- Volume 11, Issue 2, 1971

R. M. Goorha and G. E. Gifford (1971) Journal of General Virology 10, 117

Page 117. Line 8: Insert the word ‘sometimes’ after ‘Inactivation of a virus …’

A bovine enterovirus (serotype VG-5-27) was grown in BHK 21 cells and purified using gel filtration and sedimentation procedures. Infective particles sedimented at 165s and the empty capsids or procapsids at 75s. Proteins extracted from each type of component were separated by polyacrylamide-gel electrophoresis. The infective component yielded four polypeptides of molecular weight 34,000, 28,000, 26,000 and 9,000 and were present in molar ratios of 1:1:1:0.5, respectively. Three polypeptides were extracted from the procapsid. These have molecular weights 37,000, 34,000 and 26,000 and were present in molar ratios of 1:1:1, respectively. We interpret these results as indicating that the small polypeptide of the virus particle may have a specific location in the virus in relation to the three major structural proteins.

A series of 71 spontaneous temperature-sensitive mutants of vesicular stomatitis virus (type indiana) have been isolated in chick embryo cells (Flamand, 1970). In addition, 175 induced mutants have been isolated from a different wild-type strain propagated in BHK 21 cells (Pringle 1970b). These mutants have been classified into complementation groups independently.

Reciprocal complementation experiments are now described which establish the genetic homologies of these mutants. The results obtained in the two systems are in good agreement, despite differences in experimental procedure, restrictive temperature, wild-type strain and host cell. It can be concluded that complementation groups I–IV in the Glasgow classification correspond to the groups represented by ts 4, ts 52, ts 23 and ts 100 in the Orsay system. Mutant ts 45 (Orsay) belongs to a fifth group not represented among the Glasgow mutants.

On staining adenovirus-infected cells with phenanthrenequinone and with copper phthalocyanin-neutral red, characteristic structures in the form of rings and rosettes were observed. These structures could also be visualized by phase-contrast microscopy and by fluorescent antibody staining with adenovirus P antiserum. The cytochemical staining could be blocked by pre-treatment of the fixed cells with benzil, and the result suggested that the rings and rosettes were the sites of accumulation of arginine-rich basic proteins.

Temperature-sensitive mutants of adenovirus type 5 have been isolated from virus stocks mutagenized by nitrous acid, hydroxylamine and 5-bromodeoxyuridine. The frequency of such mutants in the surviving fractions of nitrous acid and hydroxylamine-treated stocks was 8.4 and 9.6%, respectively, while it was around 0.55% in stocks of virus grown in 5-bromodeoxyuridine. Most of the mutants tested so far appear to be relatively stable and show little evidence of excessive leakiness or back mutation, and will be suitable for genetic analysis. Preliminary experiments show good complementation between four of these mutants, and they have been assigned to four complementation groups.

The molecular weight of the influenza virus genome was estimated by sucrose density-gradient centrifugation, analytical centrifugation and polyacrylamide gel electrophoresis. The results indicated that the virus contained at least six species of RNA of total molecular weight approximately 3.9 × 106.

The polycation DEAE-dextran enhances the infectivity of animal virus RNA and DNA for cell cultures by up to 105 times (evidence reviewed by Pagano, 1970). It is thought to form ionic bonds with the phosphate groups of the nucleic acid which not only stabilizes it and partially protects it from nucleases, but also affects the assay cells and facilitates infection.

The apparent infectivities of plant virus nucleic acids have been increased by reconstitution with protein (Fraenkel-Conrat & Singer, 1959), by adding phosphate (Lippincott, 1961), bentonite (Fraenkel-Conrat, Singer & Tsugita, 1961; Singer & Fraenkel-Conrat, 1961), sucrose (Kongsvik & Santilli, 1970) or alkaline buffers (Sarkar, 1963), or by keeping the test plants at 37° or in darkness before inoculation (Bawden & Pirie, 1959; Kongsvik & Santilli, 1970).

When plant cells die as a result of virus infection, the activity of oxidases, particularly polyphenoloxidases and peroxidases, is altered (Martin, 1958; Farkas, Kiraly & Solymosy, 1960; Farkas et al. 1964). The in vitro activity of these two enzyme groups in extracts of infected leaves kept at 20° shows changes that are correlated with the time of appearance and number of local lesions. With most virus/host combinations, the oxidase concentration is merely increased (Van Kammen & Brouwer, 1964; Novacky & Hampton, 1968; Cabanne, Scalla & Martin, 1968), with some there is a change in the relative amount of different isozymes (Bates & Chant, 1970), and with others there is possibly the appearance of new peroxidases (Farkas & Stahmann, 1966) or of a new phenolase (John & Weintraub, 1967). Different workers have interpreted these facts to explain the formation of necrosis and virus localization in different ways (Farkas et al. 1960; Parish, Zaitlin & Siegel, 1965; Suseno & Hampton, 1966).

Nariva virus was first isolated in 1962 by Tikasingh et al. (1966) from Trinidadian rodents Zygodontomys b. brevicauda and was originally thought to be an arbovirus. It contains RNA, is ether-sensitive and haemadsorbs guinea-pig erythrocytes (Karbatsos, Buckley & Ardoin, 1969). Few of its properties are known, and since its structure and morphogenesis are also unknown, its taxonomic position is uncertain. We describe here observations on the structure and morphogenesis of Nariva virus propagated in BHK 21 cell cultures, thus providing some evidence for its classification. Monolayers of BHK 21 cells were grown in EM/TPB medium as described previously by (Macpherson & Stoker, 1962) and maintained in equal parts of EM/TPB and Basal medium Eagle (Eagle, 1955) with 3% foetal bovine serum, 0.05% of 2 m-tris and antibiotics; kanamycin and tylosin tartrate was additionally added. Nariva virus (prototype strain trvl-42520, 4th mouse brain passage) was obtained from Dr L. Spence (Trinidad Regional Virus Laboratory, Trinidad).