Journal of General Virology - Volume 12, Issue 3, 1971

Rat cells transformed by B77 virus did not produce virus as assayed by infectivity and interference tests. Each of the transformed rat cells contained the complete virus genome, since virus capable of converting chicken and rat cells could be rescued from them. Experiments are presented supporting the hypothesis that B77 virus changes genetically during residence in the rat cells. Infection of rat cells by B77 virus was followed by morphological conversion of some cells. Other cells contained the virus genome but were not transformed: this is termed ‘abortive infection’.

Some parameters of the reactivation of ultraviolet- or chemically inactivated fowl plague virus by live A2 viruses were examined. Clones of reactivated virus were obtained by two different techniques, either by isolation from plaques on chick embryo fibroblast cells or by terminal dilution in pieces of allantois-on-shell. Recombinants isolated from plaques were of one type, namely they contained only the neuraminidase antigen of the live A2 virus. In contrast, recombinants isolated in pieces of allantois-on-shell possessed a variety of A2 characteristics, and only a small proportion of these were of the type isolated by the plaque technique.

Studies in which fowl plague virus was exposed to ultraviolet or ethylene iminoquinone for various time periods enabled comparisons to be made of the rate of loss of infectivity and the rate of loss of ability to be reactivated. The results of these studies were interpreted as indicating that approximately 70% of the genome of fowl plague virus could be reactivated by A2 virus, and that this portion of the genome contained the genetic information for the haemagglutinin and neuraminidase antigens, the heat stability of the haemagglutinin, lethality for the chick embryo and efficient plaque production.

RNA from purified bovine enterovirus (serotype VG-5-27) was fractionated by sedimentation on sucrose gradients, gel filtration through Sepharose 2B and electrophoresis in acrylamide-agarose gels. Virus RNA was single stranded with a molecular weight of approximately 2.8 × 106. The RNA obtained from virus infected cells was separated into replicative intermediate and single-stranded molecules by gel filtration through Sepharose 2B. Electrophoresis of the replicative intermediate fraction on acrylamide-agarose gels gave two peaks of radioactivity, whereas after ribonuclease treatment only a single peak was present. Electrophoresis of the single-stranded molecules on acrylamide gels showed that a portion of these had slower mobilities than RNA from purified virus. This single-stranded fraction contained molecules with molecular weights in the range 5.6 to 2.8 × 106. Continuous labelling experiments showed that this size range was a function of time after infection. The existence of single strands of RNA longer than RNA found in purified virus was interpreted as supporting a cyclic model for RNA replication.

The type and concentration of sera used to support macrophages was found to influence the in vitro destruction of macrophages from genetically resistant C3H mice by the princeton strain of mouse hepatitis virus, and the accompanying conversion of mouse hepatitis virus (princeton) to the variant virus, mouse hepatitis virus (C3H). Cells incubated in 20% horse serum were more susceptible to destruction than those in 90% horse serum. Susceptibility was greatest in the presence of 10% foetal calf serum, while cells in 10% mouse sera were the most resistant. Differences in susceptibility were as great as 10,000 TCD 50. The sera had no direct effect upon either mouse hepatitis virus (princeton) inoculum virus or the released variant virus, but appeared to influence intracellular adaptation of virus. In plaque titrations using reduced concentration of horse sera, mouse hepatitis virus (princeton) produced small plaques on C3H cells. The variant virus, mouse hepatitis virus (C3H), produced both large plaques characteristic of mouse hepatitis virus (C3H) and small plaques characteristic of mouse hepatitis virus (princeton). The results suggest that a fraction of mouse hepatitis virus (princeton) virus multiplies in resistant C3H cells, and is then converted to mouse hepatitis virus (C3H), and that mouse hepatitis virus (princeton) continues to be carried in stocks of mouse hepatitis virus (C3H) during passage in C3H cells. The outcome of infection of genetically resistant C3H macrophages with mouse hepatitis virus (princeton) was greatly influenced by the type and concentration of sera used to support macrophages.

The mechanism of assembly of Newcastle disease virus in chick embryo cells was investigated in two series of experiments. When protein synthesis was inhibited by addition of puromycin, cycloheximide or fluorophenylalanine at any time during the course of infection, subsequent virus production was soon inhibited. These drugs were inhibitory even when they were added to the cultures as late as 10 hr after infection, when large amounts of virus precursor proteins were present within the cell. In the second series of experiments, the kinetics and efficiency of incorporation of radioactive amino acid into virus particles were examined by the pulse-labelling technique. A 30 min. labelling period at the 3rd or 6th hr of infection resulted in the release of highly radioactive virus during the period of 1½ hr immediately after the pulse. However, when pulse-labelling was performed at the 9th hr, the maximally labelled virus was found in the yield obtained 3 hr after the pulse, and the specific radioactivity of virus was less than 1% of that of the virus harvested from cultures labelled earlier. On the basis of these findings, possible mechanics of virus assembly are discussed.

Arginine has been shown to be essential for the replication of vaccinia virus in HeLa cell cultures. The yield of infective virus was dependent upon the concentration of arginine in the medium, maximum yield being obtained with 0.09 mm-arginine.

There were marked reductions in the synthesis of DNA, RNA and protein in infected and control cultures deprived of arginine. The yield of infective virus from cell cultures infected in the presence of sub-optimal concentrations of arginine was increased following further addition of arginine at a time after the completion of virus DNA synthesis. Arginine has been shown to be incorporated into the virus particle.

It is concluded that there are requirements for arginine demonstrable at both early and late stages in the replication of vaccinia virus in this system. While the early requirement was satisfied by 0.015 mm-arginine, later events showed a greater requirement up to a maximum of 0.09 mm-arginine.

A satisfactory purification procedure for oat necrotic mottle virus involved clarification of the plant juice by silver nitrate, followed by two cycles of differential centrifugation, and then rate-zonal density gradient centrifugation. The concentrate was finally passed through a column of agarose gel. Antiserum to clarified sap from virus-free oats did not react with the final preparation. A medium consisting of 0.1 m-sodium citrate buffer, pH 6.2, containing 0.5 m-urea, was suitable for the concentrated virus. The virus particles were filamentous with a diameter of 11 nm. and a normal length of 720 nm.

The structure of bovine parainfluenza type 3 virus (strain sf-4) was found to be indistinguishable from that of other paramyxoviruses. Negatively stained virus particles had an overall diameter ranging from 280 to 580 nm. and a nucleocapsid diameter of 17 to 19 nm. By light microscopy, sf-4-infected LLC-MK2 cells showed eosinophilic cytoplasmic inclusions, followed later by eosinophilic nuclear inclusions. By electron microscopy, aggregates of nucleocapsid-like filaments were seen in the cytoplasm and nucleus. Maturation of virus particles occurred at the plasma membrane, in a manner similar to that observed with other paramyxoviruses. Some cells containing inclusions exhibited an unusual type of particle formation in dilated portions of the endoplasmic reticulum. Bovine parainfluenza 3 virus appears to be unique among parainfluenza viruses in that its cytopathology and morphogenesis more closely resemble that of the unrelated measles subgroup of paramyxoviruses than that of the antigenically related parainfluenza viruses.

Cultures of dog kidney cells were infected with canine distemper virus at an input multiplicity of 1.9 and the subsequent events followed by electron microscopy, conventional staining methods and virus titration. Maturation and release of virus commenced within 24 hr of infection and was a slow but prolonged process, the infected cell producing small amounts of virus for at least 48 hr. After formation in cytoplasmic foci, often perinuclear, the virus nucleocapsid migrated to the cell membrane, below which it adopted a symmetrical configuration, the cell membrane at the same time acquiring a layer of fine surface projections. Maturation of virus then occurred by a protrusion and pinching off of these areas. Cytoplasmic aggregates of nucleocapsid were not detected by light microscopy. The characteristic phloxinophilic cytoplasmic inclusions, which appeared between 24 and 48 hr after infection, did not stain with acridine orange. Some consisted of a mass of amorphous electron-dense material and others of nucleocapsid-like filaments enmeshed in an electron-dense matrix but all contained a number of pockets in which membrane-bound vacuoles and tubules were prominent.

In KB cells infected with frog virus 3 at 37°, a non-permissive temperature for frog virus 3, there was a marked inhibition of host-cell RNA and DNA synthesis shortly after infection. The inhibition, which was as effective with gamma-irradiated or heat-inactivated virus as with live frog virus 3 was dependent upon the multiplicity of infection with frog virus 3. The effect of frog virus 3 could not be abolished by chemical inhibitors of protein and RNA synthesis. These findings suggested that a component of the virus particle was responsible for the inhibition of host-cell RNA and DNA synthesis.

Two established rat cell lines, WERC and TWERC, were studied for the presence of C-type RNA virus particles. WERC cells, spontaneously transformed in vitro, continuously released C-type RNA virus (WERC virus) by budding from the plasma membrane. Some of these virus particles (diameter about 100 nm.) showed an electron-lucent nucleoid of diameter 75 to 80 nm. and represented immature C-type virus. Others showed dense nucleoids of about 65 to 70 nm. diameter and looked like mature forms of murine leukaemia virus. Each cell showed 60 to 100 virus particles. The buoyant density of the virus particles in sucrose gradients was that expected for avian or murine RNA virus particles (1.16 g./ml.).

No virus particles were observed in the subline of cells (TWERC) originating from the malignant transformation with avian sarcoma virus (the prague strain of Rous sarcoma virus) of parental WERC cells.

Quantitative studies with phage 7 obtained by specialized induction of Rhizobium trifolii strain su 298 have been hampered by plaque counts showing marked random fluctuations with time and extreme variations in plaque size. The number of plaque-forming particles in phage 7 suspensions frequently fluctuated randomly by as much as a factor of 10 when stored for a week in a refrigerator. Many plaques were so small as to make accurate counting difficult. This appeared similar in many respects to the case of reversible inhibition by some part of the bacterial lysate reported by Sagik (1954) for phage T2. This paper gives results of a study which confirmed this initial suggestion and produced additional evidence on the nature of the phenomenon by the use of non-adsorbing lysogenic bacterial strains.

Several strains of Rhizobium trifolii were used: su297/32 (a small-colony variant of su297), su298/533 and su298/534 (large but distinct colony forms of su298), su298/531 (a small-colony form, also of su298) and nu18.

The different fatty acid compositions of various strains of influenza virus are derived from homologous membranes such as the cells lining the allantoic sac of the embryonated chick egg (Tiffany & Blough, 1969). The lipoprotein-complex theory of membrane structure (Benson, 1966; Green & Perdue, 1966; Wallach & Gordon, 1968), as applicable to myxoviruses, suggests that lipids are bound specifically to apolar regions of the envelope polypeptide by hydrophobic interactions (Tiffany & Blough, 1969). If this hypothesis is correct, differences in molecular composition of the apolar paraffinic chains among various species of phospholipid are to be expected in order to permit the interaction of lipid and protein with maximum release of free energy. The purpose of this study was to test this hypothesis by comparison of the fatty acids of individual phospholipids in two strains of influenza virus.

It has been reported that cellular DNA-like RNA (D-RNA) synthesis, as well as protein synthesis, is inhibited when mammalian cells are infected with adenovirus type 5 (Bello & Ginsberg, 1969). It has also been reported (Hodge & Scharff, 1968; Raskas, Thomas & Green, 1970) that adenovirus type 2 induces a marked decrease in the appearance of 18 s and 28 s ribosomal RNA species in the cytoplasm of infected cells between 10 and 19 hr after infection. The present communication provides evidence that KB cells infected with adenovirus type 7 also exhibit changed patterns of host RNA synthesis and alterations in the processing and distribution of various RNA species among the cellular compartments.

KB cells were propagated as monolayer cultures in 8 oz sealed prescription bottles in a medium consisting of Hanks's balanced salt solution (HBSS) containing 0.5% lactalbumin hydrolysate (LAH) and supplemented with 10% newborn-calf serum and antibiotics.

Disc electrophoresis in polyacrylamide gels has often been used for investigation of virus proteins (McCarthy, 1968; Sarkar & Schilde-Rentschler, 1968; Semancik, 1966), but has seldom been used to separate whole viruses (Semancik, 1966; Tiselius, Hjerten & Jerstedt, 1965). Tiselius et al. (1965) were the first to fractionate a plant virus, turnip yellow mosaic virus (TYMV) by electrophoresis in polyacrylamide gels. Semancik (1966) and Niblett & Semancik (1969, 1970) used this method for fractionation and characterization of the components of purified isometric plant viruses, but separation in polyacrylamide gel was not possible for viruses with elongated particles, such as tobacco mosaic virus (TMV) or tobacco rattle virus.

In this paper we report the electrophoresis of viruses with elongated particles in polyacrylamide-agarose gels of low polyacrylamide concentration. This method has been used before to fractionate nucleic acids and ribosomes (Peacock & Dingman, 1968; Dahlberg, Dingman & Peacock, 1969).

Interferon inducers are more active prophylactically than when given after infection. Potentially, therefore, in many clinical situations interferon inducers would have to be given repeatedly in order to maintain a continuous state of protection from virus infection. The known hyporeactivity of the serum interferon response (Ho, Postic & Ke, 1968; De Clercq & Merigan, 1970) is apparently a serious limitation to this approach. The hyporeactivity of the interferon response to Poly I:C has recently been carefully examined (C. E. Buckler, H. G. Du Buy, M. L. Johnson & S. Baron, personal communication; Du Buy et al. 1970). It was concluded that the ability to obtain high, sustained levels of interferon is hampered by hyporesponsiveness to restimulation. Adjustment of the dosage schedule, however, resulted in sustained moderate levels of serum interferon production for a period of 7 days or more.

Evidence of genetic recombination in foot-and-mouth disease virus (FMDV), a small RNA-containing virus, has been found both in crosses of mutants of the same strain (Pringle, 1968; Pringle et al. 1970), and in crosses of serologically related strains (Pringle, 1965; Pringle & Slade, 1970). In both cases the frequency of identifiable recombinants in the progeny was low. This is not unexpected because the genome of FMDV is small; the most recent estimates of the molecular weight of FMDV RNA are given as 2.8 × 106 (Wild & Brown, 1970) and 4 × 106 (Ohlbaum et al. 1970). The range of recombination frequencies observed between different temperature-sensitive(ts) mutants of FMDV was similar in magnitude, however, to that obtained by Cooper (1968) with ts mutants of poliovirus, a virus in some respects comparable to FMDV. Other RNA viruses exhibit much higher frequencies of recombination (e.g. reovirus and influenza virus), but in these cases this can be attributed to the physical substructure of the genome, rather than to its absolute size.