African horse sickness virus infects BSR cells through macropinocytosis.

Cellular pathways involved in cell entry by African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, have not yet been determined. Here, we show that acidic pH is required for productive infection of BSR cells by AHSV-4, suggesting that the virus is likely internalized by an endocytic pathway. We subsequently analyzed the major endocytic routes using specific inhibitors and determined the consequences for AHSV-4 entry into BSR cells. The results indicated that virus entry is dynamin dependent, but clathrin- and lipid raft/caveolae-mediated endocytic pathways were not used by AHSV-4 to enter and infect BSR cells. Instead, binding of AHSV-4 to BSR cells stimulated uptake of a macropinocytosis-specific cargo and inhibition of Na(+)/H(+) exchangers, actin polymerization and cellular GTPases and kinases involved in macropinocytosis significantly inhibited AHSV-4 infection. Altogether, the data suggest that AHSV-4 infects BSR cells by utilizing macropinocytosis as the primary entry pathway.

Comparative ultrastructural characterization of African horse sickness virus-infected mammalian and insect cells reveals a novel potential virus release mechanism from insect cells.

African horse sickness virus (AHSV) is an arbovirus capable of successfully replicating in both its mammalian host and insect vector. Where mammalian cells show a severe cytopathic effect (CPE) following AHSV infection, insect cells display no CPE. These differences in cell death could be linked to the method of viral release, i.e. lytic or non-lytic, that predominates in a specific cell type. Active release of AHSV, or any related orbivirus, has, however, not yet been documented from insect cells. We applied an integrated microscopy approach to compare the nanomechanical and morphological response of mammalian and insect cells to AHSV infection. Atomic force microscopy revealed plasma membrane destabilization, integrity loss and structural deformation of the entire surface of infected mammalian cells. Infected insect cells, in contrast, showed no morphological differences from mock-infected cells other than an increased incidence of circular cavities present on the cell surface. Transmission electron microscopy imaging identified a novel large vesicle-like compartment within infected insect cells, not present in mammalian cells, containing viral proteins and virus particles. Extracellular clusters of aggregated virus particles were visualized adjacent to infected insect cells with intact plasma membranes. We propose that foreign material is accumulated within these vesicles and that their subsequent fusion with the cell membrane releases entrapped viruses, thereby facilitating a non-lytic virus release mechanism different from the budding previously observed in mammalian cells. This insect cell-specific defence mechanism contributes to the lack of cell damage observed in AHSV-infected insect cells.

Utilization of cellulose microcapillary tubes as a model system for culturing and viral infection of mammalian cells.

Cryofixation by high-pressure freezing (HPF) and freeze substitution (FS) gives excellent preservation of intracellular membranous structures, ideal for ultrastructural investigations of virus infected cells. Conventional sample preparation methods of tissue cultured cells can however disrupt the association between neighboring cells or of viruses with the plasma membrane, which impacts upon the effectiveness whereby virus release from cells can be studied. We established a system for virus infection and transmission electron microscopy preparation of mammalian cells that allowed optimal visualization of membrane release events. African horse sickness virus (AHSV) is a nonenveloped virus that employs two different release mechanisms from mammalian cells, i.e., lytic release through a disrupted plasma membrane and a nonlytic budding-type release. Cellulose microcapillary tubes were used as support layer for culturing Vero cells. The cells grew to a confluent monolayer along the inside of the tubes and could readily be infected with AHSV. Sections of the microcapillary tubes proved easy to manipulate during the HPF procedure, showed no distortion or compression, and yielded well preserved cells in their native state. There was ample cell surface area available for visualization, which allowed detection of both types of virus release at the plasma membrane at a significantly higher frequency than when utilizing other methods. The consecutive culturing, virus infection and processing of cells within microcapillary tubes therefore represent a novel model system for monitoring intracellular virus life cycle and membrane release events, specifically suited to viruses that do not grow to high titers in tissue culture.

Structural insight into African horsesickness virus infection.

African horsesickness (AHS) is a devastating disease of horses. The disease is caused by the double-stranded RNA-containing African horsesickness virus (AHSV). Using electron cryomicroscopy and three-dimensional image reconstruction, we determined the architecture of an AHSV serotype 4 (AHSV-4) reference strain. The structure revealed triple-layered AHS virions enclosing the segmented genome and transcriptase complex. The innermost protein layer contains 120 copies of VP3, with the viral polymerase, capping enzyme, and helicase attached to the inner surface of the VP3 layer on the 5-fold axis, surrounded by double-stranded RNA. VP7 trimers form a second, T=13 layer on top of VP3. Comparative analyses of the structures of bluetongue virus and AHSV-4 confirmed that VP5 trimers form globular domains and VP2 trimers form triskelions, on the virion surface. We also identified an AHSV-7 strain with a truncated VP2 protein (AHSV-7 tVP2) which outgrows AHSV-4 in culture. Comparison of AHSV-7 tVP2 to bluetongue virus and AHSV-4 allowed mapping of two domains in AHSV-4 VP2, and one in bluetongue virus VP2, that are important in infection. We also revealed a protein plugging the 5-fold vertices in AHSV-4. These results shed light on virus-host interactions in an economically important orbivirus to help the informed design of new vaccines.

A reverse genetics system of African horse sickness virus reveals existence of primary replication.

African horse sickness virus (AHSV), a member of the orbivirus genus of the family Reoviridae, is an insect-vectored pathogen of horses of concern to the equine industry. Studies on AHSV replication and pathogenesis have been hampered by the lack of reverse genetics allowing targeted mutation of viral genomes. We demonstrate that AHSV single-stranded RNA synthesized in vitro (core transcripts) is infectious and that there are distinct primary and secondary stages of the replication cycle. Transfection with a mixture of core transcripts from two different serotypes or a mixture of core transcripts and a T7 derived transcript resulted in the recovery of reassortant viruses. Recovery of infectious ASHV from nucleic acid will benefit investigation of the virus and the generation of attenuated vaccines.

New generation of African horse sickness virus vaccines based on structural and molecular studies of the virus particles.

African horse sickness virus (AHSV) is a member of the genus Orbivirus, which also includes bluetongue virus (BTV) and epizootic haemorrhagic disease (EHDV) virus. These orbiviruses have similar morphological and biochemical properties, with distinctive pathobiological properties and host ranges. Sequencing studies of the capsid proteins have revealed evolutionary relationships between these viruses. Biochemical studies of the viruses together with the expression of individual proteins and protein complexes have resulted in the development of new generation vaccines. Baculovirus expressed AHSV VP2 provides protection against death caused by AHSV challenge. Similarly, BTV VP2 alone elicits protective neutralising antibodies against BTV in sheep, which is enhanced in the presence of VP5. Recent developments in biotechnology (multiple gene expression baculovirus systems) have made it possible to synthesise orbivirus particles that biochemically and immunologically mimic authentic virions but lack the genetic material. Particle doses as low as 10 micrograms elicit responses that are sufficient to protect sheep 15 months post vaccination, against virulent virus challenge. Moreover, knowledge of the three dimensional structure of these particles enables us to engineer them to deliver multiple foreign peptide components representing other viral epitopes (e.g. foot and mouth disease virus and influenza virus) in order to elicit protective immunity.

Characterization of two African horse sickness virus nonstructural proteins, NS1 and NS3.

Each of the ten segments of the African horse sickness virus (AHSV) genome encodes at least one viral polypeptide. This report focuses on the nonstructural proteins NS1 and NS3, which are encoded by genome segments 5 and 10 respectively. The NS1 protein assembles into tubular structures, which are characteristically produced during orbivirus replication in infected cells. NS1 expressed by a recombinant baculovirus in Sf9 cells also forms tubules, which were analysed electron microscopically. These tubules had an average diameter of 23 +/- 2 nm, which is less than half the width of the corresponding bluetongue virus (BTV) tubules. They were also more fragile at high salt concentrations or pH. The cytotoxic effects produced by NS3 were examined by constructing of mutated versions and expressing them in insect cells. Substitution of amino acids 76-81 in a conserved region (highly conserved amongst all AHSV NS3 proteins, as well as other orbiviruses) with similar amino acids, did not influence the cytotoxicity of the mutant protein. However, mutation of four amino acids, from hydrophobic to charged amino residues, (aa 165-168) in a predicted transmembrane region of NS3, largely abolished its cytotoxic effect. It is considered likely that the mutant protein is unable to interact with cellular membrane components, thereby reducing its toxicity.

Intracellular production of African horsesickness virus core-like particles by expression of the two major core proteins, VP3 and VP7, in insect cells.

To gain more insight into the structure of the African horsesickness virus (AHSV) core particle, we have cloned, partially characterized and expressed the two major core proteins, VP3 and VP7, of AHSV-9. VP7 was found to be highly conserved amongst different serotypes. The VP3 and VP7 genes were subsequently expressed in insect cells by means of recombinant baculoviruses. VP7 was synthesized to very high levels and aggregated into distinctive crystals. Co-expression of VP3 and VP7 resulted in the intracellular formation of core-like particles which structurally resembled empty AHSV cores.

Characterization of tubular structures composed of nonstructural protein NS1 of African horsesickness virus expressed in insect cells.

The characteristic tubules that are produced during the orbivirus infection cycle are composed of a major viral nonstructural protein, NS1. To characterize the NS1 gene and gene product of African horsesickness virus (AHSV), a full-length cDNA copy of the NS1 gene of AHSV-6 was cloned and the nucleotide sequence determined. NS1 was highly conserved within the AHSV serogroup with between 95-98% conservation of amino acids among NS1 of AHSV-6, AHSV-4 and AHSV-9. The structure of AHSV NS1 tubules was investigated by in vitro translation of the AHSV-6 NS1 gene followed by expression of the gene in insect cells. The NS1 protein assembled in tubular structures with a diameter of approximately 23 nm and lengths of up to 4 microns. The absence of a ladder-like structure and lower sedimentation value of AHSV NS1 tubules clearly distinguished them from those of bluetongue virus.

Purification and properties of virus particles, infectious subviral particles, cores and VP7 crystals of African horsesickness virus serotype 9.

Methods were developed for the purification, at high yield, of four different particle types of African horsesickness virus serotype 9 (AHSV-9). These products included virus particles purified on CsCl gradients which contain proteins apparently directly comparable to those of bluetongue virus (VP1 to VP7); virus particles purified on sucrose gradients which also contain, as a variable component, protein NS2; infectious subviral particles (ISVPs), containing chymotrypsin cleavage products of VP2; and cores, obtained by treating purified ISVPs with 1 M-MgCl2 to remove the components of the outer capsid layer (VP5 and VP2 cleavage products). Additional protein bands migrating with apparent M(r)s lower than that of VP5 were detected during SDS-PAGE analysis of virus particles. These appear to be conformational variants of VP5 and are identified as VP5' and VP5". BHK-21 cells infected with this strain of AHSV-9 produce large quantities of flat, usually hexagonal crystals of VP7, a major group antigen and core protein; these were also purified. Either 20 mg of virus particles, 20 mg of ISVPs or 10 mg of cores as well as 20 mg of VP7 crystals could be purified from approximately 8 x 10(9) infected cells. None of the preparations of particles or crystals showed any detectable contamination with BHK-21 cell proteins or antigens, as determined by SDS-PAGE or indirect ELISA. Virus particle and ISVP preparations had similar specific infectivities for BHK-21 cells (approximately 1 x 10(9) TCID50/A260 unit) but the infectivity of cores was approximately 10(5)-fold lower.

African horse sickness virus structure.

African horse sickness virus (AHSV), of which there are nine serotypes (AHSV-1, -2, etc.), is a member of Orbivirus genus within the Reoviridae family. Both in morphology and molecular constituents AHSV particles are comparable to those of bluetongue virus (BTV), the prototype virus of the genus. The two viruses have seven structural proteins (VP1-7) organized in two layered capsid. The outer capsid is composed of VP2 and VP5. The inner capsid, or core, is composed of two major proteins, VP3 and VP7, and three minor proteins, VP1, VP4 and VP6. Within the core is the virus genome. This genome consists of 10 double-stranded (ds)RNA segments of different sizes, three large, designated L1-L3, three medium, M4-M6, and four small, S7-S10. In addition to the seven structural proteins that are coded by seven of the RNA species, four non-structural proteins, NS1, NS2, NS3 and NS3A, are coded by three RNA segments, M5, S8 and S10. The two smallest proteins (NS3 and NS3A) are synthesized by the S10 RNA segment, probably from different in-frame translation initiation codons. Nucleotide sequences of eight RNA segments (L2, L3, M4, M5, M6, S7, S8 and S10) and the predicted amino acid sequences of the encoded gene products are also available, mainly representing one serotype, AHSV-4. In this review the properties of the AHSV genes and gene products are discussed. The sequence and hybridization analyses of the different AHSV dsRNA segments indicate that the segments that code for the core proteins, as well as those that code for NS1 and NS2 proteins, are highly conserved between the different virus serotypes. However, the RNA encoding NS3 and NS3A, and the two segments encoding the outer capsid proteins, are more variable between the AHSV serotypes. A close phylogenetic relationship between AHSV, BTV and epizootic haemorrhagic disease virus (EHDV), three Culicoides-transmitted orbiviruses, has been revealed when the equivalent sequences of genes and gene products are compared. Recently, the four major AHSV capsid proteins have been expressed using recombinant baculoviruses. Biochemically and antigenically these proteins are similar to the authentic proteins. Since the AHSV VP7 protein is highly conserved among the different serotypes, it has been utilized as a diagnostic reagent. The expressed VP7 protein has also been purified to homogeneity and crystallized for three-dimensional X-ray analysis.(ABSTRACT TRUNCATED AT 400 WORDS)