- Volume 25, Issue 3, 1974

Newcastle disease virus (NDV) stocks contain three types of particles which were tested for interferon inducing ability: (1) plaque-forming haemagglutinating particles, (2) non-plaque-forming haemagglutinating particles containing RNA and (3) non-plaque-forming haemagglutinating particles which contain no RNA. Single NDV-infected cells were isolated in microdrops and tested for interferon production as measured by protection of 30 ± 6 additional cells from challenge with mengovirus. This technique demonstrated that: (1) essentially every cell can be induced by plaque-forming NDV to produce interferon, (2) neither of the two types of non-plaque-forming haemagglutinating particles were capable of inducing detectable levels of interferon and (3) while infection with a single active virus was sufficient to induce interferon, the efficiency of induction increased with increasing multiplicity. It was concluded that some virus synthetic processes are probably required to initiate induction.

Eight strains of influenza virus were titrated as plaque-forming units in both monolayers and suspensions of chick embryo cells. In the absence of trypsin, satisfactory plaques were formed only by the A/WSN strain of virus. When trypsin was included in the overlay medium of cell monolayers, all the influenza virus strains tested produced plaques, and the plaque infectivities of all but one strain were close to the egg infectivities. Using cells suspended in agar in the presence of trypsin, four strains gave plaque infectivities indistinguishable from egg infectivities, two strains formed plaques with rather low efficiency and two strains did not produce plaques. Plaque formation was also enhanced by pronase or subtilisin. A preliminary investigation was made of the mode of action of trypsin.

The behaviour of lysosomal enzymes in poliovirus-infected HeLa S3 cells and vesicular stomatitis virus (VSV)-infected L cells was investigated both biochemically using enzyme assays, and histochemically using acid phosphatase dependent staining. The presence of the enzyme was shown histochemically under the light microscope by its reaction with naphthole-AS-BI-phosphate and a coupling reaction with diazotized pararosaniline. The light absorption of this stain in infected and uninfected cells was measured on a Universal Micro Spectrophotometer (UMSP-1) and recorded on-line as gray value cell images in a PDP-12 computer. These scanned images were analyzed by FORTRAN programs on a UNIVAC1108. The histochemically obtained distributions of the lysosomal enzyme are comparable to the results of the biochemical analysis. Lysosomes of poliovirus-infected cells displayed a release of lysosomal enzymes into the cytoplasm starting at 3 h after infection; VSV infection did not produce this type of effect. This investigation shows that it is possible to extract and demonstrate specific virus dependent changes using computer-aided cytophotometric techniques.

One hundred and four temperature-sensitive mutants of Sindbis virus strain AR339 have been isolated using five different mutagens. Forty-seven mutants showed no detectable RNA synthesis at the restrictive temperature (39 °C), whereas 57 showed levels ranging from 1% to over 100% of the wild-type level. The mutants did not show complementation. The ratio of the 42S:26S RNA species in cells infected with 30 mutants showing > 10% of the wild-type level of RNA synthesis at 39 °C was measured. Of these 30 mutants, none were found which were wholly defective in production of either RNA species, although the ratio was significantly different from the wild-type for some mutants. Two mutants showed production at 39 °C of polydisperse RNA of both double-stranded and single-stranded types.

Interferon induction by 11 temperature-sensitive (ts) mutants of the HR strain, and 38 ts mutants of the AR339 strain of Sindbis virus was investigated. All HR and AR339 mutants induced interferon at 30 °C. Induction by the mutants at the restrictive temperature (39 °C) was dependent on the time of incubation, and, in some instances, on the input multiplicity. At an input multiplicity of 5, and after an incubation time of 16 h at 39 °C, induction by the AR339 mutants was dependent on virus RNA synthesis. All HR mutants showing detectable RNA synthesis at 39 °C induced interferon, but of those showing undetectable RNA synthesis at 39 °C, three induced interferon and three did not. At 42 °C, RNA synthesis by the HR wild-type and all ts mutants was depressed, and only one mutant and the wild type induced interferon. Temperature shift experiments showed that interferon induction was dependent on an early virus function. It is postulated that interferon induction is dependent on a low threshold level of RNA synthesis occurring early in infection.

An assay system for fusion activity of envelope particles of Sendai virus, reassembled from NP40-solubilized envelopes, was established and conditions for the artificial assembly of NP40-solubilized Sendai virus envelope particles with haemolytic and fusion activities were investigated. Large (GP1) and small (GP2) glycoproteins and lipids seemed to be required for the expression of haemolytic and fusion activities of envelope particles. Potential haemolysin activity was associated with GP2. A relatively high proportion of GP1 was required for formation of envelope particles with a high fusion activity.

When the top lipid fraction (Hosaka & Shimizu, 1972a) was used for reassembly, the envelope particles usually exhibited both fusion and haemolytic activities but the optimal concentrations of the lipid for the two activities were different. An unidentified factor extractable with NP40 seemed to be necessary for fusion activity but not for haemolytic activity.

The cytoplasmic deoxyvirus frog virus 3 (FV3) can replicate in HeLa cells at a permissive temperature (26 °C). The effect of virus infection on the host-cell RNA metabolism was studied at both permissive and non-permissive (37 °C) temperatures. The inhibition of precursor ribosomal RNA synthesis was shown to take place soon after infection at both temperatures. However, the inhibition of synthesis of heterogeneous nuclear RNA occurs only at the non-permissive temperatures.

The major group-specific antigen (gs-a) of avian leukoviruses was detected by radioimmunoassay in extracellular medium of all embryonic cultures prepared from infected chickens. Embryos of birds not carrying an avian leukosis virus infection and of chickens from a leukosis-free flock could thus clearly be distinguished from infected ones. Release of group-specific antigen into the extracellular medium was independent of subgroup or strain of avian leukosis or Rous sarcoma virus. The radioimmunoassay of the group-specific antigen seems to be well suited to complement the COFAL (complement fixation avian leukosis) and RIF (resistance inducing factor) tests in the detection of leukosis virus in infected material.

The study was designed to examine the use of various tissue cultures for their ability to support the growth of influenza C virus; and to seek a suitable erythrocyte for haemadsorption by this virus in tissue culture.

Rat erythrocytes were found to have a strong affinity for influenza C virus, and their use demonstrated the superiority of rhesus monkey kidney cultures over several other cell cultures for the growth of this virus. Neutralization tests in rhesus monkey kidney cultures using rat erythrocyte haemadsorption proved to be a sensitive method to demonstrate antigenic cross relationships between strains of influenza C virus.

Camptothecin, an inhibitor of cellular nucleic acid synthesis, prevents fowl plague virus replication in BHK 21/13 cells. In the presence of camptothecin the synthesis of haemagglutinin and neuraminidase antigens does not occur; the ribonucleoprotein antigen is synthesized and accumulates only in the cell nucleus; and the synthesis of virus-specific RNA (of which complementary RNA predominates) is reduced considerably.

The polypeptides of BSC1 cells infected with vaccinia virus and pulse labelled with [14C]-protein hydrolysate or [35S]-methionine have been examined by discontinuous polyacrylamide gel electrophoresis followed by autoradiography. About 80 virus induced polypeptides were detected, 30 appearing before and 50 after the onset of virus DNA synthesis. These were termed pre- and post-replicative polypeptides respectively. The synthesis of most pre-replicative polypeptides was turned off shortly after the peak of virus DNA synthesis; when virus DNA synthesis was inhibited this turn-off did not occur and post-replicative polypeptides were not made. The synthesis of some post-replicative polypeptides started at about the time of maximal DNA synthesis, reached a peak about 1 h later, and was then turned off. Other post-replicative polypeptides whose synthesis started at this time were made for prolonged periods. The synthesis of most post-replicative polypeptides started about 1 h after the time of maximal virus DNA synthesis and continued thereafter for prolonged periods. The stability of pre- and post-replicative polypeptides was examined in pulse-chase experiments; most pre-replicative and ‘early’ post-replicative polypeptides were stable for prolonged periods, whereas, during a chase, eleven ‘late’ post-replicative polypeptides disappeared and seven new polypeptides appeared.

Bean leaf cells infected with bean pod mottle virus or cowpea mosaic virus contain osmiophilic globules (OG) in the cytoplasm. OG and myelinic bodies (MB) occurred between the plasmalemma and cell wall, and also embedded in the cell wall. Tubules containing virus particles were associated with OG and MB outside the cytoplasm, and membranes of some tubules were associated with MB. It is suggested that OG move from the cytoplasm through the plasmalemma, remain near the cell wall and become transformed into MB.

Intramuscular injection of concentrated human leucocyte interferon into mice, guinea pigs, rabbits, sheep and man gave a long-lasting plateau of circulating interferon. The relation between the interferon dose and the subsequent serum level was similar in all species.

Infection of thymidine kinaseless mouse cells (Cl-1D) by herpes simplex virus (type 1) was found to result in a stimulation of thymidine uptake into mitochondrial DNA (mtDNA) during a pulse interval from 1 to 6 h post-infection (p.i.). Thymidine incorporation into mtDNA by organelles isolated at 6 h p.i. from infected cells exceeded that of mock-infected controls significantly. An identifical mitochondrial reaction was observed when infected cell cultures were pretreated with 2 µg cytosine arabinoside (ara C)/ml culture medium from 1 to 6 h p.i. Administration of 25 µg cycloheximide/ml instead of ara C abolished the effect of virus infection on mtDNA synthesis. The implications of these observations are discussed with respect to regulation of mtDNA synthesis in eukaryotic cells.