F. Brown

Surface Structure of Vesicular Stomatitis Virus

Summary Trypsin (0.1 mg./ml.) reduced the infectivity of vesicular stomatitis virus by 5 log. within 5 min. and destroyed immunizing activity. It also destroyed the complement-fixing activity against antiserum to the virus but the activity against antiserum to host cells was unaffected. The external spike-like projections of the virus were removed without affecting the remainder of the surface structure. Trypsin removed radioactivity from virus labelled with [14C]amino acids, but not from virus labelled with 32P. Phospholipase reduced the infectivity to a much smaller extent than trypsin and the immunizing activity was apparently unaffected. After treatment with phospholipase, complement-fixing activity against antiserum to virus was also unaffected but the complement-fixing activity against antiserum to host cells was greatly reduced. Electron microscopy showed that the spike structure of the virus was unaffected by phospholipase C but the remainder of the surface was digested. The enzyme removed more than 50% of radioactivity from virus labelled with 32P in the phospholipid component. The results showed that the spike-like structure of the virus responsible for producing neutralizing antibodies is composed entirely of virus protein and the phospholipid component derived from the cells is located in the regions between the spikes. The spikes may be attached directly to the internal helical structure of the virus.

The Ribonucleic Acids of the Infective and Interfering Components of Vesicular Stomatitis Virus

Summary The RNA-containing components of vesicular stomatitis virus were labelled with 32P by growing the virus in a phosphate-deficient medium containing 32PO4 and Actinomycin-D. Centrifugation of the virus suspensions in a sucrose gradient gave four radioactive fractions. The most rapidly sedimenting fraction contained the infective component of the virus, whereas most of the interfering activity was in the second fraction, which contained ‘caps’, filaments and rosette-like structures. The interfering activity was associated with the ‘cap’, which could be separated from the filaments and rosettes by centrifuging in a potassium tartrate gradient. The viral RNA sedimented predominantly in the 36 to 40 S position in sucrose gradients prepared in 0.1 m-acetate buffer but frequently exhibited a more heterogeneous profile. The percentage distribution of 32P in the four nucleotides was A = 29.3, C = 21.1, G = 20.9 and U = 28.7. Ribonucleic acid from the interfering component invariably gave a sharp profile with a sedimentation coefficient of 18 to 20S and the percentage distribution of 32P was A = 27.1, C = 21.9, G = 20.3 and U = 30.7. Both nucleic acids were hydrolysed to low molecular weight materials by ribonuclease 0.01 µg./ml.

Dissection of Vesicular Stomatitis Virus into the Infective Ribonucleoprotein and Immunizing Components

Summary Treatment of the infective component of vesicular stomatitis virus with Nonidet P40 produces an infective skeleton-like structure which has the shape and approximate size of the intact virus particle. The infectivity of the skeleton is enhanced 100 to 1000 fold by mixing with DEAE-dextran. The skeleton lacks the outer envelope and fringe structure and in consequence does not produce neutralizing antibodies in guinea pigs. The density of the skeleton is 1.22 g./ml. in potassium tartrate gradients compared with 1.14 g./ml. for the virus. Sodium deoxycholate removes protein from the skeleton and releases the filamentous ribonucleoprotein in an infective form. As with the skeleton, the infectivity of the ribonucleoprotein is enhanced by DEAE-dextran. Ribonuclease has no effect on the ribonucleoprotein but trypsin destroys its infectivity. The ribonucleoprotein has a density of 1.22 g./ml. in tartrate gradients, sediments at about 140 s in sucrose gradients and does not produce neutralizing antibodies in guinea-pigs.

Application of agar gel precipitin tests to the study of the virus of foot-and-mouth disease.

Abstract Suspensions of the virus of foot-and-mouth disease prepared from infected animals or from tissue cultures give two precipitin lines with homotypic antiserum in the agar gel diffusion test. The two lines are identical with the individual lines produced when the 20 mμ and 7 mμ components, obtained by differential centrifugation of the virus, are allowed to diffuse against the homotypic antiserum. Although each component gives a precipitin line with homotypic antiserum only, formation of the line due to the 7 mμ component can be prevented by mixing it with either homo- or heterotypic antiserum. On the other hand, formation of the line by the 20 mμ component can be prevented by homotypic antiserum only. The precipitin line patterns obtained with virus suspensions which have been heated or treated with formalin are also described.

An Interfering Component of Rabies Virus which contains RNA

Summary A particle about one third of the length of the virus particle and containing single-stranded RNA has been found in BHK cells infected with rabies virus. The particle interferes with replication of rabies virus and contains RNA which sediments at 20S compared with 43S for virus RNA.

Interference as a measure of cross-relationships in the vesicular stomatitis group of rhabdo viruses.

The Indiana and New Jersey serotypes of vesicular stomatitis virus were differentiated by Cotton in 1927. They remained the only known members of the group until the Cocal (Jonkers et al. 1964), Brazil (Andrade et al. 1964, cited by Federer, Burrows & Brooksby, 1967) and Argentina (Garcia Pirazzi, Caggiano & Alfonso Fernandez, 1963) viruses were described. Federer et al. (1967) demonstrated a close relationship between the Cocal and Argentina strains, and although the Brazil, Argentina/Cocal and Indiana strains were shown to differ serologically, they were still considered as subtypes of a group distinct from that of the New Jersey serotype. Cartwright & Brown (1972) confirmed recently that only the Cocal and Argentina strains showed significant cross-reactions in complement fixation and serum neutralization tests. However, the infective skeleton-like particles produced by treatment with Tween-ether or Nonidet and the infective ribonucleoprotein (RNP) released by sodium deoxycholate showed considerable cross-reactions in both tests. On this basis the major cross-reactivity was considered to be associated with the RNP.

Efficacy of rabies vaccine prepared from virus grown in duck embryo.

Abstract Of thirteen persons each given fourteen doses of rabies duck-embryo vaccine, only four produced significant amounts of virus-neutralising activity. Comparative vaccination experiments in animals indicate that the potency of the duck-embryo vaccine is much lower than that of experimental tissue-culture or brain vaccines. These results raise doubts concerning the efficacy of duck-embryo vaccine.

A study of the interference phenomenon in vesicular stomatitis virus replication.

Baby hamster kidney cell monolayers were infected with unfractionated vesicular stomatitis virus at input multiplicities ranging from 1 to 1/105. Cells infected at multiplicities of 1/103 or less produced 10 times as much infective virus but considerably less interfering component than cells infected at multiplicities exceeding 1/100. This has been shown to be due to the presence in the unfractionated virus pool of 2 viruses having different growth rates. The faster growing virus (lag phase 2 hours) does not cause interference, even at multiplicities greater than 100, and does not produce measurable amounts of interfering component in a single passage. The slower growing virus has a lag phase of 3.5 hours and produces large quantities of interfering component in addition to infective virus. This virus will also interfere with viral replication when 10 to 50 particles are added to each cell. A similar number of interfering particles are required to produce the same amount of inhibition. The interfering particle is not self-replicating but is produced only when virus multiplies. The simultaneous presence of infective and interfering particles in a virus harvest is explained by the earlier formation and release of infective virus in the growth cycle.

Rabies virus and the problems of rabies vaccination in man

The structure of rabies virus and the importance of its glycoprotein in immunization are discussed. The improvement in vaccines for use in man, culminating in the production of the human diploid vaccine is described. Nevertheless problems remain, particularly with regard to post-exposure therapy. Possible disadvantages in the use of subunit vaccines are mentioned and attention is drawn to the discovery of the rabies-related viruses.

The Assay, Extraction and Storage of Infective Ribonucleic Acid from Foot and Mouth Disease Virus

SUMMARY: A plaque assay method is described for the titration of the infective ribonucleic acid (RNA) of foot-and-mouth disease (FMD)> virus. The method depends on the dilution of the RNA in salt solutions of high molarity and the treatment of the BHK 21 cell monolayers used with M-NaCl. The method is reproducible and is at least as sensitive as other methods of titration. The extraction of RNA in the presence of EDTA gave titres corresponding to 0.1 % of the titre of the intact virus. RNA prepared from virus suspensions containing EDTA retained about 50% of initial infectivity for 7 days at 4° but for longer periods it was best preserved in 70 % (v/v) ethanol at 20°.