Expression of largest RNA segment and synthesis of VP1 protein of bluetongue virus in insect cells by recombinant baculovirus: association of VP1 protein with RNA polymerase activity.

The bluetongue virus core particles have been shown to contain an RNA-directed RNA polymerase (1). To identify the protein responsible for the virion RNA polymerase activity, the complete 3.9 Kb DNA clone representing the largest RNA segment 1 (L1) of bluetongue virus (BTV-10) was placed under control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The derived recombinant virus was used to infect Spodoptera frugiperda cells. As demonstrated by stained polyacrylamide gel electrophoresis and by the use of bluetongue virus antibody, infected insect cells synthesized the largest protein of BTV-10 (VP1, 150 k Da). Antibody raised in rabbit to recombinant VP1 protein recognized bluetongue virus VP1 protein. The recombinant virus infected cell lysate had significantly inducible levels of RNA polymerase enzymatic activity as determined by a poly (U)-oligo (A) polymerase assay. The availability of enzymatically active bluetongue virus RNA polymerase provides a system in which we can precisely delineate the role this protein plays in the regulation of bluetongue replication.     

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.     

Bluetongue virus: Detection of antiviral immunoglobulin G by means of enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay was developed to detect antiviral IgG in the sere of sheep exposed to bluetongue virus. It was found that the enzyme-linked immunosorbent assay is a rapid and sensitive method for the detection of anti-bluetongue virus antibody. Bluetongue virus antigen prepared from extracts of virus infected BHK and Vero cells were equally effective. Antigen prepared from uninfected cells when used as coating antigen did not bind IgG from either exposed or unexposed animals. Sera raised against each of the four individual BTV serotypes, 10, 11, 13, and 17, found in the United States reacted equally with all four bluetongue virus serotype antigen preparations. Thus, any of the four serotypes can be used as the bluetongue virus antigen for the detection of anti-bluetongue virus antibody in the bluetongue virus-enzymelinked immunosorbent assay system. Antiviral IgG was readily detectable 6 days postinoculation. The anti-bluetongue virus antibody concentration continued to increase through the 35-day postinoculation test period. At 35 days postexposure, antibody titers of 1:1,600 to >1:3,200 were found. The rapid and sensitive nature of the bluetongue virus enzyme-linked immunosorbent assay indicates that this system should significantly extend serological studies on bluetongue virus.     

Bluetongue virus: (1) in pregnant white-tailed deer (2) a plaque reduction neutralization test

Six white-tailed deer (Odocoileus virginianus) were infected with bluetongue virus (BTs, vaccinal strain) approximately one-third of the way through their gestation period. One deer died of bluetongue 21 days after inoculation. Of the five surviving the infection, one had two mummified fetuses, and the others no fetuses upon euthanasia two weeks after term. Fetuses were present in two control deer and in the one which died of bluetongue. A plaque reduction neutralization test for bluetongue virus was developed and described for the first time and its sensitivity illustrated by high post inoculation titers which ranged from 1:3200 to greater than 1:16000.     

Experimental infection of Culicoides brevitarsis from south-east Queensland with three serotypes of bluetongue virus

Laboratory-reared C. brevitarsis (biting midges) were fed on sheep which had been experimentally infected with bluetongue serotype 1 (CSIRO 156), bluetongue serotype 20 (CSIRO 19) or bluetongue serotype 21 (CSIRO 154), or on cattle experimentally infected with bluetongue serotype 20 (CSIRO 19). Approximately 77 000 C. brevitarsis were exposed to sheep and 9000 to cattle. The average percentage feeding on sheep was 54% and on cattle 47%. In attempts to transmit virus by bite 3360 C. brevitarsis which had fed on viraemic sheep were held for 11-15 days before exposure to susceptible sheep. Although 11% of these insects fed, transmission of virus from sheep to sheep was not demonstrated. Estimated infection rates of C. brevitarsis for each serotype from sheep and serotype 20 from cattle were similar at 0.4% or lower. These low infection rates are one of the factors which make it unlikely that C. brevitarsis could be an efficient vector of bluetongue viruses in sheep in the field.     

Complete genome sequence of bluetongue virus serotype 9: implications for serotyping

The second complete genome of bluetongue virus serotype 9 (BTV-9) is presented in this report. The sequence analysis points to continued circulation in India of a mixed topotype virus apparently belonging to the BTV-9 serotype, and it raises questions about approaches for serotyping bluetongue viruses.     

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.     

Experimental bluetongue disease in white-tailed deer

Nine white-tailed deer and six sheep were experimentally exposed to the California BTV-8 strain of bluetongue virus. The infections were fatal for seven of the nine deer. An additional deer died from exposure to an isolate of bluetongue virus from bighorn sheep. Clinical signs and lesions of bluetongue in deer were described. The incubation period, signs and lesions of bluetongue and epizootic hemorrhagic disease of deer appear to be similar. Virus isolations were made from the blood and a variety of tissues of exposed deer and identified as bluetongue virus. Neutralizing antibodies were detected in all of the convalescent sera.     

Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana)

An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue virus (BTV) in field-collected pronghorn (Antilocapra americana) sera. To test the applicability of the ELISA to seroepizootiologic studies, pronghorn serum samples from three Wyoming counties (USA) were tested. Bluetongue virus ELISA results were compared to those of the bluetongue immunodiffusion assay. Discrepant serum samples were retested for reaction to either BTV or epizootic hemorrhagic disease virus. The pronghorn BTV ELISA gave rapid, quantitative, objective results and should facilitate testing large numbers of sera for BT diagnostic and seroepizootiologic studies.     

Serologic survey for selected microbial pathogens in bison from Kansas.

A serologic survey was conducted on an American bison (Bison bison) herd in Kansas for antibodies against Brucella spp., Leptospira interrogans serovar canicola, pomona, grippotyphosa, icterohaemorrhagiae, and hardjo, Anaplasma spp., bluetongue virus, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. There was an increase in prevalence of bluetongue antibodies from 38% in 1987 to 100% in 1989 in animals greater than or equal to 24-mo-old. Prevalences of antibodies against the other livestock pathogens were either negative or at levels associated with previous vaccination.     

蓝舌病病毒4型特异性竞争ELISA检测方法的建立

旨在建立蓝舌病病毒4型(BTV-4)特异性抗体ELISA检测方法,为蓝舌病的免疫学诊断提供新的技术。利用制备的两株抗4型BTV VP2蛋白的单克隆抗体4A-1G7和4B-1B6,建立BTV-4特异性竞争ELISA抗体检测方法。利用该方法同时对50份羊和牛BTV阴性血清进行检测,分别确定两种方法阻断率临界值为49%和40%。利用标准阳性血清检测的试验结果表明,该方法的敏感性、特异性和重复性符合OIE通用标准。同时,4A-1G7和4B-1B6两种竞争ELISA方法联合作用,可以检测感染4、18和20型BTV的血清。研究结果为建立以上各型BTV的检测方法提供了基础。     

A RNA virus in cells from Culicoides variipennis

A virus was detected in cells (designated CuVa) cultured from one laboratory colony of the biting midge, Culicoides variipennis. By electron microscopy (30 nm), nonenveloped, icosahedral virions arranged separately and in crystalline matrix arrays were seen in the cytoplasm but not in the nucleus of CuVa cells. Separation by 10% polyacrylamide gel electrophoresis revealed multiple bands of viral-induced double-stranded RNA. Inoculation of this virus onto different cell lines and intracranially into suckling mice revealed no detectable pathology. Immunoperoxidase staining using polyclonal antibody determined that the virus is infectious to toad cells, bovine endothelial cells, bovine kidney cells, mosquito cells, and cells (designated KC) initiated from another laboratory colony of C. variipennis. KC cells infected with this virus were coinfected with bluetongue virus with no decrease in bluetongue virus titer.     

Bluetongue and related viruses in New South Wales: isolations from, and serological tests on samples from sentinel cattle

Sentinel cattle at a number of localities in northern and central coastal New South Wales were sampled over the summer and autumn seasons of the years 1979, 1980 and 1981. A total of 118 orbiviruses were isolated; 99 were of the Palyam group, 15 were of the epizootic haemorrhagic disease (EHD) of deer group, and 4 of the bluetongue group. The Palyam group viruses were identified by serotype as 68 Bunyip Creek, 23 CSIRO Village, 7 D'Aguilar and one was not typed. The EHD viruses were identified as 13 type 5 and 2 type 6. All 4 bluetongue viruses were type 21. There was also convincing serological evidence that bluetongue type 1 infection occurred in 1980. Antibody to the bluetongue group, as demonstrated in a gel diffusion precipitin test, was often transient. It appeared to be mostly cross-reactive with, and induced by, other orbivirus infections, particularly those of the EHD group. Viruses of the Palyam group also seemed to be implicated in some circumstances. Where infections by viruses of the bluetongue group were demonstrated, the precipitating antibody responses to a bluetongue group antigen were not noticeably stronger than many which followed EHD virus infection. The results generally confirm previous conclusions, deduced from serological surveys, regarding the frequency of orbivirus infections, the presence of bluetongue viruses, and the transient nature of many bluetongue group antibody reactions.     

Studies on the Topography of Reovirus and Bluetongue Virus Capsid Proteins

Protein labeling experiments confirm the surface location of proteins 2 and 5 in bluetongue virus, and proteins sigma3 and mu2 in reovirus. Lambda 2 is the major surface component of the reovirus core, and proteins 1, 3, and 4 appear to be the outer components of the bluetongue virus subviral particle.     

The susceptibility of Culicoides imicola and other South African livestock-associated Culicoides species to infection with bluetongue virus serotype 8

In 2006, a strain of bluetongue virus serotype 8 (BTV-8) of sub-Saharan origin was responsible for the first outbreaks in recorded history of clinical bluetongue disease (BT) in northern Europe. In this study, we examine the oral susceptibility of Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) and other livestock-associated Culicoides species from southern Africa to infection with several strains of BTV-8. Following feeding using an artificial membrane-based method and incubation, virus was found in <1% of C. imicola individuals tested. Higher rates of susceptibility were found, however, for a variety of other South African species, including Culicoides (Avaritia) bolitinos Meiswinkel. Although these results do not preclude the role of C. imicola as a vector of BTV-8, its low susceptibility to BTV indicates that other less abundant Culicoides species may have the potential to play decisive roles in the epidemiology of this virus and should not be excluded from risk assessment studies.     

Inactivation of viral agents in bovine serum by gamma irradiation

Cell culture origin or suckling mouse brain origin viruses of Akabane disease, Aino, bovine ephemeral fever, swine vesicular disease, hog cholera, bluetongue, and minute virus of mice were each suspended in bovine serum. Aliquots (1 mL) were exposed to various doses of gamma radiation from a 60Co source while at -68 degrees C. Aliquots (100-mL) of serum from a steer experimentally infected with foot-and-mouth disease virus were similarly irradiated. The samples were assayed for infectivity in cell culture systems before and after irradiation, and the data points were analyzed by linear regression. The irradiation doses (in megarads) necessary to inactivate one log10 of viral infectivity (D10) was calculated for each virus. D10 is otherwise known as the slope of the regression line. The r2 value, a measure of association with 1.0 = perfect fit, was also calculated for each regression line. The values (D10, r2) for each virus were as follows: Akabane, 0.25, 0.998; Aino, 0.35, 0.997; bovine ephemeral fever, 0.29, 0.961; swine vesicular disease, 0.50, 0.969; foot-and-mouth disease, 0.53, 0.978; hog cholera, 0.55, 0.974; bluetongue, 0.83, 0.958; and minute virus of mice, 1.07, 0.935.     

Occurrence of Culicoides spp. (Diptera: Ceratopogonidae) in Tunisia, with emphasis on the bluetongue vector Culicoides imicola

Following the bluetongue (BT) outbreaks in Tunisia from 1999 to 2002, BTV (bluetongue virus) serotype 2 was isolated; however, no entomological investigation was performed. In the study presented here, we assessed the Culicoides species populations (particularly C. imicola) in proximity to the BT outbreaks locations, both as a retrospective analysis and to update the list of Culicoides species present in Tunisia. The insects were caught using light traps and the species identification was performed according to the standard entomological methods. This study reveaaled the presence of significant numbers of C. imicola in all the tested locations. In addition, we reported a new Culicoides species for the Tunisian fauna C. punctatus.     

Genome sequence of a reassortant strain of bluetongue virus serotype 23 from western India

The full genome sequence (19,177 bp) of an Indian strain (IND1988/02) of bluetongue virus (BTV) serotype 23 was determined. This virus was isolated from a sheep that had been killed during a severe bluetongue outbreak that occurred in Rahuri, Maharashtra State, western India, in 1988. Phylogenetic analyses of these data demonstrate that most of the genome segments from IND1988/02 belong to the major "eastern" BTV topotype. However, genome segment 5 belongs to the major "western" BTV topotype, demonstrating that IND1988/02 is a reassortant. This may help to explain the increased virulence that was seen during this outbreak in 1988. Genome segment 5 of IND1988/02 shows >99% sequence identity with some other BTV isolates from India (e.g., BTV-3 IND2003/08), providing further evidence of the existence and circulation of reassortant strains on the subcontinent.