to Brucella or Anaplasma were found. Attempts to isolate bluetongue virus were negative.

Serosurvey for selected pathogens in hunter-killed pronghorns in western Nebraska.

Exposure of pronghorns (Antilocapra americana) in western Nebraska in 1983 to selected livestock pathogens was examined by serology and attempted virus isolation. Antibodies were present to the agents of bluetongue, epizootic hemorrhagic disease, and bovine respiratory syncytial virus. There were no serologic reactors to Brucella, and attempts to isolate the viruses of bluetongue and epizootic hemorrhagic disease were negative.

Two electropherotypes of bluetongue virus serotype 2 from naturally infected calves.

The first isolation of bluetongue virus (BTV) serotype 2 in the U.S.A. was in 1982 from a sentinel herd of cattle at Ona, Florida. Electrophoretic analysis of genome RNA revealed that all 16 serotype 2 isolates obtained from this focus of infection had one of two electropherotypes (designated Ona A and Ona B). All genome segments of Ona A and Ona B had detectable differences in electrophoretic mobility, with major differences noted for segments 1, 4, 5, 7, 8, 9 and 10. Electrophoretic comparison revealed that Ona A was indistinguishable from the African serotype 2 prototype isolated 23 years earlier. In 1983, Ona B, in the apparent absence of Ona A, was isolated from two additional cattle herds in Florida. These results suggest that Ona B may be a variant of Ona A that evolved as a result of mutation or reassortment with another BTV strain, and may be better adapted to the selective pressures found in southern Florida. Comparison of the electropherotypes of Ona B with two Florida isolates of serotype 13 and 17 and the prototypes of BTV 10, 11, 13 or 17 produced no evidence for reassortment between Ona A and any of these strains as the possible origin of Ona B.

Immunologic response of sheep to inactivated and virulent bluetongue virus.

Humoral and cellular immune responses of sheep to inactivated and virulent bluetongue virus (BTV) were studied. All sheep inoculated with inactivated BTV developed BTV group-specific nonneutralizing antibodies, as determined by agar-gel immunodiffusion. The development of group-specific, nonneutralizing, complement-fixing antibodies was variable and appeared to be dependent on immunizing BTV serotype, sheep breed, and individual variation. Virus-neutralizing antibodies were never detected after inoculation with the inactivated BTV. In vitro lymphocyte stimulation to BTV soluble antigen was observed with cells from all inoculated Warhill sheep and with cells from 1 of 3 inoculated Suffolk cross sheep. Complement-fixation titers did not appear to correlate with the degree of protection observed, ie, duration of postchallenge-exposure viremia. The development of postchallenge-exposure neutralizing antibody titer was inversely correlated to protective immunity. The development of a response to BTV antigen in the lymphocyte-stimulation test associated most closely with protection. Warhill sheep were afforded better protection, by inoculation with inactivated BTV, to live virus challenge exposure than were the Suffolk cross sheep. Approximately 30% of the inoculated Suffolk cross sheep responded to challenge exposure with intensified clinical signs of blue-tongue, compared with the challenge-exposed control sheep of the same breed.

Orbiviruses from Culicoides in Florida.

Between 1980 and 1983, 45,484 Culicoides spp. collected in Florida near cattle have been examined for orbiviruses by attempted isolation in cell cultures and intravenous (IV) inoculations of embryonated chicken eggs. Bluetongue virus (BTV) serotype 2 was isolated from a pool of Culicoides insignis trapped at Ona. This is the 1st recovery of BTV from this species representing the 2nd new world species of Culicoides from which BT viruses have been isolated. C. insignis is a neotropical form which extends from northern Florida through the Caribbean region and much of South America. If this species is proven capable of biological transmission of BTV, it could help explain the distribution of antibodies to BTV in areas lacking C. variipennis. Epizootic hemorrhagic disease virus (EHDV) serotype 2 was recovered from several pools of C. variipennis collected at 2 beef cattle operations in Florida. No viruses were isolated from the small number of C. arboricola, C. stellifer and C. niger examined.

Isolation and identification of bluetongue virus: a serotype new to the U.S.

There are 5 known serotypes of bluetongue (BT) virus (BTV) in the US: 2, 10, 11, 13 and 17. The most recently discovered US serotype, BTV 2, was isolated from blood collected from cattle at Ona, Florida in 1982. Isolations were made from the September through November bleedings and from 1 pool of Culicoides insignis Lutz collected in October. Seventeen viral isolates were obtained by injecting the samples into embryonated chicken eggs (ECE) followed by serial passage onto BHK-21 cells. A single isolate was made from inoculation of pooled bovine blood into sheep. The isolates were shown to be Orbivirus-like particles by electron microscopy and were identified as BTV by indirect immunofluorescence tests. All the isolates were found to be serotype 2, however genome analysis using polyacrylamide gel electrophoresis (PAGE) identified 2 distinct electropherotypes. There were major differences between the 2 electropherotypes at least in genome segments 1, 5, 7, 8 and 9. The electropherotypes of viral isolates from bovine blood collected in September, 5 of those from the October bleedings, the insect isolate, and that from pooled blood were identical by PAGE and were designated Ona A. This electropherotype was also found to be indistinguishable from the prototype of the African serotype 2. The 2nd electropherotype, Ona B, included 3 of the viral isolates from the October bleedings and the single isolate from November. Ona A appeared to be closely related to the African prototype while Ona B may have resulted as a variant of Ona A after being subjected to a different environment. Both electropherotypes, however, appear to be stable forms of BTV serotype 2.

Epidemiologic implications of the genetic variations of bluetongue virus.

The segmented RNA genome of bluetongue (BT) virus (BTV) provides for considerable diversity. This diversity has been seen in biochemical and biophysical analyses of the numerous strains of BTV as well as in clinical and immunologic responses of ruminant animals to BTV infection. This report describes the preliminary characterization of a unique BTV serotype 11 population recovered during 4 months in 1982 from 40 naturally infected animals including cattle, sheep and a goat. The strains of BTV serotype 11 were mild in their pathogenicity for the ruminants as no clinical signs of disease were seen. Infected cattle did not always develop detectable precipitating (P) or neutralizing (N) antibody during or after the infection. A better understanding of the epidemiology of BT may result from studies that include host and vector studies along with biochemical and biophysical characterization of the infecting BTV population.

Immune response of mice and sheep to bluetongue virus inactivated by gamma irradiation.

Gamma irradiation is being tested as a means of inactivating bluetongue virus (BTV) for use in vaccines. Exposure of BTV 17 to various levels of irradiation revealed that a dose of approximately 0.6 megarad was required to reduce the virus titer by one log10, or 90%. To test the immunogenicity of irradiated BTV, mouse brain passaged virus and concentrated cell culture passaged virus were inactivated by 6 megarads of gamma irradiation, and vaccines were prepared by emulsifying the virus preparations in equal volumes of a modified incomplete Freund's adjuvant. These vaccines stimulated the production of neutralizing antibodies in mice and sheep, a cell mediated immune response in mice, and a protective immune response in sheep. The results suggest that gamma irradiation would be an effective means of inactivating BTV for the preparation of vaccines.

Potency and efficacy of inactivated bluetongue virus vaccines.

Bluetongue virus (BTV) was chemically inactivated and was shown to elicit neutralizing antibodies in vaccinated rabbits and sheep. In sheep, the neutralizing antibody was shown to be protective by immunity challenge with virulent virus. These studies have shown the feasibility of safe and effective inactivated vaccines for bluetongue (BT) disease of sheep.

Isolation of bluetongue virus serotype 2 from cattle in Florida: serotype of bluetongue virus hitherto unrecognized in the Western Hemisphere.

A serotype of bluetongue virus (BTV) hitherto unrecognized in the Western Hemisphere was isolated from cattle in the United States. Clinical disease was not seen in the cattle which were part of a sentinel herd system in Florida designed for studying the epizootiology of BTV. The isolation of serotype 2 was the first recovery of a different serotype of BTV in the United States since 1967. At least 21 serotypes of BTV have been reported worldwide; the 5 serotypes of BTV now recognized in the United States are 2, 10, 11, 13, and 17.

Venezuelan equine encephalomyelitis virus: concentration, partial purification, inactivation and immunogenicity.

Venezuelan equine encephalomyelitis (VEE) TC-84 vaccinal virus, from 10-1. quantities of infected duck embryo fibroblast cell culture fluids, was isolated by combined continuous-flow centrifugation with isopycnic banding in sucrose. Most of the recovered infectivity and hemagglutinating activity were in a single band at a buoyant density (rho) of 1.2. About 90% of the total input protein (450-520 mg) was removed with the effluent, whereas most of the remaining 10% also banded at a rho of 1.2. Infectivity was inactivated with formalin at a final concentration of 0.05% at 37 degrees C for 24 hr. Formalin-inactivated virus retained its immunogenicity and induced VEE virus-specific antibody in horses and guinea pigs. The horses and those guinea pigs that received equivalent doses of vaccine survived after a challenge of their immunity with virulent VEE virus.

Recovery of dual serotypes of bluetongue virus from infected sheep and cattle.

Dual serotypes of bluetongue virus (BTV) were recovered from field-collected samples of sheep and cattle blood. Two sheep, each infected with both BTV serotypes 10 and 17, were found in a flock with bluetongue disease associated with these two serotypes. One sheep infected with BTV serotypes 11 and 17 was found in a second flock; it was the only viremic sheep detected and was clinically ill. Dual serotype infections of one beef and two dairy cattle were found in three geographically separate herds: mixtures recovered were of BTV serotypes 10 and 17 and serotypes 11 and 17. Clinical signs of illness were absent in the cattle in two herds, but severe conjunctivitis was seen in several cows in a third herd, including the cow with a dual serotype infection (BTV 11 and 17). Two of the cattle with dual infections had no serological evidence of BTV as determined by the agar gel precipitin test; serum was not available from the other cow with a dual serotype infection. The significance of dual infections and immune tolerance are discussed.

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.

Temporal appearance, geographic distribution, and species of origin of bluetongue virus serotypes in the United States.

Beginning in 1973, all available laboratory and field strains of bluetongue virus (BTV) from the United States were serotyped. Of the viral strains serotyped, 27 were collected from 1953 through 1972; 173 were collected from 1973 through 1977. Although 20 BTV serotypes have been found worldwide, only BTV serotypes 10, 11, 13, and 17 have been found in the United States. Since 1973, serotypes 11 and 17 have been the prevalent serotypes. Samples were collected over a 24-year period in the United States and represent a wide geographic area and diverse host sources (sheep, cattle, wild ruminants, and insect vectors). The collection was not a statistical sampling.

The nucleic acid and proteins of epizootic haemorrhagic disease virus.

Purified epizootic haemorrhagic disease virus (EHDV) was shown to contain 10 double-stranded RNA segments and a double-layered protein capsid with 4 major and 4 minor polypeptides. The virus differed from bluetongue virus (BTV), the orbivirus prototype, in that EHDV had an additional minor polypeptide component. This component, together with the major polypeptides P2 and P5, formed the outer capsid layer of the virus. The extra polypeptide apparently stabilizes this layer since, unlike BTV, EHDV was quite stable on CsCl gradients at both pH 7,0 and 8,0. EHD virions were found to have a density of 1,36 g/microliter, while particles without the outer capsid layer were isolated and had a density of 1,40 g/microliter. Two non-capsid polypeptides, P5A and P6A, were identified in addition to the 8 capsid polypeptides. Polypeptide P5A was synthesized in excess of all the others. There was little homology between the nucleic acids of EHDV and BTV with only 5-10% cross-hybridization. No hybrid double-stranded RNA segments were identified. We found by cross-immune precipitation that the major core polypeptides of the 2 viruses (P7 and P3) have common antigenic determinants.