- Volume 13, Issue 1, 1971

The labelled RNAs of the three European types of foot-and-mouth disease virus (A2-spain; C-oberbayern; Oi-lombardy) have been cross-hybridized with a large excess of denatured unlabelled foot-and-mouth disease virus-specific double-stranded RNA of the respective virus types. The degree of double-strand reformation in the heterologous reactions was taken as a measure of the extent to which polynucleotide sequences were shared by the virus RNAs. Different degrees of hybridization were obtained across different pairs of virus types: A2-spain with Oi-lombardy = 65%, A2-spain with C-oberbayern = 44%, Oi-lombardy with C-oberbayern = 45%.

Extracellular rabbitpox virus released naturally from infected cells differed antigenically from intracellular virus released by artificial disruption of cells. Intracellular virus was neutralized by antiserum prepared against live rabbitpox virus and by antiserum against inactivated vaccinia virus. In contrast, extracellular virus was neutralized only by rabbitpox antiserum. The antibodies responsible for the neutralization of intracellular and extracellular virus could be absorbed separately from rabbitpox antiserum. Morphologically, extracellular virus differed from intracellular virus in possessing an outer envelope. This envelope was probably the site of the virus antigen characteristic of extracellular virus, and fluorescent antibody staining of infected cells suggested that it was derived from the modified host cell membrane. Antibody directed against extracellular virus was responsible for the ability of rabbitpox antiserum to control the spread of rabbitpox virus in tissue culture and probably for its ability to protect rabbits from rabbitpox infection. Extracellular virus should therefore be used as the test virus in titrations of neutralizing antibody if these are to assess the protective activity of an antiserum.

Inactivated vaccines were prepared from cell-associated vaccinia virus liberated mechanically from sheep dermal pulp and cultures of infected chick cells and from naturally released ‘free’ or extracellular virus. The inactivated vaccines induced dermal immunity in rabbits and protected mice against intracerebral challenge. Antibody produced by rabbits in response to inactivated cell-associated virus neutralized cell-associated but not free virus. Antibody to inactivated ‘free’ virus neutralized neither, whereas antibody to live virus neutralized both types of virus and prevented secondary spread in vitro. Antigenic differences between cell-associated and ‘free’ virus were also indicated by gel precipitation but not by complement fixation or haemagglutination-inhibition tests on the sera.

Significant virus autointerference was not observed in serial undiluted passages of vesicular stomatitis virus in Madin–Darby canine kidney cells. The ultra-structural features of vesicular stomatitis virus development into Madin–Darby canine kidney cells were studied by staining frozen-thawed cells with ferritin-conjugated antibody. The granular and fibrillar antigen could be readily identified in the cytoplasm within 4 hr after infection. Its appearance was followed closely by the differentiation of virus filaments at the cell surface. The virus antigen could be recognized in close proximity to virus particles; this material may represent virus nucleoprotein incorporated into budding virus particles.

Cultures of primary mouse kidney cells and those of L cells produced interferon in response to Newcastle disease virus and polyinosinic–polycytidylic acid. The interferon produced by L cells was made between 8 and 18 hr post-inoculation irrespective of the inducer. The interferon produced by mouse kidney cell cultures in response to Newcastle disease virus was made between 8 and 18 hr while that produced in response to polyinosinic–polycytidylic acid was made between 2 and 12 hr.

Pre-treatment of L cells with interferon inhibited interferon production by both inducers, but pre-treatment of mouse kidney cell cultures inhibited only the 8 to 18 hr interferon stimulated by Newcastle disease virus. Thus the induction process stimulated by polyinosinic–polycytidylic acid was probably different in L cells from that in mouse kidney cells. On the basis of the time required to induce interferon production and the sensitivity of the induction process to the inhibitory effects of interferon pre-treatment, the induction process stimulated by polyinosinic–polycytidylic acid must be regulated differently in L cells and mouse kidney cells. The ability to stimulate interferon production repeatedly in mouse kidney cells by polyinosinic–polycytidylic acid indicated an induction process dependent only on the presence of an effective inducer.

Primary cultures of mouse kidney cells which produced good yields of interferon with polyinosinic–polycytidylic acid lost their ability to respond to this inducer after a single passage. Secondary cultures still responded to Newcastle disease virus, however. We suggest that primary cultures of mouse kidney cells contain two types of cells each of which responds uniquely to one type of inducer and each perhaps with its own distinct induction process.

Turnip cells infected with radish mosaic virus contain characteristic large vesiculated cytoplasmic inclusion bodies, visible by light and electron microscopy. They consist of spherical aggregates of virus particles, sometimes in a crystalline array, embedded in a membranous mass. The presence of active dictyosomes and abundant endoplasmic reticulum in the outer zone of young inclusions suggests that these organelles are involved in the formation of inclusion bodies.

Phage ml 3 receptor substance was isolated from the plasma membrane of Streptococcus lactis, strain ml 3, by extraction with sodium deoxycholate at 37°. A twelvefold purification of the receptor substance was obtained by filtering the deoxycholate extract through a column of Sepharose 4B, in the presence of deoxycholate. Receptor activity was specific for phage ml 3 and was associated with a lipoprotein fraction which contained five principal and some minor polypeptide components. When phage receptor substance was aged in the absence of deoxycholate, aggregation of lipoprotein and a reversible loss of receptor substance occurred. Receptor activity was destroyed by extraction of the receptor substance with ether + ethanol at −20° and was decreased by incubation with trypsin. Activity was not affected by oxidation with sodium periodate or by digestion with lipase or phospholipases. The phage receptor substance was precipitated by antibody to the plasma membrane and activity appeared to be confined to a small proportion of the antigenic lipoprotein molecules.

Further evidence has been obtained which confirms that foot-and-mouth disease virus contains several structural proteins. By electrophoresis in urea-polyacrylamide gels, virus of type O gave six distinct bands. In sodium dodecyl sulphate-polyacrylamide gels four proteins with molecular weights of 34, 30, 26 and 13.5 × 103 were clearIy demonstrated. When virus preparations were labelled with a single amino acid, in both sodium dodecyl sulphate-polyacrylamide and urea-polyacrylamide gel electrophoresis, the fastest migrating protein contained no arginine and only traces of cysteine. This protein also stained differently from the other bands with Coomassie Blue and was absent from the 12s protein subunit prepared by mild acid (pH 6.5) disruption of the virus. This protein was separated from the 12s subunit by sucrose gradient centrifugation and by ion exchange chromatography on AmberIite IRC-50.

Two distinct immunogenic sites were present on the surface of foot-and-mouth disease virus. One site was concerned with adsorption of the virus to susceptible cells as well as the production of neutralizing antibody. Removal of this site with trypsin did not alter the gross morphology but the particles then had reduced infectivity, although the virus RNA was present in a fully infective form. The presence of a second immunogenic site was shown by the fact that trypsin-treated particles also produced a neutralizing antibody. This differed from the neutralizing antibody against the intact virus in that it was absorbed by trypsin-treated particles. Some of the neutralizing activity of antiserum produced by inoculation of intact virus particles was also absorbed by trypsin-treated particles. The relative activities of the two antibody-producing sites varied with different strains of virus.

The neuraminidase (E.C. 3.2.1.18) of A/turkey/england/69 is related antigenically to the neuraminidases of A/turkey/massachusetts/65 and A/turkey/england/66 avian influenza viruses, and to an A 2/1957 human influenza virus.

The neuraminidase of A/turkey/scotland/70 is related antigenically to the neuraminidases of A/dutch and virus ‘N’ (isolated before 1930 and in 1949 respectively), thus demonstrating the recurrence of a neuraminidase antigen in avian influenza A viruses over a period of at least 40 years.

The haemagglutinins of A/turkey/england/69 and A/turkey/scotland/70 did not cross-react specifically with each other, or with viruses representing four known avian influenza A haemagglutinin prototypes.

The kinetics of the liberation of protein components from purified poliovirus was examined under varying alkaline pH conditions at 40°. The proteins of the liberated components and of the virus capsid were analysed by sucrose gradient centrifugation and polyacrylamide gel electrophoresis.

When the virus particle was treated at pH 10.0, a minor component enriched in the capsid protein VP 4 was liberated from the virus capsid and a remaining capsid structure had the same H antigenicity as intact empty capsids free of virus RNA. A second component consisting mainly of VP 2 was released from the capsid at pH 11.0 and the residual capsid contained VP 1 and VP 3. This altered capsid still possessed H antigenicity and was stable at pH 11.0, but was degraded to smaller components at pH 12.0. This smaller component did not show H antigenicity. The results suggested that the basic matrix of the particle structure is composed of VP 1 and VP 3 and it exhibits H antigenicity.

The virus RNAs and proteins synthesized in chick embryo cells infected with different strains of Newcastle disease virus have been characterized by polyacrylamide gel electrophoresis. By comparison of the rates of electrophoretic mobility of virus RNA with those of RNA molecules of known molecular weight, the molecular weights were estimated to be 4.8 × 106 for the virus particle RNA and 2.5 × 106, 1.1 × 106 and 0.4 to 0.7 × 106 for the RNA molecules synthesized in infected cells, the latter being heterogeneous. Synthesis of a number of virus particle proteins was readily detected.

Weanling hamsters were inoculated subcutaneously in the cheek pouch with tumour tissue, or dorsally with tumour tissue or tumour homogenate, obtained from tumours induced by chick embryo lethal orphan virus. Hamsters 1 to 3 days old were inoculated subcutaneously in the dorsal region with chick embryo lethal orphan virus. Females were found to be more susceptible to tumour induction and required a lower dose of virus. There was no evidence of increased tumour resistance in hamsters up to 3 days old. Most neoplasms developed between the sixth and eighth month, usually at the site of virus deposition, although a few were noted in the following 2 months. All hamsters inoculated with transplants via the cheek pouch developed neoplasms. This incidence was well above that for tumours induced in the dorsal area. All neoplasms were well encapsulated fibrosarcomas regardless of the size of the tumour and type or site of inoculation. Most tumours were ‘kidney-shaped’, multilobulated, and of soft consistency.

Particles of henbane mosaic virus in extracted plant sap were usually straight and 900 nm. long but occasionally flexuous and 800 nm. long. The length and flexuousness of the particles depended on the composition of the extracting medium. When exposed to magnesium ions (0.05 m), the particles were long and straight but when exposed to 0.05 m-EDTA they were shorter and flexuous. Similar morphological differences were found when pepper veinal mottle virus or bean yellow mosaic virus was extracted in the two solutions. With pepper veinal mottle virus, each form of particle could be converted to the other by changing the medium.

The development and constitution of the inclusion bodies induced by Italian and Californian isolates of cauliflower mosaic virus was studied by light and electron microscopy. Cytochemical and enzyme-digestion tests revealed that the inclusion bodies are essentially proteinaceous, and contain RNA and some DNA. These inclusions have a matrix composed of densely packed, finely granular or fibrillar material and vacuole-like areas not bounded by a membrane. Virus particles are interspersed at random with the matrix and are not aggregated in regular crystalline arrays. The developmental sequence of the inclusions suggests that they are not merely structures for virus accumulation but, rather, that they are sites of virus synthesis and/or assembly.

Factors affecting the determination of buoyant density in caesium salts of several animal picornaviruses have been studied. The values obtained for the acid-labile foot-and-mouth disease virus increased with the duration of the centrifugation and were higher at elevated pH values. In contrast, the acid-stable enteroviruses had the same values irrespective of the time of centrifugation or the pH of the caesium solution. Viruses of intermediate stability, such as vesicular exanthema virus and the rhinoviruses, were affected in the same way as foot-and-mouth disease virus but to a smaller extent. The implications of these observations in respect of the structure of the picornavirus group are discussed.

A phage which attacks many motile strains of the Proteus-providence group did not replicate on aflagellate or paralysed mutants. The phage consisted of an hexagonal head, a tail and one tail fibre and resembled φx1. Electron micrographs demonstrated adsorption of the phage to the flagellar shaft of the host and the presence of phage at the flagellar base. Some of the latter phage possessed empty heads, and we postulate that the phage injects its nucleic acid at the base. This phage and ϕχ 1 had a slight serological relationship.