- Volume 43, Issue 1, 1979

Varicella-zoster virus (VZV) has been isolated and serially propagated in a continous cell line derived from a human malignant melanoma tumour. Human melanoma cells (HMC) have been further evaluated as a substrate for the production of cell-free virus and compare favourably with human embryo cells. Within 60 h after inoculation with VZV-infected cells, HMC monolayers incubated at 32 °C exhibited advanced syncytial cytopathic effect, and the overlying culture medium contained > 102 p.f.u./ml. The cell pellet from a mechanically dispersed 150 cm2 monolayer yielded 105 p.f.u. after sonic disruption, while the medium (‘scraping medium’) in which the cells had been harvested contained up to one log more infectious virus than was found in the cells from the same monolayer. When infected cells were subjected to Dounce homogenization, most of the infectivity was found in the nuclear fraction.

The concentration and purification of cell-free virus were also investigated. Concentration was carried out by three methods: ultracentrifugation, dialysis against hydrophilic compounds and liquid polymer phase separation. The first two procedures caused considerable loss of biological activity, whereas precipitation with 8% polyethylene glycol resulted in a 50-fold increase in titre. Purification of cell-free virus with retention of infectivity was achieved by rate zonal centrifugation in linear potassium tartrate gradients. Infectious virus was also recovered after sedimentation in combination equilibrium-viscosity gradients of potassium tartrate and glycerol, but not after centrifugation to equilibrium in caesium chloride gradients.

The isolation of three bacteriophages active on Hyphomicrobium is reported. These phages only infected Hyphomicrobium isolates; a variety of other Gramnegative bacteria were immune to infection with these phages. Each of these phages contained double-stranded DNA. Hyø1a had a polyhedral head and a short, non-contractile tail. Hyø22a was morphologically similar to Hyø1a. They appear to be genetically distinct however, as they could be distinguished from one another by their host ranges, their densities in CsCl, and the mol. wt. of their major protein components. Hyø32a had a polyhedral head and a long, flexible, non-contractile tail with fibres attached to its distal end. Each of these bacteriophages selectively adsorbed to daughter cells developing at the distal end of prosthecae on mother cells.

Rat embryo cells transformed by two ts mutants of HSV-2 strain HG 52 and also by u.v.-irradiated HSV-2 strain 333 were inoculated into highly inbred host rats, either newborn or which had undergone various immunosuppressive treatments. The latent period before a palpable tumour (a fibrosarcoma) was detected varied directly with the degree of immunosuppression of the host. Transformed cells could form tumours after a latent period of nearly 2 years. All tumours were invasive and in some cases metastatic. The continuing expression of HSV information in 10 tumour cell lines was demonstrated by perinuclear and cytoplasmic staining in immunofluorescence studies using a rat antiserum directed against the early polypeptides of HSV-2 HG 52 infection and a rabbit serum prepared against a 24 h cell infection with HSV-2 HG 52 ts 1. Sera from tumour-bearing rats fluoresced the surface of unfixed human or rat embryo cells 4 to 5 h after infection with HSV-2 HG 52. In addition the rabbit antiserum (4740 or 4741) fluoresced the surface of 80% of the tumour cells in culture. Transplanted tumours after 20 passages in vitro and taking up to a year to again form a tumour in a host rat also showed specific HSV cytoplasmic and perinuclear fluorescence in tests with the rat antiserum directed against early polypeptides of HSV-2 lytic infection.

Five phages which are morphologically similar to coliphage T7 but attack other host bacteria have been compared to T7 and to its relative, T3, by the following criteria: (a) cross-reactivity with antisera against T7 and T3, (b) DNA base sequence homologies, as determined by the C0t technique, (c) synthesis of two phage-coded enzymes: RNA polymerase and SAMase, (d) patterns of phage-directed protein synthesis, as determined by SDS-polyacrylamide gel electrophoresis followed by autoradiography, (e) SDS-polyacrylamide gel electrophoresis of phage coat subunits. As judged by all these criteria, Pseudomonas phage PX3 is not related to T7; thus, morphological similarity was attributed to convergent evolution. The other phages, i.e. Serratia phage IV, Pseudomonas phage gh-1, Citrobacter phage ViIII and Klebsiella phage No. 11, were considered to be related to T7 on the basis of similarities in the patterns of phage-coded proteins and because, early after infection, these phages induced, as T7 does, an RNA polymerase which specifically transcribes the DNA of the homologous phage. Phages IV and No. 11 also induced the early synthesis of SAMase (previously only known to occur upon T3 infection). With the exception of phage IV, however, DNA base sequence homologies with T7 or T3 seem to be poor or non-existent. The tested phages, again with the exception of phage IV, did not react with antiserum against T3 or T7.

It is concluded that a particular pattern of phage-directed protein synthesis (as characterized by polyacrylamide gel electrophoresis and enzyme tests) may provide evidence for phylogenetic relationships between phages, even in cases where other criteria, such as genetic recombination, serological cross-reaction, and DNA base sequence homologies, fail to indicate relatedness.

Replicative intermediate (RI), replicative form (RF) and single-stranded (SS) RNA have been isolated from BHK cells infected with a bovine enterovirus by salt precipitation and gel filtration techniques. Kinetic experiments showed that at no time up to 16 h post-infection (p.i.) did the amount of RF exceed that of RI or SS RNA.

Electrophoresis of RF on 1.5% polyacrylamide-agarose gels showed that at least three species of double-stranded RNA were present, one of which was associated with an accessible poly(A)-containing tract. All of the RF was denatured by 99% dimethylsulphoxide (DMSO), although reannealling occurred rapidly when samples were returned to aqueous conditions. No evidence for circular structures in the RF molecular population was found by use of caesium sulphate density gradients containing ethidium bromide.

Treatment of RI with ribonuclease produced double-stranded RNA molecules, some of which were smaller in size than intact RF. Denaturation with DMSO and analysis on 99% DMSO sucrose gradients showed that the RI did not contain single strands of greater length than virion RNA. A portion of the RI bound to poly(U)-Sepharose 4B columns. The poly(A) tracts involved were present only in the nascent RNA strands with greatest sedimentation coefficients (30 to 35S).

Bovine enterovirus induced SS RNA was heterogeneous with regard to both sedimentation through sucrose gradients and mobility on acrylamide gels compared to purified virion RNA. The reason for this difference has never been satisfactorily resolved. Sedimentation through 99% DMSO-sucrose gradients showed that the heterogeneity was due to aggregation rather than any variation in chain length or conformational differences.

Our results support the single-stranded template model rather than a circular model for picornavirus RNA replication.

Yields of greater than 107 p.f.u./ml at 28 or 37 °C of the alphavirus Sindbis and the flavivirus Kunjin were obtained in the Aedes albopictus (Singh) cell line, the latent periods being 4 to 6 and 10 to 12 h, respectively. Despite a high background of host protein synthesis, virtually all the virus-specified proteins of the flaviviruses Kunjin, Dengue-2 and Japanese encephalitis were labelled and resolved by slab gel electrophoresis of infected and uninfected cell proteins. In contrast, only one induced protein, of mol. wt. 30000, was identified in cells labelled during Sindbis virus infection. The envelope glycoprotein V3 of Kunjin virus was resolved as a double band in samples of infected cytoplasm labelled with 3H-glucosamine, similar to that of carbohydrate-labelled V3 in vertebrate (Vero) cells. Attempts to reduce host protein synthesis selectively during labelling periods were unsuccessful using either a hypertonic inhibition block or treatment with 0.1 µg actinomycin D per ml. The most efficient labelling of Kunjin virus-specified proteins was achieved at 37 °C in the presence of actinomycin D. The largest non-structural flavivirus protein NV5 migrated slightly faster than NV5 from infected vertebrate (Vero) cells. The small non-structural proteins NV1, NV1½ and NV2 from infected mosquito cells were successively trimmed during post translational periods exceeding 70 min, compared to much shorter periods reported previously for post translational modifications of these proteins in vertebrate cells.

Various cell lines persistently infected with para-influenza 1 virus, HVJ strain, were less susceptible to the antiviral action of interferon than the same cell lines when not infected with HVJ. When Vero cells persistently infected with a temperature-sensitive strain of HVJ were incubated at 38 °C, a non-permissive temperature, they became fully susceptible to interferon, whereas neither the haemadsorbing nor the cell-associated haemagglutinating activity of the virus was expressed. These findings suggest that the lowered interferon susceptibility of virus-carrier cells may be related to the maturation of virus in them.

It was found that the low susceptibility of virus-carrier cells to interferon is not due to blocked adsorption of interferon or to inability of the cells to respond to interferon. Studies with actinomycin D suggest that some step (or steps) before the synthesis of the messenger RNA for the antiviral protein is blocked.

The RD-114 virus rapidly induced syncytia in the human transformed cell lines, RSa, RSb and IFr. Treatment of the virus with heat or ultrasonic vibration completely eliminated the syncytium-forming activity. Irradiation with u.v.-light or treatment with β-propiolactone (BPL) reduced but did not completely destroy the activity. Pre-treatment of the cells for 16 h with 25 to 500 units/ml of human leucocyte interferon (Le-IF) or fibroblast interferon (F-IF) significantly reduced formation of syncytia by active virus or inactivated (u.v. or BPL) virus. This activity of interferon was inhibited by treatment of the cells with cycloheximide. Interferon did not increase the binding of 3H-uridine-labelled RD-114 virus to the cells. It is postulated that interferon treatment altered the plasma membrane of the cells and thus reduced their capacity to fuse.

Early after infection of cells with cytomegalovirus, the membranes were modified with respect to both glycoprotein composition and immunological specificity. Virus-specified antigens were inserted into the plasma membrane at 24 h after infection, as much as 2 days before virion and dense body maturation. Although at least four virus-induced glycoproteins were synthesized and bound to plasma and microsomal membranes between 20 and 24 h after infection, virus-specified antigen accumulated primarily on the plasma membrane. In contrast, at late times (72 h) after infection when virus nucleocapsids can be detected in the nucleus, virus-specified antigen was prominent on the plasma, endoplasmic reticulum and nuclear membranes. It is proposed that the virus-specified glycoproteins and antigens of this herpesvirus accumulate first on the plasma membrane and then on internal membranes. The appearance of virus-specified antigen on internal membranes coincides with the commencement of virion and dense body envelopment.

In Nicotiana glutinosa L. formation of local lesions on lower leaves inoculated with tobacco mosaic virus increased the susceptibility of the upper leaves to infection in a subsequent inoculation. The increase in susceptibility was detected as an increase of up to 3.5-fold in the number of lesions produced on the upper leaf and a corresponding increase in the amount of virus RNA synthesized. The concentration of endogenous abscisic acid in the upper leaves was negatively correlated with susceptibility to infection. This acquired systemic susceptibility to infection in N. glutinosa is in direct contrast to the acquired systemic resistance to infection reported to occur in hypersensitive varieties of Nicotiana tabacum under similar conditions. Mechanisms which might be involved in the acquisition of systemic resistance or susceptibility are discussed.

The tryptic peptides of aminoethylated coat protein from cowpea chlorotic mottle virus were isolated and their amino acid compositions determined. Twenty-three major peptides accounted for 177 of the 182 amino acid residues which comprise the coat protein. The tryptic peptides were identified with respect to their position on two-dimensional peptide maps of cowpea chlorotic mottle virus protein, to aid future analysis of coat protein variants.

Analysis of the infected cell polypeptides and the DNA restriction profiles of 31 HSV-1 isolates from the trigeminal, superior cervical and vagus ganglia from 17 individuals (12 U.S.A., 2 Japanese, 3 Norwegian) could be classified as 15 different virus strains. With the exception of the three Norwegian isolates which gave identical profiles, virus isolates from the ganglia of different individuals could all be distinguished from one another. In contrast virus isolates from the trigeminal, superior cervical and vagus ganglia of the same individual, or virus isolates from the left and right ganglia of the same individual or multiple isolates from different explants of a single ganglion were indistinguishable. In conclusion, a single virus strain infects each individual initially and virus descended from this event subsequently infects and becomes latent in different cells of the same ganglion as well as in different ganglia.

NIH 3T3 mouse cells were infected at very high multiplicity with murine sarcoma/leukaemia virus (MSV/MLV) and then cloned. All of the 48 clones obtained were morphologically transformed, all but one showed anchorage-independence of growth, typical of MSV-transformed NIH 3T3 cells and most (91%) produced MSV/MLV. When cells which had been pre-treated with 104 units/ml of purified interferon (IF) were infected under the same conditions and then cloned in the presence of the same amount of IF, only 6 of a total of 63 clones were morphologically transformed. All but these 6 showed a degree of anchorage-independence typical of the uninfected parental cells and very few (2.4%) produced virus. Furthermore, the MSV genome could not be rescued in any of the 23 clones tested and only 1 out of 10 clones produced tumours. The properties of these clones remained stable over a period of 10 to 20 passages in the absence of interferon. We conclude that interferon can irreversibly block an early step in the MSV/MLV infectious process.

Recombinants of human influenza type A viruses, A/Krasnodar/101/1959 (H2N2) or A/Habarovsk/15/1976 (H3N2), and fowl plague virus (FPV), strain Weybridge (Hav1Neq1) were obtained. The genome of the recombinant obtained by recombination of influenza A/Habarovsk/15/1976 virus and FPV contained the genes 4 (HA) and 6 (NA) derived from the influenza A/Habarovsk virus and all the other genes [1, 2, 3, 5 (NP), 7 (M), 8 (NS)] from FPV. The genome of the recombinant of A/Krasnodar/101/1959 virus and FPV contained the genes 2, 4 (HA) and 6 (NA) derived from influenza A/Krasnodar virus and all the other genes [1, 3, 5, (NP), 7 (M), 8 (NS)] from FPV. The recombinants, like FPV, gave high virus yields in chick embryos and could multiply at high temperatures (40 and 42 °C), but, like human influenza viruses, were non-pathogenic for chickens and did not replicate in chick embryo fibroblast culture, but did replicate in a human conjunctiva cell line, clone 1-5C-4. The virion transcriptase of the recombinants, in a number of properties determined in vitro, was similar to FPV transcriptase but not to the human influenza virus enzyme.

A host-dependent conditional lethal mutant of vaccinia virus strain DIs was rescued to form plaques in a non-permissive cell line JINET co-infected with Yaba virus, a poxvirus serologically distant from the rescued virus. The efficiency of plaque formation under optimal conditions in this system was comparable to that in Vero cells which were permissive for the mutant. The plaque number of the mutant in JINET cells was influenced greatly by the multiplicity of Yaba virus, the interval between inoculations with DIs and Yaba virus and the maintenance medium for pre-infection of cells with Yaba virus. The plaque formation by DIs in JINET cells was suppressed or inhibited when the duration of pre-infection with high multiplicities of Yaba virus was prolonged. Thus, Yaba virus possessed both rescuing and inhibitory activities on DIs and the plaque number of DIs in doubly infected cells was thought to be determined by the balance between the two activities. Ultraviolet light-inactivated Yaba virus retained rescuing activity on DIs in JINET cells.

A filterable agent, designated 13p2 was isolated from homogenized juvenile oyster tissue inoculated on to bluegill fry (BF-2) cell cultures. The oysters were from a hatchery on Long Island Sound, New York. Successive passages resulted in progressive cytopathic effects (c.p.e.) consisting of discrete plaques containing large syncytia seen within 2 to 3 days in cultures held at 15 °C. The agent was concentrated from supernatant fluids by ultracentrifugation. Negative stained preparations examined by electron microscopy revealed icosahedral particles with a mean diam. of 79 nm.

Virus replication in tissue culture occurred at both 15 and 23 °C. Susceptible fish cells in addition to the BF-2 included brown bullhead, Atlantic salmon, guppy embryo and walleye fry lines. Limited c.p.e. occurred in Atlantic salmon heart cells while rainbow trout gonad, rainbow trout spleen and fathead minnow cells were refractory to cytopathic changes.

Biochemical and physical characteristics suggested the 13p2 virus belonged to the family Reoviridae. The possibility that this virus is a known reovirus, present only as a contaminant, was ruled out on the basis of serological results and failure of avian or mammalian cells to support its growth. The 13p2 agent may be an undescribed virus. Further investigations concerning the identity of this virus and its capabilities as a pathogen in fish and shellfish are under way.

Eight ‘strains’ of myxoma virus, spanning the complete spectrum of virulence, were tested for ability to produce plaques on rabbit kidney cells at varying temperatures and pH values. A positive correlation was found between virulence in rabbits and ability to produce plaques at supra-optimal temperature and at low pH.

Temperature-sensitive (ts) mutants of adenovirus type 2 (Ad2), which are deficient in virus DNA synthesis at the non-permissive temperature, have been used to investigate whether virus DNA replication is required for the occurrence of high mol. wt. Ad2 DNA (> 100S, 50 to 90S) in human cells productively infected with Ad2. The high mol. wt. virus DNA has been previously shown to consist of virus and cellular DNA molecules covalently linked. The present data indicate that after infection with DNA− ts mutants, the production of high mol. wt. virus DNA is much less sensitive to restrictive conditions than the synthesis of unit length (34S) Ad2 DNA. This finding lends further support to the idea that the occurrence of high mol. wt. virus DNA is independent of the synthesis of unit length virus DNA.

Inhibitors of glycolysis, oxidative phosphorylation, protein synthesis, membrane Na+-K+ transport and microfilament and microtubule function have been employed to elucidate the mechanism of influenza virus uptake by CAM and CEF cells. Electron microscopy demonstrated uptake of virus by viropexis in the presence of all these inhibitors. Utilizing a pulse labelling technique, virus entering CEF cells in the presence of inhibitors was shown to initiate specific virus polypeptide synthesis after neutralization of remaining extracellular virus and removal of the inhibitors. As a consequence of these findings an energy independent mechanism of viropexis has been proposed.