Bluetongue Virus Proteins Research Articles

Recombinant virus vaccine for bluetongue disease in sheep

Bluetongue virus proteins derived from baculovirus expression vectors have been administered in different combinations to sheep, a vertebrate host susceptible to bluetongue virus, and the neutralizing antibody responses were measured. Vaccinated sheep were subsequently challenged, and the indices of clinical reaction were calculated. The results indicated that the outer capsid protein VP2 alone in doses of greater than 50 micrograms per sheep elicited protection. A dose of ca. 50 micrograms of VP2 protected some but not all sheep. However, when used in combination with ca. 20 micrograms of the other outer capsid protein, VP5, 50-micrograms quantities of VP2 not only protected all the vaccinated sheep but also elicited a higher neutralizing-antibody response. The addition of viral core proteins VP1, VP3, VP6, and VP7, the nonstructural proteins NS1, NS2, and NS3, and the outer capsid proteins VP2 and VP5 did not enhance this neutralizing-antibody response.

Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV

The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7.

Antigenic topography of bluetongue virus 17

Neutralizing monoclonal antibodies (mAbs) have been produced and used to map the topographical relationship of the surface antigenic determinants of bluetongue virus (BTV) 17 that mediate neutralization. Eight monoclonal antibodies, at least five of which were directed to the major outer coat protein of BTV 17, P2, were studied in neutralization assays using variant BTV 17 and in competition binding experiments. Five different epitopes were identified that are involved in neutralization of viral infectivity. Three of the five epitopes are clearly associated with P2, while the location of the other two epitopes is not known. The potential association of these two epitopes with one or both outer coat proteins of BTV is discussed.

Expression of two related nonstructural proteins of bluetongue virus (BTV) type 10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization of the gene product in BTV-infected BHK cells

In vitro translation of bluetongue virus (BTV) double-stranded RNA in the rabbit reticulocyte lysate system has shown segment 10 (S10) to code for two related proteins, NS3 and NS3A. The presence of both products in vivo, however, has remained unconfirmed owing to the very low level of synthesis of the S10 gene product(s) in BTV-infected BHK cells. In the present work, a cDNA copy of BTV type 10 (BTV-10) S10 RNA was inserted into Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5' coding region of the AcNPV polyhedrin gene. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized two polypeptides, which were shown to represent NS3 and NS3A by Western blot (immunoblot) and peptide map analysis. Antibodies raised to the expressed NS3 by immunization of mice detected both NS3 and NS3A in BTV-10-infected BHK cells but not in purified BTV-10 virus particles. In contrast to in vitro translation of BTV S10 RNA in which NS3 and NS3A are synthesized in equimolar amounts, NS3 was the principle product both in the baculovirus expression system and in vivo in BTV-infected cells. The results indicate the caution which should be exercised when using the rabbit reticulocyte lysate system to predict the pattern of protein synthesis from a gene with alternative start codons. The expressed NS3 and NS3A proteins reacted strongly with sera from sheep infected with homologous and heterologous BTV serotypes, suggesting that the S10 gene products are highly conserved group-specific antigens.

Bluetongue Virus Infection of Bovine Monocytes

Cultures of adherent and non-adherent bovine blood mononuclear cells were infected with bluetongue virus (BTV) serotype 10. Production of BTV proteins in mononuclear cell cultures was detected by immune precipitation of viral proteins from [35S]methionine-labelled extracts of these cells, by immunofluorescence staining of cells using monoclonal antibodies (MAbs) to BTV proteins VP7 and NS2, and by flow cytometry with MAbs to VP2, VP7, NS1 and NS2. BTV-infected cells were most numerous in cultures of adherent mononuclear cells; infected cells were initially identified as monocytes on the basis of their morphology, and size and scatter characteristics as determined by analysis with a fluorescence-activated cell sorter (FACS). The majority of adherent mononuclear cells with these scatter characteristics were confirmed to be monocytes by FACS analysis with a MAb specific for bovine monocytes. Identification of BTV-infected adherent mononuclear cells as monocytes was further established by double immunofluorescent labelling, as infected adherent cells reacted with the MAb specific for bovine monocytes, and with another MAb specific for class II antigen. Infection of adherent mononuclear cells was also confirmed by transmission electron microscopy, as BTV virions and tubules were present in lysates of cultures of BTV-infected adherent mononuclear cells and within the cytoplasm of adherent cells. In contrast, BTV proteins were detected in few cells identified as lymphocytes on the basis of their scatter characteristics, and mean fluorescence of such cells was considerably less than that of BTV-infected monocytes. Viraemia persisted until 35 days after inoculation of a colostrum-deprived calf inoculated with BTV. Virus was isolated from blood mononuclear cells at 1 week after infection of the calf, but not thereafter. BTV infection of blood mononuclear cells was demonstrated until 9 days after inoculation by indirect immunofluorescence staining of mononuclear cells. In contrast, virus was consistently isolated from erythrocyte-enriched preparations throughout viraemia in titres comparable to those in whole blood. These results indicate that although bovine monocytes are readily infected in vitro with this strain of BTV serotype 10, infection of blood monocytes is unlikely to be responsible for the prolonged viraemia that consistently occurs in BTV-infected cattle.

Recombinant bluetongue virus protein production in insect cells, e.g. Spodoptera frugiperda; application in vaccine manufacture

Recombinant bluetongue virus protein production in insect cells, e.g. Spodoptera frugiperda; application in vaccine manufacture

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 the 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 changes 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.

Expression of targest 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.

Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus Serotype 10

Four lambs and 3 calves, seronegative to bluetongue virus (BTV), were inoculated intravenously with a highly plaque-purified strain of BTV Serotype 10. A single calf and lamb served as controls and were inoculated with uninfected cell culture lysate. All BTV-inoculated lambs exhibited mild clinical manifestations of bluetongue, whereas infected calves were asymptomatic. Viremia persisted in BTV-infected lambs for 35–42 days, and for 42–56 days in BTV-infected calves. Neutralizing antibodies were first detected in sera collected at Day 14 post-inoculation (PI) from 2 BTV-infected calves and all 4 infected lambs, and at Day 28 PI in the remaining calf. The appearance of neutralizing antibody in serum did not coincide with clearance of virus from blood; BTV and specific neutralizing antibody-coexisted in peripheral blood of infected lambs and calves for as long as 28 days. The sequential development, specificity and intensity of virus protein-specific humoral immune responses of lambs and calves were evaluated by immunoprecipitation of [ 35S]-labelled proteins in BTV-infected cell lysates by sera collected from inoculated animals at bi-weekly intervals PI. Sera from infected lambs and calves reacted most consistently with BTV structural proteins VP2 and VP7, and nonstructural protein NS2, and less consistently with structural protein VP5, and nonstructural protein NS1. Lambs developed humoral immune responses to individual BTV proteins more rapidly than calves, and one calf had especially weak virus protein-specific humural immune responses; viremia persisted longer in this calf than any other animal in the study. The clearance of virus from the peripheral blood of BTV-infected lambs and calves is not caused simply by the production of virus-specific neutralizing antibody, however the intensity of humoral immune responses to individual BTV proteins might influence the duration of viremia in different animals.

In vitro transcription and translation of bluetongue virus mRNA.

Fractionation of in vitro transcribed bluetongue virus (BTV) mRNA by agarose gel electrophoresis resulted in the separation of eight of the 10 species. The relative molar ratio of the mRNAs confirmed that mRNA 5 was transcribed more frequently than would be predicted from the size of the S5 genome segment, while mRNA 10 was transcribed less frequently. In vitro translation of unfractionated BTV mRNAs resulted in the synthesis of the seven known structural proteins (P1 to P7) and two known non-structural proteins (NS1 and NS2). Two additional non-structural proteins (NS3 and NS3A) with Mr of 28K and 25K respectively were identified. The protein coding assignments for the medium- and small-sized double-stranded RNA genome segments of BTV serotype 10 were found to correspond to those reported for BTV-1 and BTV-17. The peptide maps of NS1, NS2, NS3 and NS3A synthesized in vitro corresponded to those of their counterparts synthesized in infected cells. Protein NS3A appeared to be a truncated form of NS3, since its peptide map completely overlapped that of NS3. Proteins NS3 and NS3A were present in very small amounts in the soluble fraction of the cytoplasm of infected cells, and were synthesized in variable amounts in vitro, whereas the other nine viral proteins were synthesized in constant molar ratios. A difference in the relative molar ratios in which some of the BTV proteins were synthesized in vitro and in vivo was observed. In vivo, protein NS1 was translated in the largest amount but in vitro, NS2 was the most efficiently translated protein. Conversely, protein P6 was translated much more efficiently in vitro than in vivo.

In vitro phosphorylation and purification of a nonstructural protein of bluetongue virus with affinity for single-stranded RNA.

A phosphorylated, nonstructural protein of bluetongue virus, NS2, is synthesized throughout the replication cycle in comparatively large amounts. The protein was detected in both the soluble and particulate fraction of the cytoplasm of infected cells. The particulate NS2 could be solubilized in 0.5 M NaCl. It was found that NS2 in the particulate fraction and immunoprecipitates of NS2 from the soluble protein fraction could be phosphorylated in vitro. It is not known whether the kinase involved is of cellular or viral origin, but after purification of NS2 by affinity chromatography on poly(U)-Sepharose it could still by phosphorylated in vitro without the addition of exogenous protein kinase. The affinity of NS2 for nucleic acid was also investigated. The protein was found to bind to single-stranded RNA. In the presence of purified bluetongue virus mRNA, NS2 formed a complex with an estimated S value of about 22S.

Purification and properties of virus particles, infectious subviral particles, and cores of bluetongue virus serotypes 1 and 4

Effective purification methods have been developed for virus particles, infectious subviral particles (ISVP), and virus cores of bluetongue virus (BTV) serotypes 1 and 4. The purified particles were analysed by indirect ELISA or PAGE using either silver staining, or fluorography of [ 35S]methionine-labelled preparations. No significant contamination with host cell proteins, or with the majority of BTV nonstructural proteins was detectable in any of the particle preparations. In addition to the two major outer capsid and five core proteins previously described, the purified virus particles of both serotypes were consistently found to contain small amounts of BTV protein NS2, previously regarded as exclusively nonstructural. This protein could be removed from the particle surface by treatment with a combination of chymotrypsin and sodium N-lauroyl sarcosinate, which also resulted in the cleavage of the larger of the two major outer capsid components (protein VP2). Two of the cleavage products of VP2 and the whole of the other major outer capsid component (protein VP5) formed a modified outer capsid layer in the resultant ISVP. These subviral particles were as or more infectious than the intact virus particles but had lost haemagglutinating activity. The core-associated RNA polymerase remained inactive in ISVP.

Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep

A method to purify the neutralization specific antigen of bluetongue virus P2 in large amounts has been developed. The purified protein is free from virus-specified or cellular contaminants and its immunological specificity has been preserved. The purification is based on the observation that protein P2 can be dissociated from the virion by treatment with monovalent or divalent salts. The salt concentration required to solubilize the outer capsid proteins is pH dependent and in general decreases with a decrease in pH. P2 purified by extraction from polyacrylamide gels does not induce immune-precipitating or neutralizing antibodies. The response against P5, on the other hand, is much less conformational dependent and P5 purified from gels readily induces P5-precipitating antibodies in rabbits. These antibodies do not neutralize the virus. Purified P2, immunoabsorbed with anticore serum to remove trace amounts of P7, was injected into sheep. An initial dose of 50 μg of P2 was sufficient to induce P2-precipitating antibodies as well as neutralizing and hemagglutination-inhibiting antibodies. These sheep were fully protected against challenge with a virulent strain of the same BTV serotype. Lower doses of P2 still provided a significant level of protection even though no neutralizing antibodies could be detected.

Association of bluetongue virus with the cytoskeleton

Analysis of the distribution of [ 36S]methionine-labeled virus proteins following lysis of bluetongue virus (BTV)-infected cells with nonionic detergents showed that a major proportion of the virus-specific proteins was located in the insoluble nuclear-cytoskeletal fraction. Neither the proportion nor the species of virus protein associated with the cytoskeleton was altered following treatment of infected cells with microtubule or microfilament disrupting drugs (colchicine and cytochalasin B, respectively). Electron microscopic examination of BTV-infected cells revealed cytoplasmic virus-specified tubules, viral inclusion bodies (VIB), and progeny virus particles. Whole-mount transmission electron microscopy of nonionic detergent-extracted cells demonstrated the association of VIB, virus particles, and tubules with the cytoskeleton. The identity of virus particles was confirmed with an immunogold labeling technique using a neutralizing monoclonal antibody to BTV protein VP2. Several lines of evidence indicate that virus particles, VIB, and tubules bind to intermediate filaments in BTV-infected cells. (1) These structures remained cytoskeleton associated in infected cells treated with colchicine or cytochalasin B. (2) Linear arrays of filament-associated virus particles were formed around VIB following colchicine-induced juxtanuclear aggregation of intermediate filaments. (3) Virus particles were associated with filaments approximately 10 nm in diameter. (4) Filaments associated with virus particles reacted with an anti-vimentin monoclonal antibody in an immunogold labeling procedure.

Purification of the group-specific antigen of bluetongue virus by chromatofocusing

A method for the purification of the precipitating antigen of bluetongue virus (BTV) is described. The results obtained indicate that the precipitating antigen is identical with a core protein of BTV having a molecular weight of 39,000 (P7). So far all bluetongue virus serotypes have been shown to possess this protein. It is evident therefore that a group-specific reaction could be based on the presence of antibodies against this core protein. The purification of this protein by chromatofocusing proved relatively easy to perform and immunodiffusion tests revealed a group-specific reaction.