Localization of the non-structural protein NS3 in bluetongue virus-infected cells.

The localization of the blue tongue virus (BTV) non-structural proteins NS3 and NS3a has been identified using immunoelectron microscopical techniques. NS3 and NS3a have been observed in the plasma membrane of BTV- and recombinant vaccinia virus (expressing NS3)-infected cells. The NS3 protein was associated with areas of membrane perturbation. There was a good correlation between the presence of NS3 and NS3a and BTV release. The NS3 protein was associated with membrane fragments and the inability to detect it on the extracellular aspect of intact cells suggested that the protein was not exposed extracellularly. Electron microscopical and biochemical evidence suggested that fragments of plasma membrane containing NS3 and NS3a were released from infected cells. Collectively, the data indicate that NS3 and NS3a may be involved in the final stages of BTV morphogenesis, i.e. the release of BTV from infected cells.

A characterization of the nonstructural protein from which the virus-specified tubules in epizootic haemorrhagic disease virus-infected cells are composed.

The complete nucleotide sequence of segment 6 of epizootic haemorrhagic disease virus serotype 2 (Alberta) which encodes nonstructural protein NS1 was determined from a cDNA clone containing a full-length copy of the gene. The gene was found to be 1806 bp in length, constituted by one open reading frame of 1656 bp which is flanked by 5' and 3' noncoding regions of 32 and 118 bp, respectively. The conserved 5' and 3' terminal hexanucleotide sequences were identical to those of BTV-10. The 5' noncoding nucleotide sequences of cognate genome segments 4, 6 and 8 were found to be highly conserved in EHDV-2 and BTV-10. The 3' noncoding regions are less conserved but share common characteristics. The predicted EHDV-2 NS1 gene product, a 552 amino acid polypeptide, is predominantly hydrophobic and has a net charge of +5 at neutral pH. Comparison to its BTV-10 counterpart revealed a high degree of homology. Regions of high amino acid similarity were shown to correlate with hydrophobic domains on the proteins whilst regions of lower amino acid similarity corresponded with the hydrophilic domains. Thirteen conserved cysteine residues of which the majority occurred in hydrophobic regions with more than 80% amino acid similarity were identified.

Biophysical studies on the morphology of baculovirus-expressed bluetongue virus tubules.

Bluetongue virus tubules were purified from Spodoptera frugiperda cells infected with a recombinant baculovirus containing the NS1 gene from bluetongue virus serotype 10, and expressed under control of the Autographa californica nuclear polyhedrosis virus polyhedrin promoter. These tubules were subjected to a variety of chemical and physical treatments and the resulting effects on tubule morphology were examined by electron microscopy. A number of morphological similarities were noted between bluetongue virus tubules and cellular microtubules despite a lack of homology between the component proteins at the primary sequence level. A possible multistranded helical configuration is proposed for the tubule structure.

Bluetongue virus: surface exposure of VP7.

The exposed proteins of bluetongue virus serotype 17 were determined using surface labeling and reactivity with monoclonal antibodies. Iodination of amino groups predominantly labeled VP2; however, iodination of tyrosine residues labeled both VP2 and VP5, with VP7 labeled to a significantly lesser degree. To investigate the exposure of VP7 on the intact virion further, monoclonal antibodies that reacted with this protein were used. At least two antibodies, reacting with different epitopes on VP7, bound to intact virions, as determined by adsorption of infectious particles, electron microscopic observation of antibody-bound virus, and co-sedimentation of antibody and virus. Surface iodination of viral cores was used to show that VP7 and VP3 are major exposed proteins on these particles. We conclude that a major core protein, VP7, has at least two epitopes exposed on the virus surface.

Effects of proteolytic enzymes on the infectivity, haemagglutinating activity and protein composition of bluetongue virus type 20.

The effects on virus infectivity, haemagglutinating (HA) activity and polypeptide composition of bluetongue virus type 20 (BTV 20) were determined after digestion with the proteolytic enzymes, chymotrypsin, thermolysin and trypsin. Virus infectivity increased eight to 50-fold after exposure periods which reflected the activity of the proteases. Identical maximum increases in HA activity (i.e. 4096, 1024 and 128 HAU per 0.05 ml with sheep, bovine and human erythrocytes, respectively) occurred with each of the three proteases. Peak increases in virus infectivities and HA activities occurred after similar exposure periods. Outer capsid protein VP2 was the most sensitive virus protein to proteolytic digestion, being cleaved into a number of smaller polypeptides that remained attached to the virus particle. Digestion with chymotrypsin and thermolysin yielded four common cleavage products, designated P93, P76, P54 and P25 according to their estimated molecular weight, which suggested that they shared at least three cleavage sites. VP2 cleavage products resulting from digestion with trypsin differed somewhat from those of chymotrypsin and thermolysin, although the generation of polypeptides P93, P54 and P25.5 suggested the existence of common cleavage sites for the three proteases. Possible mechanisms whereby proteolytic cleavage of VP2 may enhance the infectivity and HA activity of BTV 20 are discussed.

Selective separation of virus proteins and double-stranded RNAs by SDS-KCl precipitation.

The total viral structural polypeptides and the double-stranded genomic RNAs of bluetongue virus can be selectively separated by a single SDS-KCl precipitation step. This simple, rapid and highly reproducible method enables greater than 95% recovery and purity of both viral proteins and dsRNAs within 30 min. The serotypic identity of the separated dsRNAs can be analyzed by SDS-PAGE electrophorogram immediately. After a single phenol/chloroform extraction, the dsRNA can also be used as hybridization probes, templates for molecular cloning and direct RNA sequencing. The SDS-KCl-precipitated viral proteins could be used readily for peptide mapping and as immunogens. Polyclonal and monoclonal antibodies raised against SDS-KCl-precipitated viral structural polypeptides were useful in Western immunoblots.

Characterization of a nonstructural phosphoprotein of two orbiviruses.

A phosphorylated nonstructural protein, NS2, was detected in bluetongue and African horsesickness virus (BTV and AHSV) infected-radiolabeled-cell lysates by electrophoresis on SDS-polyacrylamide gels (SDS-PAGE). The NS2 proteins of both viruses have similar migration on one-dimensional (1D) 10% SDS-PAGE. Examination of infected cell lysates on two-dimensional (2D) gels (isoelectric focusing followed by SDS-PAGE) separated two phosphorylated isoelectric forms of BTV NS2 and four phosphorylated forms of AHSV NS2. The isoelectric points of both species of BTV NS2 were acidic relative to all forms of AHSV NS2. Nonphosphorylated NS2 polypeptides were not detected by 2D gels. A nonphosphorylated host protein, which comigrated with NS2 on 1D gels, could be distinguished from viral proteins by isoelectric focusing on 2D gels. High performance liquid chromatography (HPLC) elution profiles of NS2 tryptic peptides from the two orbiviruses were compared. Three 32P-labeled tryptic peptides were generated from both AHSV and BTV NS2 proteins, which had been isolated and eluted from SDS-polyacrylamide gels. The elution profile from reverse phase HPLC was very similar for the tryptic phosphopeptides; in contrast, 35S-labeled tryptic peptides displayed considerable differences in elution profiles for the two NS2 proteins. Phosphoamino acid analysis revealed only phosphoserine in hydrolysates of BTV and AHSV NS2.

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.

Correlation of serotype specificity and protein structure of the five U.S. serotypes of bluetongue virus.

The relationship between serotype specificity and protein structure was studied by polyacrylamide gel electrophoresis, peptide mapping and radioimmune precipitation (RIP) of structural and non-structural proteins of the five U.S. serotypes of bluetongue virus (BTV). The surface proteins, VP2 and VP5, showed the most variation in size among the serotypes. Peptide mapping of the proteins showed that VP2 is unique for each of the U.S. serotypes. The nucleocapsid and non-structural proteins showed a high degree of conservation, whereas the other surface protein, VP5, showed intermediate conservation among the serotypes. Monospecific neutralizing antiserum produced in rabbits against each serotype was used in cross-RIP against cytoplasmic extracts prepared from cells infected with each BTV serotype. There were extensive cross-reactions among those proteins which showed a high degree of structural conservation, whereas VP2 was immunoprecipitated best in the homologous RIP system. Thus, a correlation between serotype specificity and protein structure was shown among the five U.S. serotypes of BTV.

Comparison of bluetongue type 20 with certain viruses of the bluetongue and Eubenangee serological groups of orbiviruses.

The genome of bluetongue virus type 20 consists of 10 segments of double-stranded RNA each of which contains unique sequences as determined by oligonucleotide mapping. The 10 polypeptide products of the virus genome were detected in virus-infected cells, and in pulse--chase experiments there was no secondary cleavage of the primary gene products. Using stringent conditions for RNA--RNA reassociation, no significant homology could be detected between the genomes of bluetongue type 20 isolated in Australia and representative serotypes isolated in other geographic regions. The results suggest sequence divergence between geographically isolated viruses and not the recent introduction of a bluetongue virus into Australia.

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.

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.

Further characterization of the t-s mutant F207 of bluetongue virus.

Temperature-shift experiments verified that the t-s lesion of BTV mutant F207 is expressed late in the replication-cycle, that is, at a stage when all virus components have already been synthesized. All viral polypeptides were indeed found in the soluble but not in the particulate fraction of cytoplasmic extracts from infected cultures grown at the non-permissive temperature. This suggests that the t-s lesion could be a defect in one or both of the polypeptides P2 and P5, which are respectively reduced in amount and absent from the latter fraction. Alternatively, the lesion could be an inability of the core particle to bind these 2 outer capsid polypeptides.

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.

The immunological response to intact and dissociated blue-tongue virus in mice.

Antigenic fractions of bluetongue virus were separated by ultracentrifugation in Tris-buffered CsCl gradients at pH 6, 7 or 8 and the bluetongue virus polypeptide composition of the bands isolated from these gradeints was monitored by polyacrylamide gel slab electrophoresis. The immunological response to these fractions in mice was determined by a haemolytic plaque-forming cell assay, using sheep erythrocytes onto which intact bluetongue virus was adsorbed as lytic indicator cells. Isolated outer layer bluetongue virus polypeptide 2, from gradients at pH 6, and polypeptides 2 and 5, from gradients at pH 7, produced a strong primary IgM plaque-forming cell response. The subviral particles of density 1, 39 g.cm-3 and the bluetongue virus core particles of density 1,42 g.cm-3 also stimulated an IgM response at least as strong as that to intact bluetongue virus of density 1,38 g.cm-3. The isolated bluetongue virus fractions therefore appear to maintain their immunogenic integrity as effectively as those of intact bluetongue virus. The pattern of the immune response to bluetongue virus type 4 is similar to that of type 10.