- Volume 40, Issue 1, 1978

Inocula made by mixing tobacco rattle virus (strain CAM; TRV), poly-l-ornithine (PLO) and either citrate buffer or phosphate buffer were compared by measuring their turbidity, by estimating the effect of centrifugation on their infectivity and by electron microscopy. Aggregates (floccules) containing virus particles were formed in the inocula but there was no evidence of any major difference in size between aggregates formed in the different buffers. The relationship between the loss of infectivity following centrifugation and the centrifugal force applied suggested that the aggregates causing infection sedimented at an average of 1340S (mol. wt. 7 × 108). Electron microscopy showed electron dense masses in most of which 10 to 20 TRV particles were embedded. Aggregates made in citrate had virus particles arranged radially whereas aggregates made in phosphate were more randomly constructed.

Preparations of sedimented aggregates, and the supernatant fraction remaining following centrifugation were poorly infective unless PLO was added. In contrast with the addition of PLO to TRV preparations, adding PLO to these centrifuged fractions restored infectivity immediately. It is suggested that PLO can have two modes of action on infection when it is added to TRV to make inocula, one involving the formation of aggregates and the other for which aggregate formation is not essential.

Double stranded DNA, created on extraction of the plus and minus strands, which are separately packaged in densonucleosis virus particles, exists predominantly as linear monomers, although circular monomers and concatamers and other less well defined structures were observed by electron microscopy and agarose gel electrophoresis. A limited nucleotide sequence permutation, which is probably non-random, comprises about 2.7% of the genome (160 base pairs) and is considered to be the structural feature causing circularization and concatamerization. Evidence of an inverted terminal repetition observed by circularization of single stranded DNA was obtained by electron microscopy and S1 nuclease digestion. Estimates of the size of the terminal repeats varied from 60 to 380 base pairs.

CBA mice, inoculated intravenously with large doses of adenovirus type 5, showed raised levels of serum aspartate aminotransferase (SAAT; EC 2.6.1.1) and died within a few days from histologically demonstrable hepatic necrosis. After inoculation of 1 LD50, virus was rapidly taken up by the tissues where infectivity then declined greatly. Organ titres then increased about 100-fold by 48 h p.i. but, in the liver, which showed intranuclear inclusion bodies, and by electron microscopy, scattered intranuclear and intracytoplasmic adenovirions, the increase was 10000- to 100000-fold. P antigen was detected by single radial diffusion in liver extracts, and by immunofluorescence in 80% of liver cells at 36 h p.i. Hexon, penton base and fibre antigens appeared later and in fewer cells. The maximum amount of hexon, of demonstrable type 5 specificity, was shown by radioimmunoassay to be equivalent to up to 5 × 1011 whole adenovirions/g liver. It is concluded that human adenovirus type 5 undergoes an abortive but lytic infection in most liver cells but that replication may proceed to completion in a few.

A number of hitherto undescribed, serologically identical viruses have been isolated from a syndrome of depressed egg production in broiler breeder flocks (Egg Drop Syndrome 1976). One of these, 127 virus, was purified after growth in chick embryo liver cells. Three particle types B1, B2 and B3 with densities of 1.32, 1.30 and 1.28 g/ml, respectively, were separated in CsCl equilibrium density gradients. B1 and B2 particles possessed infectivity and were labelled with 3H-thymidine. However, they differed morphologically and B2 particles agglutinated chicken erythrocytes whereas B1 particles did not. B3 particles were not infectious, were not labelled with 3H-thymidine but agglutinated chicken erythrocytes. They were penetrated by stain when examined by electron microscopy and probably correspond to empty particles. B1, B2 and B3 particles differed in their polypeptide compositions; seven of the polypeptides in B1 and B2 particles had counterparts in purified fowl adenovirus type 1.

The soluble haemagglutinin from 127 virus, which did not sediment under centrifugation conditions sufficient to pellet virus particles, was purified using DEAE cellulose chromatography and gel filtration. Purified soluble haemagglutinin contained two polypeptides (mol. wt. 67000 and 65000), which co-migrated in polyacrylamide gels with two of those present in purified 127 virus particles. The soluble haemagglutinin sedimented heterogeneously (20 to 50S) in sucrose density gradients and had a density of 1.24 g/ml in CsCl. It was inactivated by trypsin, urea and pyridine. Electron microscopy of purified soluble haemagglutinin showed rod-like structures with lengths of 25 to 30 nm, which radiated from a central area measuring approx. 10 nm in diam. It is suggested that 127 virus is an adenovirus.

We have studied SA7 (simian adenovirus 7) lytic infection at the ultrastructural level. The use of cytochemical techniques which specifically stain DNA or preferentially stain ribonucleoproteins permitted the analysis of the structure of the virus-induced nuclear inclusions, and revealed presumed virus DNA before the appearance of other nuclear alterations. Correlation of these findings with high resolution autoradiography enabled the functions of virus DNA replication and transcription to be ascribed to a defined nuclear inclusion. We demonstrate that the nucleolus remains distinct from the inclusion body, contrary to the situation in other adenovirus-infected cells. The functional role of host cell chromatin and of the nucleolus are discussed.

The effect of sulphydryl reagents on haemagglutinating encephalomyelitis virus (HEV), a coronavirus of pigs, was investigated. Using increasing concentrations of dithiothreitol (DTT), 50% of the virus infectivity and haemagglutination (HA) activity could be removed by 1.5 mm and 4 to 5 mm respectively. The effect of DTT concentrations on the polypeptide composition was also examined. Of the three external glycoproteins gp 125 was found to be the most susceptible, 50% being removed by incubation of the virus with 5 to 6 mm-DTT. Of the other two glycoproteins gp 180 was unaffected by DTT concentrations up to 100 mm and the amount of gp 100 gradually declined at concentrations above 20 mm. The rates of removal of the virus HA activity and gp 125 suggested that this polypeptide was an essential part of the virus haemagglutinin. The lack of evidence for any interpeptide disulphide bonds suggested that the loss of these glycoproteins was due to an alteration in their conformation brought about by the cleavage of intrapeptide disulphide bonds. The loss of protein from the surface of the virus resulted in a change in the virus morphology with the appearance of thin fibrous projections instead of the characteristic petal-like coronavirus projections.

The fine structure of cells infected with the HG 52 strain of herpes simplex virus type 2 and 13 temperature-sensitive mutants derived from it was investigated. In cells infected with the wild-type virus, development of virions appeared to be similar to that described in previous reports. However there were two exceptions to this: (1) capsid envelopment apparently occurred de novo in the nucleus; (2) densely staining vacuolar accumulations were seen, frequently surrounding virus capsids. The 13 temperature-sensitive mutants of the virus were divided into three classes according to the type of capsid, if any, produced in cells infected and maintained at the non-permissive temperature. Class I mutants produced no capsids, Class II mutants produced empty and partial-cored capsids and Class III mutants produced empty, partial- and dense-cored capsids. Cellular alterations were also determined. Membranous tubular structures, previously unreported for herpes simplex virus, were observed in cells infected with Class III mutants and very occasionally with wild-type virus at the non-permissive temperature. Cytoplasmic particles were also found, but could not be correlated with any particular class of mutant.

NIH/3T3 cells chronically infected with the Moloney strain of murine leukaemia virus were incubated with interferon (IF). There was no effect on virus production during the first 4 h, but thereafter an antiviral state gradually developed, reaching a maximum at about 12 h. When IF was removed, the antiviral state (expressed in terms of inhibition of release of virus) remained constant for 10 h, after which there was an abrupt return to the normal rate of virus release. Analysis of IF-treated cells showed that there was a three to fourfold increase in the amount of virus RNA in the nucleus at 48 h after IF addition, and still a slight increase at 72 h. There were no increases in the amounts of virus RNA in the cytoplasm during 72 h after the addition of IF. These results agree with the postulate that IF inhibits a late stage in the maturation of virus in chronically infected cells.

Of the three major proteins associated with the rabies virus membrane, only the glycoprotein was found to be located on the external surface of the virus membrane. Glycoprotein prepared by treatment of rabies virus with Triton X-100 and purified by isoelectric focusing was found to be homogeneous with respect to size and isoelectric point. This material, which is free of phospholipids, is able to protect in vaccination experiments against a lethal challenge infection with rabies virus. The apparent mol. wt. of this component isolated under non-denaturing conditions is approx. 400000. The same material analysed by SDS polyacrylamide gel electrophoresis (PAGE) was found to consist solely of polypeptide chains of the G protein (mol. wt. 80000). A minor glycoprotein (gp 50), detected by PAGE of the Triton X-100 released material, appeared to be a breakdown product of the G-protein. Therefore the detergent released material represents homopolymers of the G-protein. Whether the antigenic determinants reside on the monomeric subunit or are a property of the polymeric form of the G-protein is discussed.

Schmidt-Ruppin Rous sarcoma virus infected chick cells injected into newborn C3H/f mice gave rise to tumours at the site of inoculation. These tumours were transplantable in adult C3H/f mice and were able to induce tumours in the wing of adult Leghorn chickens. Tumour cells from the 18th passage in mice were used to establish a cell line in tissue culture (C3HSR). These cells released C-type virus particles that produced foci and were able to propagate in chick cells. Cloning of the C3HSR cells demonstrated that the same cell expressed both avian and murine antigens. Mouse cells infected with virus released by C3HSR cells produced murine leukaemia virus-like particles as revealed by the reverse XC syncytial test and by immunofluorescence tests.

When the partially purified P65–70 proteolytic factor was added at increasing concentrations to ‘immature’ core sub-particles of Rauscher leukaemia virus (RLV), we observed an increased cleavage of P65–70 (the gag gene product) and P40 (an intermediate cleavage product containing p30) to p30, the major group specific antigen. When examined by electron microscopy the immature cores exhibited a linear decrease in number, with a concomitant increase in the number of mature cores after treatment. Various intermediate structures retaining elements of both immature and mature forms were also observed, suggesting that the in vitro conversion from immature cores to mature cores can occur on a 1:1 basis.

The structure of vesicular exanthema virus, the prototype member of the calicivirus group, has been studied in more detail. The RNA comprises 18% of the virus particle and has a mol. wt. of about 2.8 × 106, based on polyacrylamide gel electrophoresis experiments in the presence of formaldehyde. The virus contains one major polypeptide, mol. wt. 70 × 103 as determined by polyacrylamide gel electrophoresis and by chromatography on Sepharose 6B in the presence of 6 m-guanidine. Further evidence for the presence of a single major polypeptide was obtained by tryptic peptide analysis of 35S-methionine labelled virus. The mol. wt. of a protein oligomer produced by adjusting the pH of virus suspensions to 3.5 was c. 200 × 103. On the basis of these data we propose a T = 3 model for the virus capsid incorporating 180 copies of the virus protein.

The seroepidemiology of the human syncytial virus was investigated by means of an indirect immunofluorescence test on 241 sera from Kenya, Tunisia, Singapore and Britain. These included sera from patients with nasopharyngeal carcinoma, other tumours of the oro-nasopharynx, tumours of other parts of the body, and from normal donors. In this study, the virus was found to infect only Kenyan Africans and all but one of these seropositive subjects had tumours, particularly of the oro-nasopharyngeal spaces. The significance of these findings is discussed.

The synthesis of low mol. wt. RNA has been studied in KB cells infected with adenovirus type 2 after labelling with 3H-uridine or 32P. An increasing amount of virus-coded VA RNA is detected from 8 h after infection onward. The rate of synthesis of tRNA is unchanged up to 16 h after infection and thereafter decreases; from 24 to 48 h after infection, the specific activity of tRNA is about 60% of the control value. The specific activity of cellular 5S RNA increases from 12 h after infection. When the tRNA is analysed by a two-dimensional gel electrophoresis system resolving the tRNA into 42 to 47 spots, changes in synthesis of tRNA in individual spots are seen. From 8 to 12 h after infection, an increase in the relative rate of incorporation into RNA is observed in 7 spots, while a significant decrease is detected in 8 spots. From 12 to 16 h after infection, incorporation into RNA is increased in 6 spots, but is most marked (sevenfold) in 1 spot. A decrease of incorporation into RNA in 6 spots is observed at the same time. From 24 to 34 h after infection, an increase in synthesis of RNA in 8 spots is observed and a decrease also in 8 spots. Incorporation into RNA in 2 spots is virtually shut off.

The isolation and properties of three new insect viruses are described. Two were isolated from the limacodiids, Darna trima and Thosea asigna, and one from the saturniid, Philosamia cynthia × ricini. The three viruses are closely related to Nudaurelia capensis β virus and the virions of the four are serologically related to, but distinguishable from, each other. Virions of members of the group have a diam. of 35 nm, a sedimentation coefficient of 194 to 210S and a buoyant density in CsCl of 1.27 to 1.30 g/ml. The virion contains between 10 and 11% RNA of mol. wt. about 1.8 × 106, and 240 molecules of a single polypeptide species with a mol. wt. of 61000 to 63000. The mol. wt. of the virion is about 16 × 106. These four viruses superficially resemble the caliciviruses of mammals but there are some major differences between the two groups.

Although poly(I) is generally considered to be inactive as an interferon inducer, we have found several authentic poly(I) preparations to be effective inducers. Their interferon inducing ability varied considerably from one cell system to another. In human diploid fibroblasts, primed with interferon and superinduced by cycloheximide and actinomycin D, all active poly(I) samples proved nearly as effective in inducing interferon as poly(I).poly(C). In primary rabbit kidney cell cultures, the active poly(I) samples were either as active, or 3 to 30 times less active than poly(I).poly(C). In intact rabbits they were 100 times less active than poly(I).poly(C). Except for one particular sample, all active poly(I) preparations were inferior to poly(I).poly(C) when assayed for interferon induction in interferon-treated mouse L cells; in DEAE-dextran-treated L cells, they induced little, if any, interferon. The poly(I) inducers of interferon were considerably more susceptible to degradation by T1 ribonuclease, pancreatic ribonuclease and human serum nuclease(s) than was poly(I).poly(C) when assayed under the same conditions. Due to their limited half-life time in biological fluids, poly(I) analogues such as those described here may offer a greater safety margin in clinical use than poly(I).poly(C).

It has been found that 1000-fold more bovine rotavirus is obtained when trypsin is incorporated in the maintenance medium and allowed to remain throughout the growth cycle. This holds true for primary calf kidney (CK) cells and also for several continuous and semi-continuous cell lines. In the presence of trypsin it has been possible to pass the virus serially on continuous cell lines seven times. Concentrations of 1 to 10 µg/ml of trypsin are found to be effective. Preliminary results suggest that the same technique will be effective for the in vitro propagation of human rotavirus.

A host-range mutant of adenovirus type 5, which grows in human cells but not in hamster cells, has been isolated. This mutant is complemented in mixed infection in hamster cells at 38.5 °C by temperature-sensitive mutants of type 5 belonging to seventeen complementation groups, and may constitute a new group. In mixed infection of human cells at 32 °C with the host-range and temperature-sensitive mutants, recombination takes place and by a series of two factor crosses the host-range mutation has been approximately located on the adenovirus genetic map.

Direct plaque formation with representative strains of hog cholera virus (HCV) has been obtained using several pig kidney cell lines under agar overlay. HCV-infected cells appear as hazy plaques when viewed against an indirect light source, and as white plaques after neutral red staining. HCV assay by direct plaque procedure is rapid and convenient and gives infectivity titres identical to the fluorescent focus assay technique.