- Volume 31, Issue 1, 1976

Growth of phages φW and T7 was restricted in Escherichia coli lysogenic for phage P1. Only a fraction of the infected cells gave burst of phages. Cells permitting phage growth gave normal burst size. Host strains carrying P1 mutants with defective endonuclease gave no restriction of phages T7 and φ3, the latter a host-range mutant of φW. Degradation but not modification of parental phage DNA could be demonstrated. Although no DNA, RNA or protein was synthesized in φW infected P1 lysogenic cells, the parental phage DNA was found in increasingly larger complexes during the course of infection. At early times after infection, parental phage DNA was found to sediment about twice as fast as mature phage DNA. At later times during the infection the parental phage DNA was recovered as a very rapidly sedimenting material. Such material was also found in alkaline sucrose gradient centrifugation after treatment of the cell extract with sodium dodecyl sulphate, pronase digestion and phenol extractions.

Internal proteins, synthesized in T2-infected Escherichia coli b cells were recovered from bacterial membranes during the early stages of infection. Approx. 15 min after the onset of infection, T2 and T4 internal proteins were released from the bacterial membranes and sedimented along with newly synthesized phage DNA. Internal protein-DNA complexes were also obtained by chromatography on hydroxylapatite columns. Internal proteins were not released from bacterial membranes after infection with amber mutants defective in genes 21, 22 and 23. It has been suggested that these complexes are intermediates in T-even phage morphogenesis.

In addition to the four major polypeptides VP1 to VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 × 103 (P40) and 52 × 103 (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.

The aphid Cavariella aegopodii transmits anthriscus yellows virus (AYV) and parsnip yellow fleck virus (PYFV) in a semi-persistent manner and loses them on moulting. It transmits PYFV only when it is also carrying AYV. The particles of AYV have not been seen in preparations or sections from infected plants but those of PYFV are isometric, about 30 nm in diam. in negative stain. Groups of a few to several hundred virus-like particles about 20 to 28 nm in diam. were seen by electron microscopy in the foregut of C. aegopodii transmitting AYV or the AYV/PYFV complex but not in aphids fed on healthy plants or on a source of PYFV alone. The particles were surrounded by densely staining material, which was in turn embedded in a matrix of lightly staining material (M-material), overlying a 15 to 20 µm-long portion of the intima lining the ventral wall of the pharynx (anterior portion of the foregut). In this region the intima is thickened and formed into several spike-like protrusions. The M-material also occurred in aphids fed on healthy plants, but less commonly than in those fed on infected plants. In some aphids that were fixed immediately after feeding on AYV-infected plants, additional groups of virus-like particles were found free in the foregut lumen. The particles in the matrix were not found until the aphids had fed on AYV-infected plants long enough to acquire transmissible virus, but they were still present in aphids that had fed for 2 h on 10% sucrose after feeding for 24 h on a source of AYV. In moulting aphids, the matrix, with associated virus-like particles, remained attached to the old pharynx lining. It is thought that the virus-like particles are those of AYV and that the matrix is the specific site of retention within the aphid. No differences were observed between aphids carrying AYV and those carrying both AYV and PYFV, and it is not known whether some of the particles found in insects carrying both viruses are those of PYFV.

Ammonium 5-tungsto-2-antimoniate (HPA 23) protected mice partially or completely against two strains of encephalomyocarditis (EMC) virus and one strain of vesicular stomatitis (VSV) virus. The best protective effect was obtained with EMC strain vr 129 and VSV when a single i.p. injection of HPA 23 was administered shortly before virus inoculation. Mice protected by HPA 23 against EMC strain vr129 had virus titres in the blood and brain similar to those in untreated mice. A synergism between interferon and HPA 23 was observed in mice infected with EMC vr129. Our results demonstrate the in vivo activity of HPA 23 against two lethal viral infections and suggest that, at least in mice infected with EMC, death may not be related solely to virus multiplication.

Eleven temperature-sensitive mutants of herpes simplex virus type 2 strain hg52 were examined for ability to induce DNA polymerase activity in BHK 21/C13 cells. All mutants induced DNA polymerase at a permissive temperature, (31 °C) and all DNA-positive mutants at a non-permissive temperature (38 °C). Three DNA-negative mutants induced no DNA polymerase (ts 6, ts 9) or very little DNA polymerase (ts 11), at a non-permissive temperature, while ts 1, also DNA negative, induced a little more DNA polymerase than wild-type, often at both temperatures. The DNA polymerase induced by ts 6 at 31 °C was temperature-sensitive in vivo, but only slightly so in vitro. These results were confirmed immunologically and suggest that HSV-2 codes for at least part of a DNA polymerase activity, necessary for infection, and that full expression of this enzyme involves at least three viral genes.

The spectrum of tissues harbouring latent herpes simplex virus following intravenous inoculation of mice was defined by in vitro co-cultivation techniques. The virus could be detected in central and peripheral nervous systems (including adrenal medulla), but could not be found in any non-neural tissues. Spinal ganglia were the organs most commonly involved. The relationship of these findings to the natural history of herpetic infections is discussed.

A method for the purification of enveloped infectious equine herpesvirus type 1 (EHV-1) is presented. Virus from cell culture fluids harvested at 48 h post infection was concentrated by sedimentation and partially purified by differential precipitation with ammonium sulphate. The final steps of purification consisted of two cycles of flotation of virus in pre-formed CsCl density gradients. Yields of infectious virus were about 30% (18 to 44%) of that present in starting material. As judged by electron microscopy, mixed radioisotope labelling, and absence of phosphohydrolase, virus preparations possessed a high degree of purity. Sedimentation of EHV-1 into CsCl density gradients resulted in low recovery of infectious virus. Flotation of virus in CsCl gradients, however, was not deleterious to infectivity of viral preparations.

A new procedure for the purification of cauliflower mosaic virus (CaMV) which involves the use of Triton X-100 and urea results in higher and more reliable yields. CaMV particles have a s 20, w of 208S, a D 20, w of 0µ75 × 10-7 cm2/s, contain 17% nucleic acid and have a mol. wt. of 22.8 × 106. Virus precipitated by Mg2+, low pH or ethanol at moderate salt concentrations, or by polyethylene glycol, is not easily resuspended.

Deoxyribonucleoside triphosphate pools were analysed in both exponentially growing and serum starved wild type BHK C13 cells and in a derivative of this cell line which lacks both thymidine kinase and deoxycytidine kinase activities, before and after infection with herpes simplex virus. Serum starved BHK cells had low levels of all four deoxyribonucleoside triphosphates. In exponentially growing cells all pools were expanded, the pool of dCTP being largest and dGTP the smallest. The dATP and dTTP pools were of intermediate sizes. In exponentially growing deoxypyrimidine kinase free cells the pools, with respect to level and distribution, were the same as those observed in wild type cells. After infection with herpes simplex virus there were marked changes in the levels of all deoxyribonucleoside triphosphate pools; the most predominant being a 25- to 50-fold expansion of the dTTP pool. The pools of dCTP and dGTP also increased while the pool of dATP was very much reduced. These effects could be observed in both wild type and mutant cells.

Deoxyribonucleoside triphosphate pools were analysed after infection of cells with mutant herpes simplex virus which lacks the ability to induce the enzyme deoxypyrimidine kinase. After infection of exponentially growing BHK C13 cells, an increase in all four dNTP pools was observed. However, after infection of cells which themselves cannot incorporate exogenous pyrimidine deoxynucleosides only the purine deoxynucleoside triphosphate pools increased in size.

In a system which is non-permissive for virus infection, i.e. resting BHK C13 cells which have been infected with dPyK- HSV-1, there is an increase in all dNTP pool sizes except for dTTP.

A comparison of the changes in dNTP pool sizes after infection with either wild type or dPyK- mutant HSV suggests an important role for dTTP in the control of both the production of the other DNA precursors and of viral DNA synthesis.

Tilorone hydrochloride, at a concentration of 10 µg/ml inhibits the growth of herpes simplex virus type 1 in BS-C-1 cells. The growth of vaccinia virus in BS-C-1 cells is partially inhibited; however, six viruses containing RNA, including four members of the togavirus group grown in chick fibroblasts, are not affected by the drug. The inhibition of the growth of herpes virus by tilorone hydrochloride is greater when the multiplicity of infection is lower than 1 p.f.u./cell and when the drug is added early in the course of infection.

Serological tests were done to examine the relationships between twelve picornalike viruses of insects. The results of the tests indicated that the majority of the viruses are unrelated. However, cricket paralysis virus, isolated from Australian wild field crickets, appeared to be identical to Drosophila C virus, independently isolated in France. Cricket paralysis virus was infective for adults of Drosophila melanogaster and its infectivity towards Galleria melonella was neutralised by Drosophila C virus antiserum. It is therefore concluded that cricket paralysis virus and Drosophila C virus are very closely related if not identical.

Addition of 1 × 1010 p.f.u. purified poliovirus to 100 µl of a rabbit reticulocyte lysate protein synthesizing system causes a complete inhibition of initiation of protein synthesis. This inhibition is not due to the viral RNA nor to any contaminants of the preparation, but is most likely caused by the viral coat protein.

An assay is described for feline leukaemia virus pseudotypes of murine sarcoma virus which increased the virus titre by about 100-fold over conventional assays. The titre is independent of dilution and no secondary focus formation occurs. The assay may be used to study virus neutralization and to detect and type feline leukaemia virus in feline embryo cells by interference.

Three mutants of herpes simplex virus (HSV) have been isolated which form plaques in the presence of 100 µg/ml phosphonacetic acid (PPA). All three mutants (3 from HSV-1 strain 17 syn +, 14 from HSV-1 strain 17 syn, and 19 from HSV-2) induce viral DNA synthesis and viral DNA polymerase activity, and these are much less sensitive to PPA than the wild-type virus. The results support the hypothesis that PPA interacts directly with the viral DNA polymerase protein, at least part of which is virus coded.

Medical Neurology, 1975, 2nd edition, 777 pp., 127 illus. By J. Gilroy and J. S. Meyer. Published by Baillière Tindall (London). Price U.K. £14.00.