- Volume 40, Issue 3, 1978

EBNA-containing chromatin from Raji cells was solubilized by treatment with high concentrations of urea and salt and fractionated by hydroxyapatite chromatography. Fractions eluting at different phosphate concentrations were analysed for the presence of EBNA by means of an anti-EBNA-specific 125I-labelled IgG absorption assay. The antigen was found in fractions containing non-histone chromatin proteins.

By DNA-DNA reassociation kinetic analysis, less than one genome equivalent per cell of human CMV-DNA was found in two lymphoblastoid cell lines, one derived from the peripheral blood of a congenitally infected male infant at the age of 21 months (D4 cell line), the other obtained by co-cultivation of lethally X-irradiated cells from the 9-month lymphoblastoid cell line previously described by Joncas et al., (1975) with cord blood leukocytes of a female newborn (M1 cell line). Human CMV antigens could not be detected and virus could not be rescued from these cells by co-cultivation with fully permissive human fibroblasts. It may be that the CMV-DNA is defective. Epstein–Barr virus DNA as well as EBNA and EBV-EA antigens were present in these cell lines. Both lines express surface markers characteristic of thymus-independent, B lymphocytes.

The CMV-DNA of the CMV-DU strain, isolated from this infant’s urine five times successively from the age of 1 day to 30 months, appears to be closely related to the DNA of the AD-169 strain by reciprocal hybridization and by electrophoretic pattern analysis of the restriction enzyme cleavage products. Experimental attempts to transform cord blood leukocytes with this urine strain of CMV before or after u.v. irradiation have so far failed.

Twenty clones were isolated from cultured Aedes albopictus (Singh) cells in the presence of anti-Chikungunya (CHIK) virus serum. Each clone was tested for its yields of Dengue (DEN) viruses, types 1, 2, 3 and 4, and also CHIK virus. Clone C6 showed the highest yield of each virus tested. Forty-three clones obtained by recloning C6 in the presence of anti-DEN sera showed almost the same virus yields as C6. One of the clones, C6/36, showed mild to extensive cytopathic effects several days after virus infection, in contrast to the original uncloned (SAAR) cells. Fluorescent antibody staining revealed that the amount of virus antigen accumulated in the cytoplasm was almost the same in every cell in the case of clone C6/36, while it was highly heterogeneous for uncloned SAAR cells. Growth curves of the viruses indicated that clone C6/36 gave a significantly higher yield for each virus than uncloned SAAR cells up to 7 days after infection.

Virus sensitivity of the C6/36 clone did not change by growing the cells with the medium used for uncloned SAAR cells, nor did the virus sensitivity of uncloned cells increase in medium used for clone C6/36. However, the C6/36 clone became resistant to CHIK virus, but not to DEN or Sindbis viruses, after incubation with the medium used for another A. albopictus cell line (SAAK). The transfer of the specific resistance to CHIK may be mediated by some latent virus related to CHIK.

The sequence of events in the infection of TN-368-10 and TN-368-13 cells by Autographa californica nuclear polyhedrosis virus (AcMNPV) was investigated by using the indirect immunoperoxidase technique. Antisera raised against enveloped nucleocapsids detected homologous antigens at 6 to 8 h post infection which was about 2 h before the appearance of both intracellular and extracellular infectious virus. Similar tests using polyhedrin antiserum showed that polyhedrin is first synthesized at 12 h post infection, 2 to 4 h after the appearance of infectious non-occluded virus. The immunoperoxidase technique was also applied to four other invertebrate cell lines after inoculation with AcMNPV. The most significant result was that 90% of AcMNPV-inoculated Bombyx mori 5 cells produced enveloped nucleocapsid antigens and infectious virus but only 1% or less of the cells produced polyhedrin. This disparity emphasizes the need for assays for NPV infection that are independent of polyhedron production.

The growth of Herpes simplex virus type 1 (HSV-1) in human lymphocytes of adult and foetal origin was studied. Virus DNA synthesis, antigen and particle production and the yield of infectious progeny were determined in cultured lymphocytes with or without exposure to stimulating concentrations of the mitogens phytohaemagglutinin and pokeweed mitogen. Separated sub-populations of cells were examined and the conclusion reached that only the stimulated T-lymphoblast was permissive for full virus expression.

Stimulation of cell DNA synthesis in response to infection was observed in cultures of adult and foetal lymphocytes under conditions which were nonpermissive for virus growth. Morphological change and prolonged culture survival were a feature of foetal lymphocytes exposed to u.v. irradiated HSV-1.

Poly (A) polymerase activity has been measured in crude cytoplasmic extracts of mouse L cells infected with encephalomyocarditis (EMC) virus. After infection there is first a decrease in enzyme activity followed by an increase which itself precedes detectable virus RNA and protein synthesis. The activity of the enzyme then declines before the release of mature virions and cell death take place. The early inhibition of poly (A) polymerase activity is correlated with the virus-induced shut-off of cellular protein synthesis but it is not due to inhibition of the synthesis of cellular enzyme and occurs in the absence of virus replication. The poly (A) polymerase is not synthesized after infection and modification of its activity can be reversed late in the virus cycle. These results indicate that host poly (A) polymerase activity can be regulated by the virus and further show that there is a correlation between the modification of poly (A) polymerase activity and the biosynthesis of poly (A).

Translation of the satellite RNA (RNA-3) of tomato black ring virus (TBRV) in wheat germ extracts or reticulocyte lysates resulted in the synthesis of a polypeptide of mol. wt. about 48000, both in the presence and in the absence of RNA-1 and RNA-2. The RNA-3 specific polypeptide of TBRV strain G was slightly larger than that of strain S. A polypeptide of the same electrophoretic mobility as the in vitro translation product of RNA-3 was found in extracts of protoplasts infected with an isolate of TBRV-S possessing RNA-3 but not in extracts of protoplasts infected with an isolate lacking RNA-3. The lack of phenotypic effect of RNA-3 raises the question of the function of this protein in the infected plant.

IgM and IgG neutralizing antibody responses were estimated in the sera of rabbits and mice immunized with different doses of Semple, duck embryo and cell culture rabies vaccines. IgM synthesis was prolonged in animals immunized with multiple doses of either Semple or duck embryo vaccines but not those immunized with cell culture vaccine.

Mice were passively protected by IgG antibody against subsequent challenge but not by IgM of equivalent neutralizing titre. Mice challenged when the antibody response was solely IgM were not protected if the transition to IgG synthesis was prevented by cyclophosphamide treatment.

DNA hybridization kinetic analysis of cellular DNA following high multiplicity infection of non-permissive XC cells by herpes simplex virus type 1 showed that HSV DNA penetrates to the nucleus of the cell but that the number of virus DNA copies present in each cell quickly begins to decline. There did not appear to be any net virus DNA synthesis and the loss of virus DNA copies continued until there was approximately one per haploid genome equivalent. HSV-2 likewise did not show any detectable virus DNA replication. The residual virus information was stable for more than 48 h. CsCl density gradient analysis of the infected cell DNA suggested an association between the HSV DNA and that of the cells. Network analysis also supported the suggestion that a stable association between the virus DNA and host DNA begins shortly after infection. Cell division resulted in the segregation of the virus DNA but not its loss from the cell population. Virus-specific RNA synthesis was easily detectable and 40 to 50% of a labelled DNA probe was converted to an RNA:DNA hybrid.

A close relationship between three temperate phages (gd, ge and gf), specific for three strains of the newly described species Pseudomonas pseudoflava was revealed by an identical pattern of structural proteins, by serological cross-reactions and by the finding of cross-immunity and cross-resistance. In addition attachment of the phages to the pili of their hosts and the presence of a restriction and modification mechanism was indicated.

The OK 10 virus complex was isolated from a liver tumour of a chicken which, as an embryo, had been inoculated intravenously with a field isolate of an avian leukosis virus. The OK 10 virus complex contains at least two viruses: the interference assay and serum neutralization test indicate that the helper virus belongs to subgroup A. One of the viruses, OK 10 V, induces distinct foci in chick embryo cells under agar overlay and cells from the foci form colonies in soft agar. These properties allow in vitro assay of the virus. Injection of virus or infected cells into chicks induces acute leukaemia but no local tumours. Another virus, OK 10 AV (associated virus), comprises about 99% of the OK 10 complex. This virus does not induce foci in chick embryo cells. In chickens it causes leukosis 17 months after injection. Electron micrographs of OK 10 virus stocks show typical C type virus particles. These particles have a density of 1.16 g/ml and contain 70S RNA which, after heat denaturation, releases type b RNA subunits. The OK 10 virus complex apparently represents a strain of acute leukaemia viruses.

Morphological revertants have been isolated from one line of adenovirus type 12-transformed hamster cells. This line, T637, is oncogenic in hamsters and contains multiple copies of the virus genome per cell. Different parts of the virus genome are represented in non-stoichiometric amounts and the virus DNA persists in the cells in an integrated form. The pattern of integrated virus genomes has been determined by the blotting technique.

In the T637 line, morphological revertants arise spontaneously at relatively high frequency. Two of these revertants have been cloned. In contrast to the T637 line, the revertants F10 and G12 exhibit fibroblastic morphology. The patterns of integrated virus genomes in the revertants differ markedly from that of the T637 line; one of the revertant cell lines, F10, appears to have lost all virus DNA sequences. The morphological revertants continue to express the oncogenic phenotype, although the time required to produce tumours in animals appears to be prolonged compared to the parental BHK21 and the T637 cell lines. A number of biological parameters of the revertant lines have also been investigated.

The interaction in mesophyll protoplasts of two strains of raspberry ringspot virus, RRV-S and RRV-E, was studied using fluorescent antibody to detect strain-specific antigen. Staining with fluorescent antibody was weak and generalized unless the protoplasts were also infected with tobacco rattle virus (TRV), which induces RRV to form antigen aggregates and was therefore used routinely to make RRV antigen more easily detectable.

When tobacco protoplasts were inoculated simultaneously with equal amounts of the two RRV strains, both strain-specific antigens were produced in more than half the protoplasts. The proportion of protoplasts producing antigen aggregates of both strains depended on the ratio of particles of the two strains in the inoculum, but RRV-S had a greater specific infectivity than RRV-E and tended to dominate unless this difference was compensated for. Adding an equal amount of RRV-S to an inoculum of RRV-E decreased the proportion of protoplasts producing RRV-E antigen aggregates. When one RRV strain and TRV were inoculated before the other strain, the strain inoculated second produced antigen aggregates in fewer protoplasts than when it followed a first inoculation with TRV only. Exclusion of the second strain increased with increasing interval between inoculations and was not overcome by increasing the inoculum virus concentration. It was less strong in RRV-E-inoculated than in RRV-S-inoculated protoplasts, in which it was total by 12 h.

Nicotiana benthamiana plants systemically infected with RRV-S did not develop additional symptoms after their recovered leaves were inoculated with RRV-E, but some RRV-S-infected protoplasts from recovered leaves produced RRV-E antigen after inoculation with RRV-E and TRV. This shows that some stages of RRV-E replication can occur in cells long infected with RRV-S. However, RRV-E antigen aggregates were produced in only 11% of the challenge-inoculated protoplasts, 99% of which became infected with TRV from the same inoculum, suggesting that partial protection exists and is virus-specific.

The phenomena of interference and cross protection seem best explained by competition in cells for virus-specific sites or material, possibly RNA polymerase, that can be used in the replication of either strain.

A plaque count infectivity assay was developed in which chick cells infected with the Mount Elgon bat virus were completely resistant to superinfection with large doses of Sendai virus. Several variables markedly affected the assay sensitivity. The defined plaque assay was simple, highly reproducible and sensitive. It allowed determination of virus neutralizing antibody in a simple and reproducible test. Both actinomycin D and 5-iododeoxyuridine were without effect on plaque formation.

In vitro multiplication of rabies virus was inhibited by a condensed mineral ion, ammonium-5-tungsto-2-antimoniate (HPA 23). The inhibitory effect was evaluated by two different methods, plaque reduction and one step virus growth. Plaquing showed 50% inhibition with 4.5 µg/ml of HPA 23 and complete inhibition with 12.5 µg/ml. A reduction of two logs in virus yield was obtained in BHK21C13S cells in suspension treated with 50 µg/ml of HPA 23. Inhibition also occurred when treatment with HPA 23 was started 18 to 24 h after infection in the plaque assay but no effect was seen when HPA 23 was added 48 h after virus inoculation. All these inhibitory effects of HPA 23 on rabies virus multiplication were observed at non cytotoxic doses. Therefore HPA 23 contrasts with other antiviral drugs which do not inhibit rabies virus multiplication without affecting the viability of cells.

A new model for the adsorption of bacteriophage P22 to its host Salmonella typhimurium is proposed. The main feature of this model is that only three of the six tail proteins found on the mature phage function during adsorption. This model explains why there is a difference in the specific endoglycosidase activity of the tail protein of mature virions as opposed to unattached tail protein. It also accounts for the cubic relationship between p.f.u. and tail protein concentration in in vitro assembly experiments.

Intranuclear crystalline inclusions with leaf-like striated appearance, i.e. zebra structures, were observed by electron microscopy in adenovirus type 5-infected human cervical and bladder carcinoma cells at 4 to 6 days after infection.

Human fibroblast interferon has been labelled with 125iodine using the Bolton-Hunter reagent. Purification of the interferon after iodination gave a product which had stable biological activity for 1 month and had a specific radioactivity of 2 to 4 µCi/µg.

A variant of adenovirus type 12 (R-Ad12) has been isolated by infecting a clonal line of normal rat kidney cells (NRK) with wild-type adenovirus 12. Although R-Ad12 failed to produce infectious progeny virions when cycled a second time through NRK cells, it nevertheless retained the ability to code for virus structural proteins that accumulated in the cytoplasm.