- Volume 3, Issue 3, 1968

Measurements of the neuraminidase content of two recombinant influenza viruses [X-7 and X-7 (F1)] possessing A0 (nws) haemagglutinin and A2 (r1/5+) neuraminidase showed that particles of X-7 (F1) virus had more than twice as much A2 neuraminidase as those of X-7 virus.

Antibody to A0 haemagglutinin reacted to high titre in antihaemagglutinin, neutralization and complement-fixation tests with both viruses. Antibody to A2 neuraminidase (at the same concentration) did not inhibit haemagglutination by either virus and did not prevent X-7 virus from infecting cells. It did, however, reduce the size of X-7 virus plaques. Antineuraminidase neutralized, but only to a low titre, the infectivity of X-7 (F1) virus for cells of the chick allantonic membrane and completely inhibited plaque formation by this virus in tissue cultures. Antineuraminidase and antihaemagglutinin antibodies reacted with both viruses in complement-fixation tests to similar titres.

The biological ineffectiveness of antineuraminidase antibodies on the haemagglutinin activity or on the infectivity of these viruses was not due to lack of combination of antibody with the virus. The average avidity of the antineuraminidase antibody was high and not different from the antihaemagglutinin antibody (K≃2 × 1011 c.g.s. units). X-7 virus has approximately one quarter and X-7 (F1) virus one half of the number of enzyme antigenic sites as haemagglutinin sites.

Sarcomas induced in 13 BALB/c mice, one Sprague-Dawley rat and five Syrian (golden) hamsters by the murine sarcoma virus-harvey (MSV-h) were grown in cell culture and the cell lines maintained for a maximum of 194 days. The majority of the tumour lines formed three-dimensional colonies of densely packed cells which eventually broke free from the glass and floated in the medium. Infective MSV-h was obtained from the mouse and rat tumour cultures. Filtrates of the culture fluids induced typical MSV-h lesions when injected into newborn mice, and those from cultures maintained for 4 to 6 months were as effective as those from cultures maintained for shorter periods. The newborn mice receiving undiluted culture fluids rapidly developed sarcomas and erythroblastic splenomegaly, while recipients of limiting dilutions developed lymphocytic leukaemia which never appeared earlier than 2 months after injection. Thus, the MSV-h derived from cell cultures behaved in the same way as that derived from infected animals.

However, hamster tumour-cell culture filtrates did not contain active MSV-h when tested in mice, but one such filtrate induced typical sarcomas and other characteristic MSV-h lesions when injected into newborn hamsters.

Infection of BHK 21 (C 13) or HEp-2 cells with herpes virus was followed by a marked increase in the activity of alkaline DNase which was prevented by actinomycin D and puromycin. Activation of a latent enzyme was not responsible for the increase. The properties of the DNase appearing after virus infection in both cell types were the same, but they differed from those of the enzyme from uninfected cells in specificity towards the secondary structure of the DNA substrate, heat-stability, requirement for thiol groups, inhibition by K+ and Na+ ions and response to various concentrations of Mg2+ and Mn2+. The activities of acid DNase and alkaline phosphomonoesterase were not significantly altered after herpes infection. Further, the activity of alkaline RNase was not altered by infection, and this implies that the induced DNase was specific for DNA. The new DNase could be separated from the DNase present in uninfected cells and from alkaline phosphomonoesterase by chromatography on columns of DEAE-cellulose; its enzymic properties were the same as those observed with soluble extracts of cells infected with herpes virus.

A number of recent reports concerning the inhibitory action of sulphated polysaccharides on d strains of poliovirus prompted a reinvestigation of the factors involved. It was found that polyglucose compounds in agar are not released in concentrations sufficient to inhibit viruses under the overlay conditions used for suppressing d strains of poliovirus. Furthermore, sulphated polyanions in agar cannot be the critical factor, since d strains of poliovirus are also suppressed under other solidifying agents (starch gel and methylcellulose) which are free of sulphated polyglucose. Purified agar free of sulphated polysaccharides, used with medium containing a low bicarbonate concentration attains the same high pH after equilibration as regular agar (containing polyanions) made up with high concentrations of bicarbonate, and it is this high pH which appears to explain the ready growth of d strains of poliovirus.

Virus-free buffer extracts of vaccinia-infected sheep dermis were fractionated by elution from DEAE-cellulose with increasing concentrations of NaCl. The fraction eluted with 4.0m-NaCl was serologically homogeneous when examined in precipitation-in-gel tests. Further fractionation on Sephadex G-200 gave a material which was also physically homogeneous when examined by both ultracentrifugal and electrophoretic techniques. A sedimentation coefficient of 3.47 S and a molecular weight of 31,000 were calculated from ultracentrifugal data. The isolated material contained 74.3% DNA, 9.4% protein estimated as bovine serum albumin and 14.9% carbohydrate estimated as glucose. The DNA contained the bases adenine, thymine, guanine and cytosine in the ratios 1.0:1.1:0.8:0.8. Paper chromatography of formic-acid hydrolysates of the material resolved seven substances reacting with ninhydrin. Qualitative colorimetric tests indicated, apart from deoxyribose, the presence of a hexose and a hexuronic acid.

The serological activity of the isolated material was heat-stable and identical with that of a previously described heat-stable extract of vaccinia-infected rabbit dermis. Treatment with enzymes indicated the presence of two different, serologically active sites. Failure to elicit an antibody response following injection into rabbits suggested that the isolated material was a hapten. Serum absorption studies showed serological identity with an antigen present on the surface of the vaccinia virus particle.

Plaques were produced in mouse-embryo monolayers by infection with polyoma virus DNA in the presence of diethylaminoethyl-dextran. Optimum conditions for plaque assay were established and dose-response relationships for component I and component (II+III) polyoma DNA determined. Efficiencies up to 6 × 105 p.f.u./µg. were obtained for component I DNA.

Chick embryos from the inbred Reaseheath I line of chickens contained a complement-fixing antigen which reacted in the COFAL test, whereas embryos from the inbred Reaseheath C line lacked the antigen. In cross-bred embryos between the I and C lines the antigen segregated in accordance with the hypothesis that the presence of the antigen was controlled by a single autosomal dominant gene. The antigen appeared to be identical to the group-specific antigen of the avian leukosis-sarcoma group of viruses. No infectious virus was detected in I-line embryos, and the presence of the antigen was not correlated with response to the A and B subgroups of avian leukosis-sarcoma viruses. The possible relationship between the I-line antigen and viral group-specific antigen is discussed.

Several derivatives of benzimidazole were tested for antiviral activity; of these the 1-β-d-2′-deoxyriboside of 5,6-dichlorobenzimidazole and the α and β anomers of 5,6-dimethyl-1-(2′-deoxy-d-ribofuranosyl)benzimidazole inhibited the reproduction in vitro of two DNA viruses, herpes simplex virus and polyoma virus; 5,6-dichloro-1-(2′-deoxy-β-d-ribofuranosyl)benzimidazole (dDBZ) was the most inhibitory compound. Pretreatment of the host cells with dDBZ, subsequently removed by washing, did not affect the yield of these viruses. dDBZ did not have any direct inactivation effect on herpes simplex virus. Growth curves of herpes virus in the presence of dDBZ indicated that a reduced yield of the virus was obtained after a considerable lag, the first progeny virus being detected 24 hr after infection. Marked inhibition could be demonstrated when the compound was added as late as 15 hr after infection. Attempts to prevent the inhibition by dDBZ by addition of the following compounds failed: deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, adenine and adenosine. 5,6-Dichlorobenzimidazole, its ribonucleoside (5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (DRB)) and the corresponding deoxyribonucleoside (dDBZ) were compared for their relative antiviral activity against a DNA virus (herpes simplex virus) and RNA viruses (three strains of polio virus). All three compounds inhibited herpes simplex virus; the 5,6-dichlorobenzimidazole inhibited two strains of polio virus; and DRB inhibited all three strains of polio virus. dDBZ had the least inhibitory activity for two of the three strains of polio virus and did not inhibit the lsc 1 2ab strain of polio virus. dDBZ at a concentration required to inhibit viral replication inhibited the synthesis of RNA and to a lesser extent that of DNA and protein of uninfected green monkey kidney cells.

The RNA bacteriophage µ2 was grown efficiently under controlled conditions in a 2 l. fermenter and labelled with [32P]orthophosphate and [5-3H] uridine. The RNA was extracted and digested completely with pancreatic ribonuclease and the resulting solution of oligonucleotides was fractionated by DEAE-cellulose chromatography in 7 m-urea at pH 7.9. Eleven fractions were obtained which were shown to contain fragments with chain lengths 1, 2, …, 11 respectively. Fractions 10 and 11 were recovered in yields corresponding to one oligonucleotide in each fraction per original viral RNA molecule, and the nucleotide compositions of these oligonucleotides were measured. The distribution of purine nucleotide sequences that might reasonably occur by chance in an RNA molecule of finite length was calculated and compared with the observed distribution in µ2 viral RNA. The use of these procedures for identification and characterization of viruses was demonstrated.

Separations of A-protein of tobacco mosaic virus by electrophoresis on polyacrylamide gels of various concentrations gave clear resolution of several components. The pherograms which were obtained varied according to the particular A-protein preparation employed, but corresponded well with estimates of the state of aggregation of the preparations made by electron microscopy or by sedimentation in the analytical ultracentrifuge. These estimates together with the results of immunodiffusion studies and electron-microscopic observations on material eluted from the gels following electrophoresis enabled the various components in the pherograms to be correlated with the known stable aggregates of A-protein. The electrophoretic mobilities of the various aggregates differ and increase with increases in size of the aggregates.

Preparations of alfalfa mosaic virus made by precipitation with polyethylene glycol were as infective and contained the same relative proportions of components as virus preparations made by ultracentrifugation. A column chromatographic procedure using a continuously decreasing concentration gradient of polyethylene glycol was employed to fractionate the nucleoprotein components of the virus. By this procedure a partial sorting of the virus components into three groups according to particle length was achieved. Particles in the three groups are thought to correspond to top component a, top component b, and a mixture of middle and bottom components.

Photoinactivation of vaccinia virus was complete but slower with rose bengal than with methylene blue as the sensitizing dye. Photoinactivation was inhibited and changes in dye absorption spectra occurred when protein but not when nucleic acid was added to rose bengal + virus mixtures; with methylene blue + virus mixtures nucleic aid but not protein was inhibitory. Photoinactivation with methylene blue did not alter the amino acid content of the virus, but with rose bengal, there was a substantial decrease in the histidine content. Antigenicity of the virus decreased more rapidly on exposure to light with rose bengal than with methylene blue as the sensitizer. Despite the apparently greater affinity of rose bengal for protein and of methylene blue for nucleic acid, no specific action on these components of the virus was demonstrated in cross-reactivation tests.

Most strains of influenza A virus contain sialidases which are distinct from those of the host cell (Laver, 1963; Kelly & Grieff, 1965) and are presumed to be coded by the virus genome. However, the neurotropic variant nws, derived from the A0 strain ws (Stuart-Harris, 1939), did not elute readily after adsorption to red blood cells, or destroy the haemagglutination-inhibiting (HI) activity which various sialo-mucoids exhibit against sensitive test strains of influenza virus (Burnet & Lind, 1951). In contrast, the parent ws strain was active in both these tests. A decrease in viral sialidase activity may thus be a genetic marker or determinant of neurovirulence. On the other hand, these two viral characteristics could have arisen separately during the long series of chick embryo and animal passages by which nws was selected from ws, and have merely been accidentally associated in early allantoic fluid pools of this strain.