- Volume 45, Issue 2, 1979

The development of cytopathogenic changes in chicken embryo fibroblasts infected with the herpesvirus of the turkey, strain PB-THV1, and the release of virus particles into the supernatant of infected cultures is accelerated at temperatures higher than 37 °C and is fastest at 41 °C, the normal body temperature of chickens. The growth rate of HVT in CEF cultures was followed by determination of the number of virus genome equivalents within infected cells at various time intervals p.i. A temperature-dependent increase in the amount of virus DNA per infected cell could be detected, which is highest at 41 °C. At all temperatures tested (34, 36, 41 and 43 °C) the number of virus genome equivalents per infected cell ultimately reaches the same level. In the course of infection, virus DNA in CEF cultures at 37 and 41 °C becomes associated with the cellular DNA, as determined by neutral CsCl gradients of the total cellular DNA and hybridization of each fraction with 32P-labelled virus-specific complementary RNA. Association of virus to cellular DNA occurs earlier at 41 than at 37 °C. However, the same proportion (45%) of the total virus DNA appears ultimately to be associated with cellular DNA at both temperatures. A temperature-shift of CEF cultures infected with PB-THV1 from 41 to 37 °C 24 h p.i. resulted in the same replication kinetics of virus DNA as was found at 41 °C.

The pathogenic strain Italien and the apathogenic strain Ulster of Newcastle disease virus have been compared with respect to organ tropism and spread of infection in 11-day-old chick embryos. After infection of the endodermal layer of the chorioallantoic membrane by intra-allantoic inoculation with strain Italien, high virus titres are found in all extra-embryonic membranes and fluids and in the embryo itself. Infection results in early death of the embryo. In contrast, after infection with strain Ulster by the same route of inoculation, high virus titres are found only in the allantoic sac and embryos are not killed. Inoculation with strain Italien on to the ectodermal layer through an artificial air sac results in rapid spread of infection in the chorioallantoic membrane and the embryo dies before the virus invades other tissues including the embryo. Under the same conditions of infection, strain Ulster neither spreads within chorioallantoic membrane nor does it kill the embryo. Virus spread in each germinal layer of the chorioallantoic membrane was analysed by immune fluorescence. These studies showed that endoderm as well as mesoderm and ectoderm allowed the spread of strain Italien, whereas only the endoderm is permissive for strain Ulster. These differences in host range are based upon differential activation of the virus glycoproteins by proteolytic cleavage. The glycoproteins of strain Italien are cleaved in each germinal layer, whereas those of strain Ulster are cleaved only in endoderm. These studies demonstrate that, in the system analysed here, spread of infection and organ tropism are important factors for pathogenicity and both of these factors are determined by the susceptibility of the virus glycoproteins to proteolytic cleavage.

The new antiviral substance phosphonoformate (PFA) has been tested in a cell-free system for its effect on reverse transcriptases from an avian retrovirus (avian myeloblastosis virus, AMV) and from mammalian retroviruses (Rauscher leukaemia virus, RMuLV; bovine leukaemia virus; baboon endogenous virus; simian sarcoma virus; visna virus). The observed inhibitory effect of PFA has been compared with that of a structurally related substance, phosphonoacetate (PAA). Phosphonoformate, at a concentration of 100 µm, reduced the activities of all the above mentioned polymerases by 90% when (rA)n.(dT)10 was used as a template/primer. The dose-response curves for AMV and RMuLV polymerases primed with (rA)n.(dT)10 showed PFA to be a 1000-fold more active than PAA; the RMuLV polymerase activity was reduced to 50% after incubation with 0.7 µm-PFA and 0.7 mm-PAA, respectively. There was no difference in PFA inhibition of virus-associated and purified reverse transcriptase activity. Results with various synthetic templates showed that both the RNA- and the DNA-dependent polymerase activities of reverse transcriptase were inhibited by PFA. The endogenous polymerase activity of AMV was inhibited to 50% at 100 µm-PFA, while PAA had no effect. The PFA inhibition was dependent on whether Mg2+ or Mn2+ was used as divalent cation in the assay. Phosphonoformate arrested DNA synthesis immediately after being added to the assay system. The mechanism of inhibition of the AMV polymerase was non-competitive with respect to substrate and template and the apparent inhibition constants were 16 µm and 9 µm, respectively.

Antiserum to pure M-protein extracted from PR8 virions neutralized the infectivity and inhibited the haemagglutinating activity of various influenza A virions. It agglutinated concentrated suspensions of these virions and fixed complement in their presence. Antibodies to M-protein were readily absorbed by intact virions or by spikeless particles obtained after proteolytic treatment, giving clear evidence that M-protein is exposed at the surface of the virus envelope. The data suggest that when antibodies to M-protein occupy specific ligands exposed at the surface of the virion they interfere with sites critical for infectivity and haemagglutinating activity.

We describe in vitro conditions for packaging of exogenous DNA of Salmonella phage P22 which has terminally redundant, circularly permuted DNA. The method is a modification of the Kaiser-Masuda procedure. The most important aspect is to prepare all components (proheads, enzymes and concatemeric DNA) in end − cells. The influence of several factors such as DNA- and Mg2+ concentration and kinetics has been investigated.

Priming by mouse interferon pre-treatment resulted in an accumulation of interferon mRNA in poly(rI).poly(rC)-treated L cells, starting early in the period of interferon synthesis. On electrophoresis, the priming activity of an interferon preparation co-migrated with the antiviral activity, which suggests identity of the functional principle(s) for these activities.

Forty-two primary human-mouse cell hybrids, derived in two separate experiments, were treated with Newcastle disease virus (NDV): eight hybrids were found to produce human interferon and this was shown in every case to be predominantly of the fibroblast type. An extensive analysis was made in terms of karyotype and marker enzymes on all the eight hybrids producing interferon and also on five hybrids which did not produce interferon, five randomly selected hybrids and eleven subclones resistant to diphtheria toxin. The results suggest that, contrary to previous reports, a gene on chromosome 5 is not involved in production of human interferon. Its production was however correlated with the presence of chromosome 9 in the hybrids. Analyses of two sets of human-Chinese hamster hybrid subclones from two different crosses were also consistent with the assignment of a human interferon gene to chromosome 9.

Demethylchlortetracycline (DMCT), doxycycline and, to a lesser extent, chlortetracycline were capable of mediating the in vitro photoinactivation of Venezuelan equine encephalitis (VEE) virus. Other tetracyclines tested were found to be inactive in this respect. However, no correlation between chemical structure and photosensitizing activity could be established. The photoinactivation of VEE virus by DMCT proceeds through a photodynamic mechanism as shown by the absolute requirement of O2 for the inactivation to take place. The photoinactivating effect of DMCT was also exerted upon other animal viruses tested, i.e. vesicular stomatitis virus, herpes simplex virus and poliovirus, even when, in the case of poliovirus, the capsid seems to be impermeable to the tetracycline. The fact that the two most effective photosensitizing tetracyclines for VEE virus are also the drugs more frequently associated with drug-induced phototoxicity in humans, suggests that virus photoinactivation could be used as a screening procedure for potentially phototoxic drugs developed for human application.

The physical state of the Epstein-Barr virus (EBV) DNA in three cell lines which spontaneously produce virus has been characterized. Circular EBV DNA molecules have been found in P3HR-1, B95-8 and M81 cells. The size of the intracellular M81 circular EBV DNA molecules is comparable with the linear virus genome isolated from virus particles but the circular P3HR-1 and B95-8 DNA molecules are shorter than the virion DNA. In addition to the circular form, some EBV DNA with physical properties indicative of integrated sequences was found in all three producer cell lines. There was no marked change in the amount of either the circular of integrated forms of EBV DNA when these producer cell lines were grown in the presence of phosphonoacetic acid to suppress the spontaneous virus production which occurs in a small percentage of the cells in untreated cultures.

We have directly tested the hypothesis that single-stranded cytoplasmic A particle-associated DNA (ss CAP DNA) is a murine mammary tumour virus (MMTV) proviral intermediate by hybridizing 125I-labelled ss CAP DNA to MMTV RNA or to MMTV complementary DNA (cDNA). 125I-labelled CAP DNA did not form duplexes with either MMTV RNA or MMTV cDNA. In contrast, CAP RNA hybridized readily with MMTV cDNA. CAP RNA contained all the MMTV virus sequences, but at lower concentrations than in MMTV virus particles.

Single-stranded CAP DNA hybridized readily with mouse DNA from several sources. A study of the rate of hybridization of CAP DNA to cell DNA at various driver to probe ratios showed that its rate of hybridization is similar to that of tumour cell DNA reassociation. Further, in reassociation studies accelerated by using the phenol emulsion reassociation technique (PERT), CAP DNA originally isolated as single-stranded DNA was shown to reanneal (70%), to protect 125I-cell DNA to the same extent (67%) and to do so with kinetics of reassociation equivalent to that of mouse DNA. Although CAP DNA isolates were slightly enriched for MMTV specific sequences when compared to total cellular DNA, we conclude that the majority of ss CAP-associated DNA is equivalent to a random sample of total tumour cell DNA.

Two polypeptides (imm-a and imm-b) which are not induced by an immunity mutant T4Dimm2 but by a wild-type strain T4D were identified by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Their mol. wt. were 77000 and 45000, respectively. These polypeptides exhibited a similar kinetic pattern of synthesis. Within a few minutes p.i. the primary phage established the system that inhibited imm gene expression of superinfecting phage. This was shown by measuring both the phenotypic expression of immunity and the synthesis of imm gene polypeptides. The expression of two other immediate-early genes, namely genes s and 30, and the early gene 33, was not affected by primary infection.

Cucumber mosaic virus (CMV) strains S and WT were selected as reference viruses to provide the double-stranded and 3H-labelled single-stranded RNAs in competition hybridization experiments which determined the degree of nucleotide sequence homology among 18 CMV isolates of different origin. A group of 3 and a group of 14 isolates (S and WT groups, respectively) were found to have extensive, if not total, sequence homology among their members and little, if any, homology between the groups. Only one isolate obtained from Ixora (CMV-Ix) was partially homologous (43%) with the WT group and had no homology with the S group. The finding of two main groups of CMV isolates, based on nucleic acid homology, parallels the divisions on the basis of symptomatology and serological properties proposed previously by other workers.

Capsule components of two strains of a granulosis virus (GV) which infect the armyworm (Pseudaletia unipuncta) consist of capsule proteins and, in one strain, a synergistic factor, which had higher affinities for hexylamine than the viral components of the enveloped nucleocapsids. The synergistic factor in the capsule of the Hawaiian GV strain was separated from the capsule components using agarose gel coupled with antibody against the synergistic factor. Analysis by peptide mapping showed that the capsule proteins of the two viruses were similar, but that the synergistic factor differed markedly from them in its constitution.

Highly infective nucleic acid preparations were made from Nicotiana clevelandii leaves infected with carrot mottle virus (CMotV). The infective material had the properties of a single-stranded RNA but was less than 1% of the total RNA in the preparations. It had an apparent mol. wt. of about 1.5 to 1.6 × 106 in agarose-polyacrylamide gels and a sedimentation coefficient of about 28 to 30S in linear-log sucrose gradients. The infective RNA did not contain any considerable poly-adenylate sequence, nor did it require an associated protein for infectivity.

This report deals with physical characterization of the generalized transducing Proteus mirabilis phage 5006M. The morphology of the phage is presented, the buoyant density was determined (1.491 g/ml) and the G + C content of the phage DNA was found to be 44%. The phage genome has a length of 14.8 µm and mol. wt. of 30.7 × 106. Denaturation mapping revealed non-random circular permutation of the phage DNA. The genome exhibits 3.6% terminal redundancy as shown by homoduplex analysis. The existence of concatemeric precursors of phage 5006M DNA is inferred and the results are interpreted in terms of a sequential headful packaging mechanism.

In cells infected with herpes simplex virus a protein associated with the small subunit of ribosomes became phosphorylated. It was not detectably labelled with 14C-amino acids added after infection and is therefore probably a cellular protein. The phosphorylated ribosomal proteins from HSV-1- and HSV-2-infected cells were indistinguishable electrophoretically and had an apparent mol. wt. of about 48000.

Phosphorylation of the 48K protein was detected 2 to 3 h after infection and reached a maximum rate at 4 to 5 h. It was prevented by adding cycloheximide at 2 h, or actinomycin at 1.5 h p.i., or azetidine at the beginning of infection. The phosphorylation did not occur on reversal of a cycloheximide block in the presence of actinomycin, confirming that it is not caused by a virus α-polypeptide.

Virus that had been irradiated with u.v. light, although still able to suppress synthesis of cellular protein and DNA, did not induce phosphorylation of the 48K ribosomal protein. Therefore the phosphorylation is not responsible for the suppression of host synthesis.

The α polypeptides ICP 4, 0, 22 and 27 are also phosphorylated but, in contrast to that of the ribosomal protein, their phosphorylation does not depend on the synthesis of β and γ polypeptides. It is probably mediated by a host enzyme.

Two African green monkey kidney (AGMK) cell lines, 37RC (interferon-producing) and Vero (non-interferon-producing), were infected by egg-grown Sendai virus passaged in eggs at high or low m.o.i. The appearance of haemadsorption, and cytopathic effect (c.p.e.) as well as the presence of haemagglutinating virions in the supernates were much more pronounced with a virus seed obtained with 10−3-diluted passages than with a seed obtained with undiluted inoculum. They were also independent of interferon production (data obtained in 37RC and Vero cells were almost superimposable).

In studies carried out with the virus seed prepared at high dilution the establishment of infection was maximal when monolayers were infected as soon as 5 h after trypsinization and cell seeding, regardless of cell density. Virus in supernates obtained from cultures infected 5, 20 or 50 h after seeding exhibited a greatly reduced infectivity for monkey cells, but not apparently for chick embryos. Trypsin treatment of the virus supernates restored their infectivity for AGMK cells more efficiently for virus released from cells infected 5 h after seeding than for virus released from cells infected later after seeding. In keeping with these observations, virus in supernates from cultures infected 5, 20 or 50 h after seeding contained increasing amounts of auto-interfering virions. The decreased infectivity obtained in cell supernates was not accounted for by major differences in virus RNA synthesis. Similarly, the optimum infection established in cells seeded only for 5 h was not due to increased virus adsorption.

Several lines of cells surviving first infection with egg-grown Sendai virus have been obtained and kept in cultures for 3 to 18 months. Virus release and c.p.e. apparently become reduced in the cells surviving the first infection until the newly repopulated monolayers must undergo trypsinization. Weekly protease treatments of the cells reactivate all parameters of virus infection which again will tend to disappear slowly and then reappear following each trypsinization (‘intermittent’ carrier state). The establishment and the ‘intermittent’ reactivation of these lines were not prevented by the inclusion in the medium of anti-Sendai virus serum. Temperature-sensitive ts functions do not seem to play an important role in this virus-host relationship.

Parma wallaby herpesvirus (PWHV) has been characterized by a number of properties. Electron microscopic examination of thin sections of infected cell nuclei revealed virus nucleocapsids with various morphologies characteristic of herpesviruses; enveloped particles were seen in cytoplasmic vacuoles and outside cells. Negatively stained virus preparations from the medium of infected cell cultures contained particles with the typical appearance of naked and enveloped herpesviruses. The DNA of PWHV had a mean buoyant density in preparative caesium chloride gradients of 1.712 g/ml, giving an estimated base composition of 51% guanosine plus cytosine. The virus was able to replicate in all marsupial cells, but not in most eutherian cells tested, and a single cycle of infection lasted about 25 h. Infectivity was destroyed by a number of agents including lipid solvents, acid pH and heat. The observed properties support the classification of this virus as a new member of the herpesvirus family.

Tests to detect complementation between ts mutants of herpes simplex virus types 1 and 2 by infectious centre and yield of virus assay were investigated. Progeny analysis of both intratypic and intertypic complementation showed a considerable proportion of recombinant or ts + virus in the progeny; this was more marked in the infectious centre tests. Virus of intermediate temperature-sensitivity was produced in intratypic as well as intertypic complementation. Reduction in the input multiplicity of one of the two mutants in the test to extremely low levels did not prevent complementation, suggesting that non-infectious particles probably contribute to complementation. Demonstration of virus DNA synthesis in mixedly-infected cells at the non-permissive temperature was used to detect complementation between DNA-negative mutants.

A heterogeneous population of virions is generated by measles virus-infected cells. These particles are partially separable by sucrose density centrifugation into three peaks. Each population is stable and contains infectious particles. The particles of all three populations contain at least six polypeptide species that differ between particle populations only in quantity. All three populations contain a 50S RNA species, and the heaviest density peak also contains an additional species of 43S RNA. The difference between these results and previous studies with measles virions will be discussed.