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 conjuctivitis 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.     

Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21

Genetic reassortment studies of bluetongue virus (BTV) Types 20 and 21 have revealed a reassortant genotype that was not neutralized serotype-specifically. In reciprocal neutralization tests, BTV 20 and 21 were neutralized specifically by homologous antiserum. Similarly, reassortants that possessed both outer capsid proteins (i.e., VP2 and VP5) from the same parent virus reacted with that antiserum specifically. However, two reassortants, 16(9) and 19(1), with VP2 of BTV 20 and VP5 of BTV 21 had intermediate neutralization characteristics. These reassortants were neutralized to high titres by antiserum to BTV 20 and to lower, but significant titres by antiserum to BTV 21. In addition, antiserum to BTV 20 induced 10-16-fold higher titres in plaque reduction neutralization (PRN) tests with these two reassortants compared with BTV 20 itself. Evidence of the serological cross-reactivity of Reassortants 16(9) and 19(1) was also found with respect to reductions in plaque sizes observed in the PRN tests. The average plaque sizes of these reassortants were reduced to differing extents by antiserum to BTV 20 and 21, while those formed by the parent viruses were reduced in size by homologous antiserum only. Immunoblotting analysis of the structural proteins of BTV 20 and 21 demonstrated that VP2 alone was antigenically distinct, therefore confirming its role in determining serotype specificity in virus-neutralization tests. Electrophoretic analysis revealed considerable migrational differences between VP2 and VP5 of the parent viruses, suggesting that there was some divergence in their molecular weights, intrinsic charges or structural compositions. Taken together, the data suggest that the intermediate neutralization characteristics of the reassortants that contain VP2 and VP5 from different parent viruses are due to conformational alterations in their outer capsid structure which allow antibody recognition of common neutralizing epitopes that are not exposed on BTV 20 or BTV 21.     

Serological differentiation of five bluetongue virus serotypes in indirect ELISA.

The serological reactivity in indirect ELISA of five different bluetongue virus (BTV) serotypes (4, 10, 15, 16 & 20) was compared using polyclonal antisera raised against virus particles and an outer structural protein, VP2. Rabbit and sheep antisera against BTV-10 produced higher ELISA values with their homologous antigens than with heterologous serotypes. A hyperimmune rabbit serum specific for virus particles was able to distinguish heterologous serotypes from each other, but a sheep serum from an infected animal was not. An antiserum directed against VP2, the protein responsible for serotype specificity in neutralization tests, was not serotype-specific in ELISA and cross-reacted with other serotypes. The discriminatory ability of a BTV-4 antiserum was improved by cross-absorption with heterologous antigens. This greatly reduced the ELISA signals with heterologous serotypes and produced an antiserum that was effectively serotype-specific.     

Serogrouping of United States and some African serotypes of bluetongue virus using RT-PCR

The diagnostic potential of RT-PCR for detection of bluetongue virus (BTV) ribonucleic acid (RNA) sequence in cell culture and tissue samples from infected ruminants from United States, Sudan, South Africa and Senegal, was evaluated. The non structural protein 1 (NS1) gene of North American BTV serotype 11 was targeted for PCR amplification. The United States BTV serotypes 2, 10, 11, 13 and 17 and the Sudanese BTV serotypes 1, 2, 4 and 16 and BTV serotype 4 from South Africa and BTV serotype 2 from Senegal were studied. RNAs from all BTV field isolates used in this study, propagated in cell cultures, were detected by the described RT-PCR-based assay. The first specific 790bp BTV PCR products were amplified using a pair of outer primers (BTV1 and BTV2). Specificity of the PCR products was confirmed by a nested amplification of a 520bp PCR product using a pair of internal (nested) primers (BTV3 and BTV4). The BTV PCR products were visualized on ethidium bromide-stained agarose gels. Amplification products were not detected when the RT-PCR-based assay was applied to RNAs from closely related orbiviruses including, epizootic hemorrhagic disease virus (EHDV) prototypes serotypes 1, 2, 4; RNA from Sudanese isolate of palyam orbiviruses serogroup and total nucleic acid extracts from uninfected Vero cells. Application of the nested BTV RT-PCR to clinical samples resulted in amplification of BTV RNA from blood and serum samples from goats experimentally infected with BTV4 and from naturally infected sheep, goats, cattle and deer. The results of this study indicated that this RT-PCR assay could be applied for rapid detection of BTV, in cell culture and clinical samples from susceptible ruminants during an outbreak of the disease, in the United States and African.     

A comparison of an australian bluetongue virus isolate (CSIRO 19) with other bluetongue virus serotypes by cross-hybridization and cross-immune precipitation

No major differences in size were observed when both the double-stranded RNA and the polypeptides of the Australian bluetongue virus (BTV) isolate CSIRO 19 (BTV-20) were compared with those of other BTV serotypes such as BTV-10 and BTV-4. Minor capsid polypeptide P6 of both BTV-20 and BTV-4, which electrophoreses as a single band on continuous phosphate buffered gels, in separated into 2 distinct bands on discontinuous glycine-buffered gels. This was not the case with BTV-10. Cross-immune precipitation of BTV-20 with BTV-10, BTV-17, BTV-4 and BTV-3 indicated strong immunological cross-reaction of the group-specific antigen P7 of the different serotypes. There was also some cross-immune precipitation of the serotype-specific polypeptide P2 of BTV-20 and BTV-4. This result is in agreement with the observed cross neutralization of these 2 viruses. The main distinction between BTV-20 and the other BTV serotypes was observed in cross-hybridization experiments. The homology between the nucleic acid of BTV-20 and other BTV serotypes was less than 30%, whereas homology normally found between BTV serotypes is at least 70%. The hybridization products of the different BTV serotypes were analysed by electrophoresis and fluorography. Two main hybrid segments were observed in all heterologous hybridizations with BTV-20 as a compared with 7 hybrid segments in hybridizations between BTV-4 and BTV-10. In order to determine from which genome segment of BTV-20 these 2 hybrid segments were derived, the hybridizations were carried out with individually purified double-stranded RNA segments. These results indicate that the 2 segments of BTV-20 that show the largest homology to corresponding segments of a heterologous BTV serotype are No. 7 and 10.     

Nontransmission of bluetongue virus by embryos from bluetongue virus-infected sheep

Donor sheep were infected either by bites of bluetongue virus (BTV)-infected (serotype 11, "Texas Station strain") Culicoides variipennis or by inoculation with 100,000 median chicken embryo intravascular lethal doses of BTV (serotype 11) from a suspension made from infected C variipennis. Fourteen embryos from 4 BTV-infected ewes bred by rams not infected with BTV were transferred to 8 BTV-seronegative recipient ewes, and 35 embryos and 4 unfertilized eggs from 14 BTV-infected ewes bred by BTV-infected rams were transferred to 19 BTV-seronegative recipient ewes. Eleven pregnancies and 12 lambs resulted. None of the recipients or lambs seroconverted, and BTV was not isolated from the pregnant recipient ewes or their lambs at slaughter 30 days after parturition.     

Identification of the serotype-specific and group-specific antigens of bluetongue virus

The bluetongue virus (BTV) core particle contains 2 major polypeptides, P3 and P7, and is surrounded by an outer capsid layer that is composed of the 2 major polypeptides, P2 and P5. Analysis of the immune precipitates from soluble 14C-labelled BTV polypeptides and hyper-immune rabbit and guinea-pig sera indicated that polypeptide P2 precipitates only with homologous BTV sera. This would indicate that P2 is the main determinant of serotype specificity. It was also found that in sheep infected with BTV the P2-precipitating antibodies in the serum correlate with the neutralizing antibody titres, whereas the appearance and subsequent decline of P7-precipitating antibodies correspond well with those of the complement fixing antibodies. This suggests that BTV group specificity, as measured by a complement fixation tests, is determined by the core protein P7. This result was supported by the observation that mouse ascitic fluid, which contains a high titre of BTV-specific complement fixing antibodies and a very low titre of neutralizing antibodies, contains almost exclusively antibodies that precipitate P7.     

Recombinant cDNA probe from bluetongue virus genome segment 10 for identification of bluetongue virus

Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.     

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.     

Interferon induction in bovine and feline monolayer cultures by four bluetongue virus serotypes.

The interferon inducing ability of bluetongue viruses was studied in bovine and feline monolayer cultures inoculated with each of four bluetongue virus serotypes. Interferon was assayed by a plaque reduction method in monolayer cultures with vesicular stomatitis virus as challenge virus. Interferon was produced by bovine turbinate, Georgia bovine kidney, and Crandell feline kidney monolayer cultures in response to bluetongue virus serotypes 10, 11, 13 and 17. The antiviral substances produced by the bluetongue virus infected cultures had properties of interferon.     

Peptide mapping of the group-specific antigens from the Australian bluetongue virus (BTV-20) and serotypes from southern Africa and North America

The Australian bluetongue virus (BTV-20) was compared with six serotypes isolated in southern Africa and North America by peptide mapping of the virus proteins with group antigen properties. The p7 group antigens from each of the seven serotypes analysed did not have identical primary structures and a comparison of shared and unique tryptic peptides has been used as a means of estimating virus relationships. Whereas serological studies have suggested that BTV-20 is closely related to BTV serotypes 4 and 17, comparative peptide mapping of p7 indicates a different set of relationships with viruses from both southern Africa and North America. In contrast with cross-immune precipitation results, peptide mapping of p3 suggest that this protein is not a group specific antigen.     

Clinical syndromes associated with the circulation of multiple serotypes of bluetongue virus in dairy cattle in Israel

From 2008 to 2011, seven distinct bluetongue virus (BTV) serotypes (BTV-2, BTV-4, BTV-5, BTV-8, BTV-15, BTV-16 and BTV-24) have been identified to be circulating in diseased sheep and cattle in Israel. This paper describes the array of clinical manifestations caused by BTV in cattle in Israel. Each set of clinical manifestations has been categorised as a syndrome and six distinct clinical syndromes have been observed in dairy cattle: 'footrot-like syndrome', 'sore nose syndrome', 'subcutaneous emphysema syndrome', 'red/rough udder syndrome', 'bluetongue/epizootic haemorrhagic disease systemic syndrome' and 'maladjustment syndrome'.     

Comparative studies on the growth of Australian bluetongue virus serotypes in continuous cell lines and embryonated chicken eggs

The replication of cell culture passaged Australian bluetongue virus (BTV) isolates, serotypes 20 (CSIRO19) and 1 (CSIRO156), and an untyped BTV (CSIRO154) was assessed in eight continuous cell lines (one derived from baby hamster kidney cells, BHK-21; three derived from monkey kidney cells, Vero, LLC-MK2 and CV-1P; a foetal ovine lung and a mouse fibroblast cell line, CSL503 and L929, respectively, a Super-Vero-Porcine stable cell line, SVP; and a mosquito cell line, Aedes albopictus cells) and in 11-day-old embryonated chicken eggs (ECE) at different multiplicities of infection. All three viruses replicated in the cell lines tested, maximum extracellular virus yields being attained from BHK-21 cells at high multiplicities of infection (approximately 10 PFU per cell). Also BHK-21 cells produced much higher yields of virus than the other cell lines tested when low multiplicities of infection were used (approximately 10(-4) PFU per cell). All BTV serotypes multiplied in Singh's Aedes albopictus cells with no cytopathogenic effects over the 4 day period tested. The viruses also replicated in 11-day-old ECE; however, the sensitivity of ECE for growth of the Australian serotypes was not as high as has been reported for BTV isolates in other countries. In all cell culture systems and in ECE, BTV1 and BTV20 replicated more efficiently than did CSIRO154 virus.     

Role of wild ruminants in the epidemiology of bluetongue virus serotypes 1, 4 and 8 in Spain

ABSTRACT: Although the importance of wild ruminants as potential reservoirs of bluetongue virus (BTV) has been suggested, the role played by these species in the epidemiology of BT in Europe is still unclear. We carried out a serologic and virologic survey to assess the role of wild ruminants in the transmission and maintenance of BTV in Andalusia (southern Spain) between 2006 and 2010.A total of 473 out of 1339 (35.3%) wild ruminants analyzed showed antibodies against BTV by both ELISA and serum neutralization test (SNT). The presence of neutralizing antibodies to BTV-1 and BTV-4 were detected in the four species analyzed (red deer, roe deer, fallow deer and mouflon), while seropositivity against BTV-8 was found in red deer, fallow deer and mouflon but not in roe deer. Statistically significant differences were found among species, ages and sampling regions. BTV RNA was detected in twenty-one out of 1013 wild ruminants (2.1%) tested. BTV-1 and BTV-4 RNA were confirmed in red deer and mouflon by specific rRT-PCR.BTV-1 and BTV-4 seropositive and RNA positive wild ruminants, including juveniles and sub-adults, were detected years after the last outbreak was reported in livestock. In addition, between the 2008/2009 and the 2010/2011 hunting seasons, the seroprevalence against BTV-1, BTV-4 and BTV-8 increased in the majority of provinces, and these serotypes were detected in many areas where BTV outbreaks were not reported in domestic ruminants. The results indicate that wild ruminants seem to be implicated in the dissemination and persistence of BTV in Spain.