- Volume 32, Issue 3, 1976

Purified preparations of grapevine fanleaf virus consist of three serologically indistinguishable centrifugal components, T, M and B, with sedimentation coefficients of 50, 86 and 120S respectively, and containing 0, 30 and 42% RNA. At equilibrium in CsCl gradients, T, M and B components precipitate. Their buoyant densities are 1.31, 1.41 and 1.49 g/ml, respectively. Infectivity is associated mostly with B component. RNA is single-stranded and occurs as two species with mol. wt. of 1.4 × 106 (RNA-2) and 2.4 × 106 (RNA-1). M particles contain only RNA-2 whereas B particles contain both species. RNA-2 is non-infective, some infectivity is associated with preparations of RNA-1, and infectivity can increase tenfold when RNA-1 and RNA-2 are mixed. The capsid contains a single protein species with mol. wt. of about 54000. Comparable data for arabis mosaic and raspberry mosaic viruses are similar. The present cryptogram of grapevine fanleaf virus is R/1:2.4/42 + 1.4/30:S/S:S/Ne.

Baby hamster kidney cells were persistently infected with lymphocytic choriomeningitis (LCM) virus (BHKpi cells). After 21 passages of the BHKpi cells infectious virus could no longer be detected; however, the cultures continued to produce LCM virus particles which interfered with the replication of infectious LCM virus in BHKpi cells and protected mice from a subsequent intracranial inoculation of infectious LCM virus. Cultures of BHKpi cells appeared to consist of three cell populations: uninfected cells, infected cells containing infectious LCM virus, and infected cells releasing interfering particles of LCM virus.

SDS-polyacrylamide gel electrophoresis showed that purified sowthistle yellow vein virus (SYVV) contains four major and one minor structural polypeptides. The mol. wt. were estimated to be approx. 150000, 83000 (G), 60000 (N), 44000 (M1) and 36000 (M2). Covalently bound carbohydrate was detected in the 150000 mol. wt. species and the G protein.

Saponin and Nonidet P40 treatment removed the projections and membrane; however, the resultant bullet-shaped nucleocapsid structures, which contained N protein, were unstable and readily uncoiled into strands. Trypsin and thermolysin treatment removed the surface projections, and the G protein and high mol. wt. protein. Enzymically liberated G protein had a mol. wt. 5000 to 7000 smaller than attached G protein, suggesting that a 5000 to 7000 mol. wt. part of the G protein was left embedded in the membrane.

Lactoperoxidase- and chloramine T-catalysed iodination of intact SYVV particles labelled the G protein first, confirming its external location. The M1 and M2 proteins were the next labelled and were considered to be membrane associated. The N protein and the high mol. wt. protein were the last to be labelled.

Foot-and-mouth disease virus (a member of the picornavirus group) RNA could be translated effectively in an S-30 extract from Ehrlich ascites tumour cells. This translation was inhibited by aurintricarboxylic acid, cycloheximide, puromycin and RNase. Cell-free products of translation were identified by disc gel electrophoresis and immunoprecipitation with specific antisera. Gel electrophoresis of the products without prior immunoprecipitation suggested the synthesis of some of the non-capsid proteins and capsid proteins VP1, VP2 and VP3 of the virus. Immunoprecipitations with antisera against whole virus and VP3 indicated the synthesis of VP3 and of at least two additional peptides of 100000 and 56000 daltons containing antigenic sites of VP3. Gel electrophoresis after immunoprecipitation with antiserum against virus infection-associated antigen indicated the synthesis of a different 56000-dalton protein appearing to resemble non-capsid protein NCVP5. The amount of foot-and-mouth disease virus and VP3-specific peptides in the virus RNA-directed products were measured by immunoprecipitation.

A purification method for Semliki Forest virus-specified RNA-dependent RNA polymerase from BHK cells is described. The procedure entails (i) the preparation of a crude cell lysate by Dounce homogenization of cells 3.5 h post-infection, (ii) differential centrifugation to give a 15000 g ‘mitochondrial’ pellet, (iii) equilibrium centrifugation on discontinuous sucrose gradients (Friedman et al. 1972) to give a membranous band of density 1.16 g/ml, (iv) solubilization with Triton N-101 and velocity centrifugation to give a 25S solubilized polymerase complex and (v) affinity chromatography through an oligo (dT)-cellulose matrix bearing immobilized 42S virus particle RNA. The overall purification was approx. 360-fold with a 5% recovery of activity.

Of the various intermediate fractions in the purification procedure, only the relatively crude post-nuclear supernatant fraction was competent to synthesize the major single-stranded RNAs found in infected cells. Other fractions incorporated precursor only into replicative intermediate (RI) or replicative form (RF). Analysis of the product RF showed that it was of the same size and could bind to the same extent to oligo (dT)-cellulose as the RF isolated directly from lysates of infected cells. Displacement hybridization and ribonuclease digestion suggested that the purified polymerase could only complete previously initiated progeny positive strands using negative strands as template and, even in its most highly purified form, was still tightly bound to its template.

Analysis on polyacrylamide slab gels revealed the presence of three 35S-labelled polypeptides in the purified polymerase preparation, but a polypeptide which had identical electrophoretic mobility to the lowest mol. wt. polypeptide of the purified polymerase was also present in material from mock-infected cells which had been taken through the purification procedure. From these results we conclude that only two virus-specified polypeptides are present in the polymerase. A scheme for the synthesis of these polypeptides is presented in the accompanying paper.

Two previously undescribed stable polypeptides (referred to as nsp 90 and nsp 63) appear in mammalian and avian cells infected with Semliki Forest virus. They are distinguishable from the virus structural proteins and their known precursors by their molecular weights and tryptic peptide maps, and are identical in size to two polypeptides found in purified preparations of virus-specific RNA polymerase. Data from pulse-chase experiments and from the use of inhibitors of proteolytic cleavage indicate that nsp 90 and nsp 63 are synthesized via a series of post-translational cleavages from three larger polypeptides, p200, p184 and p150. The labelling kinetics after synchronous initiation of protein synthesis are also consistent with the synthesis of nsp 90 and nsp 63 from a common initiation site, and show that nsp 63 is located close to this site. It is concluded that nsp 90 and nsp 63 are components of the virus-specific RNA polymerase, and are synthesized via a post-translational cleavage scheme entirely separate from that leading to the synthesis of the virus structural proteins.

Vero cells were persistently infected with canine distemper virus by continuous undiluted passage of virus harvests. The cells were refractory to superinfection by both measles virus and canine distemper virus. These persistently infected cells produced and released into the medium a labile component which had a potent and selective inhibitory effect on the replication of canine distemper and measles virus. The inhibitory agent was not inactivated by u.v.-irradiation or sedimented by ultracentrifugation. Antisera against canine distemper virus or SSPE sera were able to block this inhibitory effect. We propose that these persistently infected cells produce an excess of a virus-induced regulatory protein.

The segregation of resistant and susceptible phenotypes in response to infection by RSV(RAV 2), RSV(RAV 50) and RSV(RAV 0), of avian RNA tumour virus subgroups B, D and E, respectively, was analysed in several test-crosses using chickens from the RPRL line 7-2, HPRS-synthetic line E and the Rease-heath line C. The results were fully consistent with our view reported previously that the genes at the tve and tvb loci segregate independently and recombine under the Mendelian second law of independent assortment. The dominant susceptibility es gene is expressed phenotypically when associated with the dominant susceptibility bs gene, but its expression is suppressed when associated with two doses of the recessive resistance br gene. Genetic causes such as lack of penetration, recessive epistasis, and/or complementary interaction between the tvb and tve genes have been discussed to account for the modified phenotypic expression of brbreses cells, i.e. resistance to subgroup E virus.

Also, as reported previously, it was observed in this study that the tvb genes control the cellular response to subgroup D virus.

The presence of Epstein-Barr virus (EBV) antigens in human palatine tonsil-derived lymphocytes (TDL) was investigated using the indirect fluorescent antibody (FA) technique. The TDL were screened for the presence of EBV early antigen (EA), virus capsid antigen (VCA), and EBV nuclear antigen (EBNA).

In 76% of the patients diagnosed as recurrent exudative tonsillitis, and in 33% diagnosed as recurrent tonsillitis and/or serous otitis media, EBNA was demonstrated in the purified TDLs. No EA- or VCA-producing cells were found in either the glass adsorbed or TDL cell preparations from all of the patients. These data suggest that in our patient sample, the tonsils may serve as a reservoir for EBV carrying lymphocytes and a basis for recurrent disease.

Owl and African green monkey kidney cell cultures have been infected with 1 p.f.u./cell of herpesvirus saimiri and sample cultures have been taken for examination by electron microscopy at 3 to 6 hourly intervals over a period of 7 days; the experiments were repeated several times. The peculiarly slow replication cycle of Herpesvirus saimiri has enabled distinct cytoplasmic and nuclear phases in virus maturation to be clearly distinguished; the overall fine structural features were similar in both cell types. Immature particles were first detected in the nucleus and cytoplasm 63 h after infection. Thereafter, abundant cytoplasmic immature particles matured by budding through cytoplasmic membranes until about 100 h, whereas nuclear immature particles budded through the inner nuclear membrane or intranuclear invaginations of it later, from about 100 h until cytolysis was complete at 160 h. Morphological differences were also observed between particles budding at cytoplasmic membranes and the nuclear envelope. At the former site the membrane overlying the bud showed an electron opaque thickening which imparted to the mature particle an asymmetrical appearance. Such thickenings of the envelope were not observed in mature particles of nuclear origin.

Unusual tubular and laminated nuclear structures were seen towards the end of the replicative cycle corresponding with the phase of nuclear virus maturation by budding; the morphology of the latter structures is described.

A haemadsorption microtest for African swine fever (ASF) virus is described. This assay is as sensitive and its response is faster than the conventional assay which uses buffy coat cultures in Leighton tubes. The method can also process a larger number of samples by using smaller amounts of swine blood and laboratory space. A plaque assay for ASF virus adapted to grow in VERO cells gives a titre similar to that obtained using the haemadsorption microtest. In both the micromethod and the plaque assay infection may be produced by a single infective particle.

African swine fever (ASF) virus was grown either in swine macrophages or in VERO cells and purified free of cell DNA. Virus DNA was isolated from virions as a molecule with a sedimentation coefficient of 60S and a contour of 58 ± 3 µm. These two values give a mol. wt. of 102 ± 5 × 106 and 107 ± 5 × 106, respectively, for the genome of ASF virus. Denatured DNA fragments from ASF virus reassociate with a C 0 t ½ value of 0.60 ± 0.05 ms, which compared with the corresponding value for T4 DNA gives for the molecular mass of ASF virus DNA a value of 102 ± 8 × 106 daltons. Only virus DNA is synthesized in ASF virus-infected swine macrophages.

LMTK- cells infected with u.v.-irradiated herpes simplex virus type 1 have been selected for the presence of the enzyme thymidine kinase. These cells have an altered morphology compared to the control cells and contain herpes-specific antigens in their cytoplasm.

The thymidine kinase activity present in these cells has been shown, on the basis of a number of biochemical properties, to be identical to the herpes virus specified deoxypyrimidine kinase found during lytic infection of this virus. In addition it has been possible to detect herpes simplex-specific RNA sequences in the transformed cells and this occurs in both the polyadenylated and non-polyadenylated cytoplasmic and nuclear fractions.

Methods for the purification of African swine fever virus (ASFV) and its dissection into two fractions are described. The difficulties which have been encountered previously in attempts to purify the virus, namely contamination with large amounts of cellular constituents and aggregation of the virus particles, have been overcome by treatment with Tween 80 and by the use of 1m-NaCl in the sucrose gradients. Five major polypeptides, mol. wt. 103 × 125 (VP1), 76 (VP2), 50 (VP3), 44 (VP4) and 39 (VP5) were found in the purified particles. The virus was dissected by treatment with Nonidet NP 40 into (a) a fraction which had the appearance of an empty capsid shell and contained the polypeptides VP2 and VP3 and (b) a structure containing VP1 and VP4. The location of VP5 was not ascertained.