- Volume 26, Issue 1, 1975

Nodamura virus multiplied in mosquito cell lines, as determined by infectivivity assays in adult honey bees (Apis mellifera) and wax moth larvae (Galleria mellonella). Titres of more than 107 and 105 bee LD50/ml were obtained in culture fluids of Aedes albopictus and Aedes aegypti cells respectively after 10 days. Comparable titres were obtained after several months, during which the cultures were subdivided up to six times. Nodamura virus also multiplied in BHK cells and yielded titres of 104.8 to 106.6 mouse LD50/ml and 105.1 to 107.1 wax moth LD50/ml in culture fluid 1 to 4 days after infection. No c.p.e. was observed in infected cells.

Three classes of mammalian ribosome-like structures were found to be associated with purified Pichinde virus preparations. These structures, possessing sedimentation coefficients of 80S, 60S and 40S, were sensitive to EDTA dissociation and possessed a buoyant density in caesium chloride of 1.61 g/ml, a value analogous to that displayed by BHK21 cell ribosomes. Analyses of RNA components associated with ribosomal particles of Pichinde virus established that 28S and 18S RNAs could be extracted from 80S monosomes. Similarly, 28S and 18S RNAs were derived, respectively, from 60S and 40S ribosomal subunits. Virus-specific 31S and 22S RNAs, as well as 28S and 18S RNAs, could be isolated from the heavy region of sucrose gradients. An additional 15S type of RNA, which was subsequently shown to be a component of intact virus particles, was also isolated from the heavy region.

Four Agrobacterium tumefaciens temperate phages (PB2A, PB6(Ω), PV-1(LV-1) and PS8) were shown to have the same genome size. Moreover hybridization experiments by the heteroduplex method and electron microscopy showed a 100% homology between these four phage genomes. Indications for lysogeny were found by direct means for the Agrobacterium tumefaciens strain 396, Agrobacterium radiobacter strain 8149 and Agrobacterium species 0362 and by the electron microscope negative staining technique for the A. tumefaciens strains B6-806, B6-6, B6S3, B2AS, CV-1, 4452, 11156, 11158, 396 and 925; for A. radiobacter strains TR-1 and 8149, the latter being bi-lysogenic, and for the A. species 0362. These isolated phage particles, most of which appear to be defective, could be grouped into different classes. No particles could be detected in the lysates of A. tumefaciens RV3, A. radiobacter strains 4718 and S1005, and A. species 0363.

Further characterization by genome size was carried out for the defective temperate phages PB6-806, P4452, P814911 and P0362. No evidence for homology between PB6-806 and PB6(Ω) could be found. The defective phages PB6-806 and P4452 showed the same morphology but a different genome size, whereas the two phages P0362 and P814911 had a very different morphology and genome size.

SV40 stocks prepared by successive undiluted passages in African green monkey kidney (GMK) cells contain particles which produce T antigen but not capsid antigen (T particles). The DNA of the T particles was shown to replicate during abortive infection of GMK cells, as indicated by immunofluorescence assay for antigen forming capacity of virus DNA. It was also shown that the synthesis of T particle DNA was induced in heterokaryons formed by fusion of 3T3 cells transformed by T particles with susceptible GMK cells.

Polyoma DNA replication is initiated predominantly at a site which is 29% from the EcoRI cleavage site. Molecules replicating from this site, after digestion with EcoRI, appear as linear structures with a double stranded loop centred at the origin of replication. These forms constitute 90% of all replicating intermediates.

Approx. 10% of the replicating intermediates of polyoma and SV40 DNAs occur as Y-forms after treatment with EcoRI. These structures have probably resulted from unidirectional replication initiated at an additional origin of DNA replication which is located near the EcoRI cleavage site on the genomes of these viruses.

The replication of frog virus 3 in primary chick embryo fibroblasts has been studied by examination of thin sections with the electron microscope and the assay of infectious virus. Uptake of frog virus 3 by the cells was observed to occur by pinocytosis and this may be the method of entry. Early in infection (1 h p.i.) marked margination of the nuclear chromatin occurred and the chromatin remained in this condition throughout the infection. Foci of infection were first detected in the cytoplasm of cells 24 h p.i. when production of infectious virus commenced. These foci appeared as electron translucent areas containing fine grains, surrounded by degenerate mitochondria. The foci usually contained virus particles. At this time budding of virus particles at the plasma membrane occurred. Later in infection at 36 and 48 h p.i. large numbers of virus particles were detected in the cytoplasm of cells either scattered loosely throughout the cell, arranged as clusters or in paracrystalline arrays. Extensive budding at the plasma membrane then took place. Virus particles were detected in the nucleus of the cells at these late stages and it is possible that the virus may infect and replicate at this site. Throughout the productive stages of infection aberrant forms of the virus, namely particles devoid of cores, incompletely assembled particles and elongated bacilliform particles were noticed.

Satellite or defective bacteriophage particles can appear in extensively recycled stocks of coliphage M13. These particles, herein known as miniphage, replicate using the wild type bacteriophage as a helper. Their physical properties (u.v. spectra, sedimentation of DNA and bacteriophage, electrophoretic mobility) are described and a method for the isolation of specific satellite bacteriophage is presented.

The purification and characterization of the type, nasturtium ringspot and petunia ringspot strains of broad bean wilt virus are described. The use of sucrose throughout purification reduced losses due to aggregation of the virus particles. Electron microscopy of virus preparations revealed particles which were apparently hexagonal in outline and approx. 27 nm in diam. Each virus had three sedimenting components, top (T), middle (M) and bottom (B), indistinguishable from those of cowpea mosaic virus (CPMV) in co-sedimentation experiments. The sedimentation coefficients, s 0 20, w of PRSV were 56S (T), 95S(M) and 116S(B). The buoyant densities of M and B components of BBWV, NRSV and PRSV in CsCl + 2.34% (w/v) Igepon T73 were approx. 1.40 and 1.44 g/ml respectively. B components of the viruses contained a single RNA species (RNA-1) of mol. wt. 2.0 ± 0.1 × 106 and approximate molar percentage base ratio of 26% guanine, 30% adenine, 17.5% cytosine and 26.5% uracil. M components of the viruses contained a single RNA species (RNA-2), mol. wt. 1.5 to 1.6 × 106. The base ratios of RNA-1 and RNA-2 of BBWV were very similar. Mixtures of RNA-1 and RNA-2 (or M and B components) were more infective than the separate RNA species (or virus components). Mol. wt. of the formaldehyde-denatured RNA species of BBWV, NRSV and PRSV were very similar to those of CPMV. BBWV, NRSV and PRSV contained two species of polypeptide, mol. wt. 42000 and 26000.

These properties in conjunction with serological evidence indicate that the viruses are closely related strains. The similarity between some of the properties of BBWV, NRSV and PRSV and those of CPMV is discussed.

Although the infection of BHK cells with foot-and-mouth disease virus did not cause a marked inhibition of cellular protein synthesis, the proteins synthesized gradually changed from host-specific to virus-specific. The synthesis of at least thirteen virus-induced proteins was demonstrated by polyacrylamide electrophoretic analysis of the infected cells. Only a small proportion of the virus-induced proteins was incorporated into the mature virus particles. The addition of amino acid analogues caused an accumulation of the larger mol. wt. proteins, suggesting that in infected cells some cleavages occurred, giving rise to the smaller mol. wt. proteins. The operation of the precursor-cleavage mechanism was more clearly shown in pulse-chase experiments, in which it was found that the proteins were degraded from larger to smaller mol. wt.

The mol. wt. of a [32P]-labelled bovine enterovirus RNA has been determined by digesting with pancreatic RNase and separating the resulting oligonucleotides using a two-stage fractionation method on DEAE-Sephadex in 7 m-urea at pH 7.6 and pH 3.0. We have estimated the number of nucleotides as 8612 ± 55. This corresponds to a mol. wt. of 2.93 ± 0.02 × 106 which is in agreement with estimates obtained by sedimentation and gel electrophoresis techniques.

A bacteriophage which infects and lyses Bacillus megaterium atcc 19213 was isolated from the soil. The phage produces lysis on nine strains of B. megaterium tested but did not lyse a Bacillus cereus or Bacillus licheniformis strain, nor any of eight Bacillus subtilis strains tested. Physical characteristics of the phage including morphology, size, thermal and pH stability, and buoyant density were examined. The nucleic acid is a double-stranded DNA of mol. wt. 41.7 × 106 and 36 to 38.5 mol percent guanine plus cytosine (G + C).

9-O-methyloxime erythromycin A and its analogue inhibited reverse transcriptase and blocked focus formation of Rous sarcoma virus. These chemicals inhibited neither DNA-dependent DNA polymerase nor DNA-dependent RNA polymerase from bacterial sources. However, they inhibited reverse transcriptase with an apparently different mechanism than that by rifamycin ABDP.

Bovine adenovirus type 3 (BAV-3), strain WBR-1, quantitatively transforms hamster embryo carcass cells with a maximum efficiency of 4.7 × 104 p.f.u./focus-forming units (f.f.u.). This frequency is significantly inhibited by the presence of 1.8 mm of Ca++. The transformed cells are highly tumourigenic in hamsters and possess T- and tumour-specific transplantation antigens. BAV-3 is one of the most oncogenic of the adenoviruses and an ideal model virus for the study of transformation.

Three-week-old mice were inoculated in the right ear pinna with pseudorabies virus. Ears were surgically removed at various times after inoculation and changes from the normal pathogenesis were observed. Virus replication in the ear tissue and cervical dorsal root ganglia was also monitored. Following inoculation with a small dose of virus, local multiplication of the virus was necessary before the infection spread to the nerves. With larger infecting doses there was probably direct uptake of virus from the inoculum into the nerve endings. After these larger doses virus was first detected in the dorsal root ganglia 17 h after infection, suggesting a retrograde axonal flow rate of at least 1.7 mm/h.