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.

Conformational alteration in foot-and-mouth disease virus virion capsid structure after complexing with monospecific antibody.

A mechanism of neutralization of virus infectivity by antibody is described and related to the immune defences in vivo. The interaction of a particular monoclonal antibody with homologous foot-and-mouth disease virus alters the conformation of the virions to permit penetration of staining reagents. A consequence of this structural alteration is that the RNA genome becomes susceptible to dissociation from the capsid proteins. This mechanism of virus neutralization is irreversible and therefore provides an effective in vivo defence measure against virus attack, complementing the enhanced phagocytosis effected through opsonization of virions. With viruses that can replicate in phagocytes, such a mechanism of virus neutralization could provide a major 'specific' immunological defence against virus invasion.

Electron microscopy as an aid to the rapid diagnosis of virus diseases of veterinary importance.

The use of electron microscopy to assist in the rapid diagnosis of virus diseases of veterinary importance is reviewed. Electron microscopy can be used to assist the laboratory diagnosis of a virus disease at two stages during the investigation; either by demonstrating virus in clinical material or by identifying isolates from tissue culture or similar systems. Direct electron microscopy and immunoelectron microscopy are particularly useful for rapid diagnosis. The advantages of electron microscopy lie in speed and flexibility, and the disadvantages in the high particle concentration needed and the presumptive nature of a diagnosis.

A model for vesicular exanthema virus, the prototype of the calicivirus group.

The structure of vesicular exanthema virus, the prototype member of the calicivirus group, has been studied in more detail. The RNA comprises 18% of mol. wt. of about 2.8 x 10(6), based on polyacrylamide gel electrophoresis experiments in the presence of formaldehyde. The virus contains one major polypeptide, mol. wt. 70 x 10(3) as determined by polyacrylamide gel electrophoresis and by chromatography on Sepharose 6B in the presence of 6 M-guanidine. Further evidence for the presence of a single major polypeptide was obtained by tryptic peptide analysis of 35S-methionine labelled virus. The mol. wt. of a protein oligomer produced by adjusting the pH of virus suspensions to 3.5 was c. 200 x 10(3). On the basis of these data we propose a T = 3 model for the virus capsid incorporating 180 copies of the virus protein.

Relationship of a virus from Tellina tenuis to infectious pancreatic necrosis virus.

The physicochemical and serological properties of a virus isolated from the bivalve mollusc, Tellina tenuis, have been examined. The virus has a diam. of 59 nm, sediments at 430S in sucrose gradients and bands at a density of I-32 g/ml in CsCl. The virus contains RNA with a mol. wt. about 2-8 X 10(6) as extimated by polyacrylamide gel electrophoresis but in sucrose gradients the RNA sediments at 14S. The virus RNA is resistant to ribonuclease under conditions in which ribosomal RNA and the single stranded Mengo virus RNA are completely hydrolysed. Two major polypeptides, mol. wt. 67 and 40 X 10(3), and minor polypeptide, mol. wt. 110 X 10(3), are present in the virus particle. These properties are similar to those found in different serotypes of infectious pancreatic necrosis (IPN) virus. Although there was only a very low level of cross-neutralization between Tellina virus and IPN virus. there was some cross-reaction in immune electron microscopy tests and in immunofluorescence tests with infected tissue culture cells. This cross reaction, together with the close similarity in morphology and physiochemical properties, suggests that Tellina virus and IPN virus belong to the same virus group.

Model for vesicular stomatitis virus.

Vesicular stomatitis virus contains single-stranded ribonucleic acid of molecular weight 3.6 x 10(6) and three major proteins with molecular weights of 75 x 10(3), 57 x 10(3), and 32.5 x 10(3). The proteins have been shown to be subunits of the surface projections, ribonucleoprotein, and matrix protein, respectively. From these values and from estimates of the proportions of the individual proteins, it has been calculated that the virus has approximately 500 surface projections, 1,100 protein units on the ribonucleoprotein strand, and 1,600 matrix protein units. Possible models of the virus are proposed in which the proteins are interrelated.

Infective virus substructure from vesicular stomatitis virus.

Treatment of suspensions of vesicular stomatitis virus with Tween-ether results in a rapid and considerable loss of infectivity (ca. 4 logs in 2 min), but the residual infectivity is comparatively stable to further treatment with ether. The infectivity remaining after the short exposure to Tween-ether is not due to virus for the following reasons. (i) It is much less infective for tissue cultures than for mice, whereas the intact virion is equally infective for both hosts. (ii) The residual infectivity is much less stable than virus infectivity in both sucrose and tartrate gradients. (iii) Virus immune serum does not neutralize its activity. (iv) The infectivity is associated with material which sediments further in sucrose gradients and has a greater buoyant density in tartrate gradients than the virion. Experiments with (32)P-labeled virion showed that the infective substructure contains ribonucleic acid with the same sedimentation characteristics as that extracted from the virion. Electron microscopy shows that the infective component has the same overall bullet-like structure as the virion but lacks the outer envelope and fringe structure.