Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

Genetic diversity of bluetongue viruses in south east Asia.

Bluetongue viruses (BTV) were isolated from sentinel cattle in Malaysia and at two sites in Indonesia. We identified eight serotypes some of which appeared to have a wide distribution throughout this region, while others were only isolated in Malaysia or Australia. Nearly half of the 24 known BTV serotypes have now been identified in Asia. Further, we investigated the genetic diversity of their RNA segments 3 and 10. Using partial nucleotide sequences of the RNA segment 3 (540 bp) which codes for the conserved core protein (VP3), the BTV isolates were found to be unique to the previously defined Australasian topotype and could be further subdivided into four distinct clades or genotypes. Certain of these genotypes appeared to be geographically restricted while others were distributed widely throughout the region. Similarly, the complete nucleotide sequences of the RNA segment 10 (822 bp), coding for the non-structural protein (NS3/3A), were also conserved and grouped into the five genotypes; the BTV isolates could be grouped into three Asian genotypes and two Nth American/Sth African genotypes.

Functional expression and membrane fusion tropism of the envelope glycoproteins of Hendra virus.

Hendra virus (HeV) is an emerging paramyxovirus first isolated from cases of severe respiratory disease that fatally affected both horses and humans. Understanding the mechanisms of host cell infection and cross-species transmission is an important step in addressing the risk posed by such emerging pathogens. We have initiated studies to characterize the biological properties of the HeV envelope glycoproteins. Recombinant vaccinia viruses encoding the HeV F and G open reading frames were generated and glycoprotein expression was verified by metabolic labeling and detection using specific antisera. Glycoprotein function and cellular tropism were examined with a quantitative assay for HeV-mediated membrane fusion. Fusion specificity was verified through specific inhibition by anti-HeV antiserum and a peptide corresponding to one of the alpha-helical heptad repeats of F. HeV requires both F and G to mediate fusion. Permissive target cells have been identified, including cell lines derived from cat, bat, horse, human, monkey, mouse, and rabbit. Fusion negative cell types have also been identified. Protease treatments of the target cells abolished fusion activity, suggesting that the virus is employing a cell-surface protein as its receptor.

Quantification of recombinant core-like particles of bluetongue virus using immunosorbent electron microscopy.

Immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (BTV) core-like particles (CLP) in either purified preparations or lysates of recombinant baculovirus-infected cells. The capture antibody was an anti-BTV VP7 monoclonal antibody. The CLP concentration in purified preparations was determined to be 6.6 x 10(15) particles/l. CLP concentration in lysates of recombinant baculovirus-infected cells was determined at various times post-infection and shown to reach a value of 3 x 10(15) particles/l of culture medium at 96 h post-infection. The results indicated that immunosorbent electron microscopy, aided by an improved particle counting method, is a simple, rapid and accurate technique for the quantification of virus and virus-like particles produced in large scale in vitro systems.

The attachment protein of Hendra virus has high structural similarity but limited primary sequence homology compared with viruses in the genus Paramyxovirus.

The complete nucleotide sequence of the attachment protein gene of Hendra virus, a new member of the subfamily Paramyxovirinae, has been determined from cDNA clones derived from viral genomic RNA. The deduced mRNA is 2565 nucleotides long with one open reading frame encoding a protein of 604 amino acids, which is similar in size to the attachment protein of the members of the subfamily. However, the mRNA transcript is >600 nucleotides longer than others in the subfamily due to the presence of long untranslated regions at both the 5' and 3' ends. The protein is designated G because it lacks both hemagglutination and neuraminidase activities. It contains a hydrophobic transmembrane domain close to the N terminus, eight potential N-linked glycosylation sites, and 18 cysteine residues. Although the HeV G protein had low sequence homology with Paramyxovirinae members, the predicted folding pattern of its extracellular globular head was very similar to that of members of the genus Paramyxovirus, with the location of seven potential pairs of sulfide bonds absolutely conserved. On the other hand, among the seven residues known to be critical for neuraminidase activity, only one was conserved in the Hendra virus G protein compared with at least six in HN proteins of paramyxoviruses and rubulaviruses and four in H proteins of morbilliviruses. The biological significance of this finding is discussed.

Use of a gene-targeted phage display random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein VP5.

We describe the use of a gene-targeted random epitope library for the mapping of antigenic determinants. A DNA clone encoding the target antigen was digested randomly with DNase I to generate a population of DNA fragments of different sizes and sequences. After size fractionation, small DNA fragments (100-200 bp) were isolated and cloned into the phage expression vector fUSE2 to form an expression library displaying random polypeptide sequences as fusion proteins at the N terminus of the phage gene III protein. This library, termed a gene-targeted random epitope library to distinguish it from totally random synthetic epitope libraries, was then screened by affinity selection for recombinant phages which were specifically bound by the antibody of interest. Using this approach, we have mapped a monoclonal antibody (mAb)-defined epitope on the bluetongue virus outer capsid protein VP5. This epitope is not accessible on the intact virus surface, but is recognised by the immune system of sheep and cattle during virus infection. Although the example given here utilised a DNA fragment of known sequence and the library was screened for a mAb-defined epitope, the strategy described should be equally applicable to genes of unknown sequence and for screening of epitopes using polyclonal antibodies. The approach can also be extended to identify immunodominant epitope from much more complex genome-targeted random epitope library for virus, bacteria and eukaryotic organisms. Other applications of recombinant phages expressing defined immunodominant epitopes include serodiagnosis and vaccine development.

Characterization of virus inclusion bodies in bluetongue virus-infected cells.

A combined qualitative and quantitative approach has been used to examine the role of virus inclusion bodies (VIBs) in the morphogenesis of bluetongue virus (BTV). VIBs were detected as early as 4 h post-infection (p.i.), and their number and profile areas increased significantly between 12 and 16 h, and 20 and 28 h p.i. respectively. Core- and virus-like particles were found within and at the periphery of the VIB matrix, respectively, and their numerical density (number per area of VIB matrix) decreased during the course of infection whereas the numerical density of virus particles in the cytoplasm increased. Virus-like particles had a diameter of 57 +/- 8 nm and core-like particles appeared to fall into two size ranges, 32 +/- 3 nm and 38 +/- 3 nm in diameter. Both pre- and post-embedding immunoelectron microscopy procedures were used to localize BTV structural and non-structural proteins within the VIBs. The VIB matrix was labelled with antibodies to structural proteins VP5 and VP7 and non-structural proteins NS1 and NS2. Cores within VIBs contained proteins VP5, VP7 and NS1 but not VP2. Virus-like particles at the periphery of VIBs contained VP2, VP5, VP7 and NS1. The results suggest that BTV particles are synthesized, assembled and released from the perimeter of VIBs and not from within the matrix. Cores embedded in the VIBs are likely to have been trapped there during expansion of the matrix during replication.

Competitive ELISA for serodiagnosis of bluetongue: evaluation of group-specific monoclonal antibodies and expressed VP7 antigen.

The performance of 2 competitive enzyme-linked immunosorbent assays (C-ELISA) was compared with the reference C-ELISA I for the detection of antibodies to bluetongue virus (BTV). One of the assays (C-ELISA II) used a group-specific monoclonal antibody (MAb) to BTV, obtained from the American Type Culture Collection (8A3B-6) and tissue culture (TC)-derived BTV antigen (Ag), and the other assay (C-ELISA III) used BTV core protein VP7 (expressed in yeast) and the reference MAb (Pirbright Laboratory, 3-17-A3). Test sera were obtained by sequential blood samples from 22 calves, each inoculated with a different serotype (T) of BTV (South African [SA] T-1-T-16 and T-18-T-20 and USA T-11, T-13, and T-17). Sera were also obtained from 4 calves and 4 sheep inoculated with USA BTV T-10 and from several groups of calves exposed to single or multiple doses of epizootic hemorrhagic disease virus (EHDV) T-1-T-4 grown in TC (BHK-21) or suckling mouse brain (SMB). A total of 618 bovine and ovine field sera collected from BT-free and BT-endemic areas were also tested. The C-ELISA III was more sensitive than the C-ELISA II in the detection of anti-BTV antibody in sera from cattle and sheep early after infection with BTV. Seroconversion was demonstrated by the 3 C-ELISAs in all animals inoculated with BTV by 20 days postinfection (DPI), except in calves that received SA T-3 or USA T-13, which became positive at 40 DPI.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

High level expression of the major core protein VP7 and the non-structural protein NS3 of bluetongue virus in yeast: use of expressed VP7 as a diagnostic, group-reactive antigen in a blocking ELISA.

The major core protein VP7 and a non-structural protein NS3 of bluetongue virus serotype 1 have been synthesized from recombinant plasmids using both an in vitro transcription/translation system and a yeast expression system. Bluetongue virus genes were transcribed under the control of the bacteriophage SP6 promoter and the regulatable yeast metallothionein promoter. An indirect ELISA showed that expression of NS3 in yeast was inducible with 1 mM CuSO4 and VP7 synthesis was constitutive but could be further induced. The preferred procedure for antigen extraction from yeast was sonication for VP7 and SDS/NaOH treatment for NS3. Yeast-expressed VP7 antigen and a monoclonal antibody were used in a blocking ELISA to distinguish sera raised against bluetongue virus serotypes from those generated to viruses of the epizootic haemorrhagic disease serogroup.

A bluetongue serogroup-reactive epitope in the amino terminal half of the major core protein VP7 is accessible on the surface of bluetongue virus particles.

Immunoelectron microscopy has been used to confirm that the core protein VP7 is accessible on the surface of bluetongue virus (BTV) particles. Monospecific antibodies generated to vaccinia virus-expressed VP7 and an anti-VP7 monoclonal antibody (MAb 20E9) bound to native virus particles and were localized by protein A-gold. In contrast, MAb 20E9 labeled directly with gold failed to gain access and bind, suggesting that VP7 is neither adventitiously adsorbed to the virion surface nor exposed in a manner such as protrusion through the outer capsid. Thus the surface layer of BTV may be considered as a net which only partially obscures the underlying core particle. Sequencing of VP7 revealed it to be an extremely hydrophobic protein, 350 amino acids in length with cysteine residues at positions 15, 65, and 154. Examination of VP7 in the cytosol of cells infected with either BTV or a vaccinia virus recombinant expressing VP7 indicated that the protein may exist as an oligomer, whose constituent monomers are not linked by intermolecular disulfide bonds. The cysteine residues in sodium dodecyl sulfate (SDS)-denatured, dithiothreitol (DTT)-treated VP7 were labeled with the fluorescent iodoacetamide AEDANS and the protein was cleaved by V8 protease. The size of the labeled peptides and knowledge of the location of potential V8 cleavage sites suggested that the enzyme cleaved VP7 at three locations (glutamic acid residues at positions 61, 104 (or 108), and 132 (or 134 or 135). Analysis of the fluorescent peptides generated by V8 protease cleavage of VP7 labeled with AEDANS in the absence of DTT (i.e., with any putative intramolecular disulfide bonds intact) suggested that the cysteine at position 154 was the only one accessible to AEDANS. The cysteines at positions 15 and 65 may therefore be linked via a disulfide bond. Denaturation of VP7 with SDS did not eliminate the capacity of the protein to bind MAb 20E9. However, the sensitivity of the epitope to reduction and acetylation and its resistance to either of these processes alone suggest that it may be located near a disulfide bond linking cysteines at positions 15 and 65. Confirmation that the epitope lay in the amino-terminal half of the VP7 came from immunoelectron microscopy experiments in which thin sections of bacteria expressing the complete VP7 and the amino-terminal half were probed with MAb 20E9 and protein A-gold.

Association of bluetongue virus with the cytoskeleton.

Analysis of the distribution of [35S]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. These structures remained cytoskeleton associated in infected cells treated with colchicine or cytochalasin B. Linear arrays of filament-associated virus particles were formed around VIB following colchicine-induced juxtanuclear aggregation of intermediate filaments. Virus particles were associated with filaments approximately 10 nm in diameter. Filaments associated with virus particles reacted with an anti-vimentin monoclonal antibody in an immunogold labeling procedure.

The grid-cell-culture technique: the direct examination of virus-infected cells and progeny viruses.

We describe a method for the structural analysis and identification of viruses, without purification or concentration steps which could alter virus morphology. Virus-infected cells grown on carbon-Parlodion-coated electron microscope grids release large numbers of progeny viruses which adsorb to the surface of the grid and are revealed by negative staining. The technique is rapid, sensitive and can be used at three levels. Negative staining of whole cell preparations revealed both extracellular and intracellular viruses or nucleocapsids beneath the plasma membrane; non-ionic detergent extraction of cells infected with certain viruses reveals cytoskeleton-associated, virus-specific structures normally only observed after thin sectioning; cultures prepared by either procedure are suitable for colloidal gold immunological studies. Extracellular and cytoskeletal-associated viruses were heavily and specifically labelled with gold. The results indicate that the technique may be used to rapidly identify unknown viruses on the basis of size, topography, morphology and mode of maturation from the infected cell, as well as the presence of characteristic intracellular cytoskeletal-associated structures. The technique also has potential use in the sero-grouping and sero-typing of viruses with specific monoclonal antibodies.