- Volume 4, Issue 4, 1969

Semliki Forest virus preparations of different degrees of purification were sprayed into atmospheres of different relative humidity. Inactivation of airborne virus was greatest at high relative humidities and it decreased gradually as the relative humidity was decreased. Removal of salts from the sprayed suspension resulted in improved survival over the whole range of relative humidity tested. Extraneous protein was essential for survival at high relative humidities. Polyhydroxy-compounds protected airborne virus very well in conditions of low relative humidity.

Unfractionated harvests of foot-and-mouth disease virus grown in baby hamster kidney cells fixed complement with both heterotypic and homotypic antisera but the freshly prepared intact virus (25 nm. component) from these harvests fixed complement only with the homotypic antiserum. Storage at 4° or heating at 37° released an antigen from the 25 nm. component which fixed complement with heterotypic serum. This antigen could also be prepared by mixing the 25 nm. component with baby hamster kidney cells but it was obtained in greatest yield by disrupting with guanidine. It had a sedimentation coefficient of 14S in sucrose gradients. Serum from hyperimmunized infected guinea pigs which had been absorbed with excess homotypic 25 nm. component fixed complement with the disrupted virus but not with intact virus. The disrupted virus also reacted with heterotypic antiserum produced by inoculation of guinea pigs with inactivated 25 nm. component, providing further evidence that the antigen is a structural component of the virus particle.

Infection of a thymineless strain of Bacillus subtilis with either the temperate phage φ3 or its clear plaque mutant φ3c enabled DNA to be formed in the absence of added thymine. Growth of phage φ3c under these conditions was associated with an enhanced rate of DNA synthesis compared with uninfected cultures growing in the presence of thymine; when phage φ3 became a prophage the lysogenized cells synthesized DNA at the same rate as the uninfected bacteria with thymine. The thymine independence of phage-infected bacteria was due to the acquisition of the enzyme thymidylate synthetase absent in the uninfected mutant bacteria. Phage φ3c growth caused a rapid rise in the specific activity of the enzyme after a short lag, and by the time the cells lysed there was approximately ten times more activity than in uninfected wild-type B. subtilis. In thymineless B. subtilis lysogenized with phage φ3 the amount of thymidylate synthetase was the same as in wild-type B. subtilis. Phage DNA was able to transform thymine-requiring B. subtilis to thymine independence, showing that it contained the gene for thymidylate synthetase. Plaque-forming particles could not be separated by density gradient centrifugation from those possessing the thymidylate synthetase gene. This result, together with the failure to get ‘thymineless’ mutants of the phage to regain their ability to promote thymine synthesis, suggested that phage φ3 was a converting phage. However, the results of transformation implied that the phage thymidylate synthetase gene was closely related to that of the recipient cells, and may have originated from the bacterium.

The plaque titre of rabbitpox and early passage vaccinia viruses was enhanced by diethylaminoethyl-dextran. Both viruses have low initial plaquing efficiency. Later passage vaccinia virus of high plaquing efficiency was little enhanced either before or after exposure to u.v., X or γ rays or to heat at 56°. The dextran made an insignificant improvement in adsorption of the virus particles to the cells. It formed flat, round bodies visible in the electron microscope but these were not found to form complexes with the virus particles nor to induce them to aggregate. This eliminated synergistic infection by aggregation of virus particles as a mechanism for plaque enhancement by dextran but suggested a protective action of the dextran particles upon freshly uncoated DNA within the cell vesicle.

Small concentrations of actinomycin D increased the yields of both virulent and attenuated strains of measles virus in cultures of the stable simian BSC-1 cell line by as much as five- to tenfold. The edmonston virulent strain of measles virus did not induce detectable levels of interferon in these cells but interferon was induced by the schwarz vaccine (attenuated) strain of virus over a wide range of virus input. Actinomycin D suppressed the induction of interferon in BSC-1 cells. Interferon produced in BSC-1 cells by the attenuated virus was able to protect both these cells and Vero (another stable line of green monkey kidney) cells against vesicular stomatitis virus.

Plaques produced by the virulent and attenuated virus in Vero cells were approximately the same size, in contrast to variations observed in BSC-1 cells. This was correlated with the failure of the Vero cells to produce interferon when inoculated with either the virulent or attenuated strain of virus and the failure of actinomycin D to affect yields of infectious virus. The results suggest that the effect of actinomycin D on virus yields and on plaque size in BSC-1 cells is a result of inhibition of induction of interferon.

The rescue of Rous sarcoma virus in mixed cultures of virogenic Chinese hamster cells transformed by sr-RSV (RSCh cells) and chick fibroblasts treated with Sendai virus was studied. When the ratio of RSCh to chick fibroblasts was 1:64, or less, the amount of rescued Rous sarcoma virus decreased proportionately with the dilution of RSCh cells. Over this range treatment with Sendai virus increased rescue of Rous sarcoma virus 100 times as compared with untreated cell mixtures. About 600 RSCh cells were necessary for obtaining 1 f.f.u. of Rous sarcoma virus under these conditions. No infectious virus was found in tissue culture fluid or extracts from RSCh cells when both were incubated with chick embryo cells which afterwards underwent treatment with Sendai virus.

Using different conditions of incubation after treatment of cell mixtures with Sendai virus, a varying frequency of heterokaryon formation was obtained. There was good correlation between heterokaryon formation and rescue of Rous sarcoma virus measured in parallel cultures. The effect of Sendai virus on mixtures of chick embryo and RSCh cells under various conditions was examined by electron microscopy.

Antibody prepared against HEp-2 cells inhibited the replication of echovirus 1. The number of infective centres was reduced and the cells which were infected produced less virus per cell than control cells. The attachment of virus to cells was not affected. The inhibition was reduced by increasing the multiplicity of infection. Application of antibody 5 min. after virus adsorption at 4° reduced the inhibition and there was little inhibition when it was applied 10 min. after adsorption. The antiserum had no inhibitory effect when infection was initiated by virus RNA; the antiserum may act by preventing uncoating of the virus.

Twenty-six strains of foot-and-mouth disease virus have been studied by sucrose gradient zone electrophoresis and a broad range of mobility is evident. It has been established that the electrophoretic mobilities of strains are a unique heritable property of the virion. No covariation is evident with several in vivo and in vitro characteristics of these strains but there is a relationship between mobility and immunological type. This relationship is unconnected with serological specificity and probably reflects the phylogenetic divergence of the seven immunological types of foot-and-mouth disease virus.

Two types of stable electrophoretic mutant have been isolated by serial selection, one of which, however, exhibits phenotypic instability. The other is stable but can be shown to be at a selective disadvantage in competition with the normal virus.

It has not been possible to demonstrate phenotypic mixing in progeny from cells infected with electrophoretically different strains or mutants. The biological implications of this are discussed.

The herpes-type virus of Marek’s disease showed a gradual increase in the rate of development of cytopathic effect on passage in cell culture. By the 60th passage macroscopic plaques were produced after 6 days under fluid overlay, but no cell-free virus could be recovered after filtration of culture medium.

A loss of pathogenicity for chicks was noticed in the virus after 33 passages in cell culture. Furthermore, an antigenic change occurred between the 20th and 30th passage which was characterized by the loss of an antigen which could normally be found in the supernatant medium of cultures infected with low passage virus.

The possible origin of the attenuated virus is discussed.

In chick fibroblast cell cultures infected with Newcastle disease virus an RNA-dependent RNA nucleotidyl transferase (RNA polymerase) has been detected which was not found in non-infected cells. The enzyme mainly synthesizes RNA complementary to viral RNA as shown by nearest neighbour analysis and hybridization experiments. In contrast to the influenza virus-induced enzyme the Newcastle disease virus enzyme cannot be inhibited by dextran sulphate. Its synthesis is not inhibited by actinomycin. It is first detectable 3 hr after infection and it has reached its highest activity at 5 hr after infection.

The multiplication of fowl-plague virus was much less sensitive to actinomycin D in chick cells infected with avian myeloblastosis virus and in hamster cells, transformed by Rous sarcoma virus than in comparable normal cultures. In addition, reproduction of fowl-plague virus was less effectively suppressed by u.v.-irradiation of cells carrying avian myeloblastosis or Rous sarcoma virus than of control cultures. These findings could indicate that avian myeloblastosis and Rous sarcoma viruses induce a function of host-cell DNA, whose products can be utilized in reproduction of fowl-plague virus.

Bacteriophage PL25 was purified by centrifugation in caesium chloride. It has S°20,w = 485; D°20,w = 0.68 × 10−7 cm.2/sec. and m.w. = 54.3 × 106. An equation was derived relating S°20,w and molecular weight of bacteriophages.

Using Earle’s L cells, the amino acid requirement for the propagation of vaccinia virus was found to coincide with the amino acids found by Eagle (Eagle, 1955; Eagle et al. 1956) to be essential for the L cells themselves. There was no evidence that vaccinia virus particles harbour enzymes involved in amino acid synthesis.

Phage 34.13 adsorbs well to Proteus mirabilis strain n but does not form plaques on it. The DNA of the phage is severely degraded in strain n. Phage which does emerge plates on P. mirabilis strain 13 but not on n. The phenotype of strain n is r+m−. Restriction by stationary phase organisms is weaker than in early log. phase cells. The acceptor ability of n str-r for a p-lac + plasmid from strain 13 is less than that of strain 13str-r. Spheroplasts of 13 plate ϕ34.13 DNA with an efficiency of 10−7. The efficiency on strain n spheroplasts is <10−11. The transduction rate of markers by ϕ34.13 into n is only reduced to about 10−1 of the rate into strain 13. Transductants are non-lysogenic for the phage, are stable and may be retransduced. Small doses of ultraviolet radiation do not increase the transduction rate. This is interpreted to mean that ϕ34.13 transduces strain n by integration of the bacterial exogenote, which like ϕ34.13 DNA and possibly strain 13 p-lac + DNA, is degraded in strain n. nh mutants of strain n were isolated on which ϕ34.13 has an e.o.p. of 5 × 10−1 and into which ϕ34.13 transduces markers at the same rate as into strain 13. Phage 34n mutants plate on strain n. A strain n-induced host specificity was discovered which must be carried by ϕ34n for it to form plaques on n. Strain n and its nh mutants are lysogenic for ϕ13vir and produce a phage tail-like bacteriocin but neither of these factors account for the restricting and modifying properties of the strains. Phages 34.13 and 34n−1.n do not affect one another in mixed infections of strain n.

T antigen (neoantigen) of Simian Virus 40 has been localized at the ultrastructural level in infected MA 104 cells in culture. The cells were fixed 15 min. to 1 hr in 1% formaldehyde, freshly prepared from paraformaldehyde, in 0.2 m cacodylate buffer, pH 7.3, + 0.25 m sucrose. This allowed adequate conservation of ultrastructure while permitting subsequent penetration of reagents into the cells. The antigen was revealed by successive exposure to unlabelled hamster serum containing antibody against the T antigen and to labelled rabbit antibody against hamster immunoglobulin. The latter conjugate of rabbit antibody was labelled with peroxidase for which the cytochemical reaction results in an osmiophilic, electron dense reaction product which can be visualized by electron microscopy. The T antigen of SV40 first appeared in a reticular network throughout the nucleus, except the nucleolus, in a position which seems to correspond with that of the chromatin network.