- Volume 39, Issue 2, 1978

The structural polypeptides of two strains of measles virus grown in Vero cells were analysed in SDS-PAGE slab gels. Six major polypeptides were identified with mol. wt. of 79000, 72000, 60000, 43000, 40000 and 36000. The largest polypeptide was sensitive to trypsin digestion and was the dominant glycosylated polypeptide identified when the virus was grown in medium containing 3H-fucose or 3H-glucosamine or when the virus was treated with galactose oxidase and labelled with 3H-sodium borohydride. It is concluded that the 79000 mol. wt. polypeptide represents the haemagglutinin. Treatment with non-ionic detergent removed this polypeptide and also the 40000 mol. wt. polypeptide from the virus envelope. The 40000 mol. wt. polypeptide is probably associated with haemolysin and cell fusion activities and is analogous to the F1 of paramyxoviruses. A polypeptide of mol. wt. approx. 20000 detected after glycoprotein labelling may represent the F2 of measles virus. The 43000 mol. wt. polypeptide co-migrates with cellular actin and is the only major measles polypeptide that is heavily labelled when the virus is grown on Vero cells prelabelled with 35S-methionine. Thus it may represent cellular actin incorporated into the virus during maturation. The quantity of the 72000 mol. wt. polypeptide relative to the other major polypeptides varied considerably in different virus preparations. The role of the polypeptide could not be defined. By analogy with previously published data the 60000 and 36000 mol. wt. polypeptides are inferred to represent nucleocapsid and membrane proteins, respectively.

Adult mice inoculated with Semliki Forest virus (SFV) were protected from a lethal infection of the central nervous system by intranasal administration of defective-interfering (DI) SFV. DI SFV was prepared by eight passages at high m.o.i. in BHK 21 cells. Mice were treated with unpurified, unconcentrated tissue culture fluid which had been u.v.-irradiated to inactivate the infective virus present. Prevention of death was maximal when the DI virus was administered simultaneously with the infecting inoculum, and under the same conditions multiplication of infective virus in the brains of treated mice was reduced by 105-fold. It was shown that DI SFV was propagated in mouse brains following intranasal inoculation and it was concluded that protection was brought about through the intrinsic interfering capacity of the DI virus.

Leukaemia-resistant C57BL/6 mice inoculated with the Friend complex (FLC) present a transient but definite depression of the ability to develop IgM and IgG antibody-producing cells to sheep red cells (SRC). In this paper it is shown that this immunodepression cannot be attributed solely to the lymphatic leukaemia virus (LLV) component of FLC which is immunodepressive in susceptible mice. This conclusion was reached by investigating the immunological reactivity of C57BL/6 mice following inoculation with two isolates of LLV. Circumstantial evidence obtained by examining the replication of FLC and LLV, the antibody response to E. coli lipopolysaccharide (LPS) and the ability of syngeneic macrophages administered together with the antigen to restore the response to SRC points to the same conclusion. There were also indications that the low sensitivity to depression by viruses of the Friend complex exhibited by the anti-LPS antibody response is due to the mitogenic activity of this antigen.

Phosphonoacetic acid (PAA) inhibits the multiplication of African swine fever (ASF) virus in VERO cells. The observed inhibition of the in vivo DNA synthesis could be related to the in vitro inhibition of a virus-induced DNA polymerase activity present in cytoplasmic extracts from infected VERO cells.

The ordering of restriction endonuclease fragments of HSV-2 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by EcoRI, Hind III, Bgl II, Xba and Hpa I have been constructed. The mol. wt. of the various regions which constitute HSV-2 genome are very similar to the corresponding mol. wt. in the HSV-1 genome.

The absence from the medium of any of the 13 amino acids essential for cell growth has an inhibiting effect on the multiplication of adenovirus 9–15 and adenovirus 1 in HeLa cell cultures. The inhibition is accentuated by previous amino acid starvation of the cultures. Whereas with arginine deprivation, the arginine pool inside the cells is at a minimum within 30 min, the cells are assumed to adapt slowly to the new metabolic state, which is characterized by an increased ‘turnover’ of protein synthesis. With arginine deficiency and in Hanks’ BSS some synthesis of virus and capsid proteins takes place. Quantitative and possibly qualitative differences between the influence of the various deficient media were observed.

The experiments rule out DNA synthesis as a primary cause of the amino acid deficiency effect. They lead to the hypothesis that arginine deficiency inhibits the formation of an essential protein which is synthesized very late in the infectious cycle under complete MEM.

Serum obtained from mice 3 to 5 weeks after the third i.p. dose of dengue type 2 virus (DV) protected recipient mice against intracerebral challenge with DV, whereas the serum obtained after 1 and 2 weeks provided minimum protection. Adoptive intravenous transfer of immune spleen cells obtained from mice 1 to 5 weeks after immunization did not protect recipient mice against even a small dose (10 LD50) of DV. Depletion of T-cells by treatment of mice with anti-thymocyte serum did not potentiate DV infection. Development of a cell-mediated immune response (CMI) against DV was noted only at two periods by the leucocyte migration inhibition test (LMI), with borderline values of 20 and 21%. Dengue virus did not cause illness or death in mice when given by i.p. or i.v. routes and this was not affected by pre-treatment of mice with silica to damage local macrophages. It is concluded that humoral antibody plays a critical role in recovery from primary dengue virus infection of mice whereas CMI and macrophages appear to have no protective role.

The mechanism for regulating interferon production was investigated in relation to accentuation of production in serum-free human diploid cells (strain WI-38) treated with N 2, O 2-dibutyryl guanosine 3′, 5′-cyclic monophosphate (db-cyclic GMP).

Interferon production in serum-free WI-38 cell cultures in response to Newcastle disease virus (NDV) was greatly reduced. In these cells, there was decreased incorporation of 5-3H-uridine into the acid-insoluble fraction, but unimpaired incorporation of U-14C-l-leucine, as compared with serum-containing cultures. When serum-free cell cultures were treated with 0.2 mm-db-cyclic GMP, incorporation of both 5-3H-uridine and U-14C-l-leucine was increased and there was an 8-fold enhancement in the yield of interferon in response to NDV. Induction of db-cyclic GMP-treated cells by NDV in the presence of cycloheximide and actinomycin D suggests that db-cyclic GMP enhances transcription of the interferon gene, and thereby augments interferon production.

Proteolytic digestion of the M-protein of Sendai virus produces a product with a mol. wt. approximately 5000 less than that of the intact protein. In the case of digestion with chymotrypsin this cleavage is quite specific and the cleaved protein can be isolated. The smaller fragment appears to be physically removed from the larger (30000 mol. wt.) fragment, rather than remaining in non-covalent association with it. The cleavage is likely to be near the N-terminus of the protein. At the present time there is no indication of the biological function of this fragment.

The nature of the restriction of herpes simplex virus replication in C 1300 neuroblastoma cells was studied. A low rate of adsorption was observed, probably due to the relatively few receptors for HSV on plasma membranes of C 1300 cells. The penetration rate of HSV to the nucleus was slow with an impaired processing of attached virus from plasma membrane to cell nucleus. Even at a high multiplicity of infection only a low percentage of the C 1300 neuroblastoma cells was permissively infected as determined by infectious centre assays. The yield of infectious HSV per virus-producing C 1300 cell was 1% of the yield from GMK control cells. The restriction in neuroblastoma cells of HSV infection could not be accounted for by sensitivity of cells to interferon or by an efficient induction of interferon. Evidence was obtained for the presence in C 1300 cells of an inhibitor of HSV replication not compatible with classical interferon. Observations on C 1300 cells maintaining many characteristics of differentiated neurons suggest that these cells may be useful as a model for studies on HSV-neuron interactions.

Optical diffraction studies of electron micrographs of potato virus T particles have been made to try to understand the variation in appearance of the particles under different staining conditions. These studies show that the particles are constructed from a helical arrangement of protein subunits lying on a primary helix of pitch 3.4 nm. They also suggest that the different surface patterns observed on the virus particles are due to stain lying in secondary helical grooves which are formed by aggregation of the structural protein subunits. The optical diffraction patterns indicate a variation in the periodicity of the secondary helix, which implies a variation in the number of structural protein subunits per turn of the primary helix.

The b1 protein, produced in leaves of Nicotiana tabacum cv. Xanthi-nc following infection with tobacco mosaic virus, has been purified to homogeneity by a procedure which involves gel chromatography and absorption on to DEAE-cellulose. One gel chromatography step was sufficient when the procedure was applied to leaf extracts made in an acid buffer, whereas two were necessary with extracts made at pH 8. The final product migrates as a single protein band on electrophoresis in both acrylamide and SDS-acrylamide gels. Its mol. wt. is estimated to be 15000 by electrophoresis and 14200 by ultracentrifugation. Amino acid analysis suggests that it contains about 136 residues of which 39 are potentially acidic, 13 basic and 16 aromatic. The absorbance coefficient is estimated to be 18.9. No evidence was found for the presence of a nucleotide component.

The infectivities of several strains of herpes simplex virus type 1 and type 2 were measured by plating on established human and Chinese hamster cell lines. Whereas all the type 1 strains reproducibly showed a 104-fold lower titre on the Chinese hamster cells, the type 2 strains showed only a 10- to 100-fold lower titre. This host range difference has been used in the analysis of recombinants between type 1 and type 2 viruses.

Exhaustion type hybridization was used to measure the amount of nuclear virus RNA complementary to the early (E) and late (L) polyoma virus DNA strands. At 36 h after infection between 2.5 and 7.3% of the newly synthesized virus RNA was complementary to the E-strand (-strand) and between 92.7 and 97.5% was complementary to the L-strand (+ strand). This proportion was independent of the labelling time, indicating similar accumulation of the E- and L-RNA transcripts in the nucleus. The nuclear E- and L-RNA transcripts sedimented in a similar manner through sucrose gradients.

X-ray diffraction studies of oriented specimens of white clover mosaic virus particles suggest that the particles have a helical structure of pitch 3.25 ± 0.05 nm. The X-ray diffraction patterns indicate that there is an integral, or near integral, number of subunits in four turns of the helix and that the number is 4q + 3. Optical diffraction from micrographs of the virus particles are in agreement with the X-ray results and suggest that the number of subunits in four turns is close to 27.

In human KB cells permissively infected with adenovirus type 2, the high mol. wt. forms of virus DNA have been characterized. These size classes of virus DNA sediment at > 100S and 40 to 90S in alkaline sucrose density gradients. Considerable evidence from a series of earlier communications supports the notion that the high mol. wt. forms of virus DNA represent virus DNA sequences covalently linked to cellular DNA. 3H-labelled high mol. wt. adenovirus type 2 DNA from productively infected cells can be shown to hybridize to virus DNA fixed to filters. In the present paper we demonstrate that on alkali elution of the DNA from the filters used in the first step of the hybridization experiment, the labelled DNA re-hybridizes to cellular DNA in the second step of a sequential hybridization experiment. The order of performing the two successive hybridization experiments can be reversed and very similar results are obtained. These data provide conclusive evidence for the covalent linkage of virus and cellular DNA sequences in KB cells productively infected with adenovirus type 2.

Ultraviolet irradiation of herpes simplex virus (HSV) did not affect the transfer of uncoated virus DNA to the nuclei of infected cells but the synthesis of virus DNA was suppressed. The virus-specific DNA polymerase was synthesized in cells infected with the u.v.-irradiated HF strain of HSV. In cells infected with the u.v.-irradiated KOS strain, the virus DNA polymerase activity was hardly detectable. The two strains of HSV differ in the sensitivity of the virus DNA polymerase gene to u.v.-irradiation.

Bacteriophage ϕ6 nucleic acid was present as a torus after chromic acid-formaldehyde-OsO4 fixation and acetone and propylene oxide dehydration. A herpes virus, infectious bovine rhinotracheitis virus, had its DNA mostly as a torus, collapsed in the centre, or as a network, after glutaraldehyde-OsO4 fixation, but in an uncollapsed torus or network formation after chromic acid-formaldehyde-OsO4. This fixative stabilized nucleic acids, allowing acetone dehydration and plastic embedding without collapse of nucleic acid to the centre of the virion.

Post-exposure protection of rabies-infected mice was observed by proximal application of axonal flow inhibitors, particularly vinblastine, to the local nerve(s). These observations indicate that rabies virus is transported by the axonal flow of the peripheral nerves to the central nervous system. Both a fixed virus (CVS) and a street (sylvatic) virus were used.

This model in mice could be used to develop an additional post-exposure local treatment of rabies infection in man, by infiltrating local nerves or ganglions with axonal flow inhibitors, with the advantage that it would not interfere with subsequent vaccination as is the case with the administration of hyperimmune serum or immunoglobulin.