- Volume 5, Issue 1, 1969

Cell cultures were established from the lungs of hamsters which had been previously infected intranasally with the judith strain or the HPV 77 vaccine strain of rubella virus. The hamster lung cells were cultured in vitro and produced infective rubella virus for at least 12 subcultures. The persistently infected cells had a lag phase of 48 hr compared to a lag phase of 24 hr in uninfected cells. Morphologically, the persistently infected cells were indistinguishable from uninfected cells. Treatment of the persistently infected cells with 25 µg./ml. amantadine, 0.15 µg./ml. actinomycin D or 250 µg./ml. hydrocortisone had no detectable effect on rubella virus yields. Infective cell counts indicated that between 5 and 50% of hamster lung cells were releasing infective rubella virus. The persistently infected cells were resistant to challenge with vaccinia virus and, to a lesser degree, with herpes simplex virus.

H-1 virus underwent an abortive cycle of replication in secondary human embryonic lung-cell cultures, characterized by formation of immunofluorescent H-1 antigen, without the production of infective virus. After mixed infection with adenovirus 12, approximately 30 to 40% of cells yielded infective H-1. A single infective adenovirus particle was capable of acting as helper. Comparison of the growth cycles of H-1 and ‘helper’ adenovirus showed that the two viruses had a similar latent period of about 24 hr. Inoculation of cells 24 hr before with adenovirus shortened the latent period for H-1 by at least 12 hr. Particles with serological characteristics of both viruses (mixed coats) were not found in virus yields from mixed infections.

H-1 inhibited yields of adenovirus p.f.u. as well as virus antigens, provided that a low adenovirus input was used. The decrease in adenovirus yield was due to a reduction in the number of cells producing virus. With a high adenovirus input (10 or more p.f.u. per cell), no interference by H-1 was found. Maximal or nearly maximal potentiation of H-1 growth apparently occurred even when the H-1 caused virtually complete interference with formation of its ‘helper’ adenovirus.

Lipopolysaccharide was isolated from a Proteus vulgaris strain susceptible to the killing action of P. vulgaris bacteriocin 45, from two resistant mutants and from a wild P. vulgaris strain, none of which adsorb the bacteriocin. The carbohydrate composition of the lipopolysaccharide of the sensitive organism differs from that of the resistant strains. Neutralization tests and electron microscopy showed that this phage-tail-like bacteriocin only adsorbed to the cell wall or the lipopolysaccharide fraction of the sensitive P. vulgaris strain. Adsorption was accompanied by triggering of the bacteriocin particle.

A procedure for the purification of rubella virus from infected suspensions of BHK 21 cells resulted in preparations containing 1 to 3 × 1010 p.f.u./ml. In sucrose gradient centrifugation the area of maximal infectivity (buoyant density of 1.175 g./ml.) coincided with peaks in haemagglutinating activity, and in E 260. It also coincided with presence of virus-like particles, and a sharp band visible by naked eye. Specific activities of the order 30 × 106 p.f.u./µg. protein, 106 HAU/µg. protein, and 5 to 10 × 105 p.f.u./HAU were achieved.

Glutaraldehyde-fixed negatively stained rubella virus preparations showed round particles with rough surfaces measuring 50 to 73 nm. (average 61 nm.) in diameter. Unfixed viruses had a greater size variation, 55 to 89 nm. (average 74 nm.) in diameter, apparently due to deformability and fragility of the particles. Spontaneous and deoxycholate-induced breakdown of the particles showed rupture of the envelope but revealed no characteristic inner structure.

The growth of rubella virus in suspended BHK 21 cells was studied by electron microscopy under conditions of one-step growth. After a latent period of 15 hr the first new virus particles were found with their number increasing successively till about 25 hr. This corresponded well with the infectivity assays obtained from these samples.

The virus is formed through two pathways; first, by budding into vesicles of the Golgi-apparatus which then are emptied outside the cells; second, to a lesser extent by budding directly from the marginal cell membrane. No accumulation of nucleoids in the cytoplasm is detected, but they are formed at the site of budding.

Except for herpes virus, a number of enveloped viruses have previously been reported to be sensitive to white light in the absence of photoreactive dyes. We have shown that herpes virus, like measles, Sindbis and vesicular stomatitis viruses, can also be rendered photosensitive if the virions are removed from the protective effects of organic compounds contained in the virus harvest. Under the very same conditions, non-enveloped viruses (vaccinia, polio and adenoviruses) are completely photoresistant. The photosensitivity of enveloped viruses can be enhanced by the presence of salts or increased pH values. Enveloped viruses are photosensitive even when replicated in cells grown and maintained in the absence of serum and riboflavin. Human herpes virus strains obtained from herpetic lesions and never passaged in cultures were also readily inactivated by white light. Experiments with monochromatic light showed the 425 nm. wavelength to be most effective in inactivating the virus. The virus envelope behaves like a functional membrane, since neutral red, which is taken up only by living cells, markedly increases the photosensitivity of enveloped viruses, but has no effect on naked viruses.

L cells infected with the virus of lymphocytic choriomeningitis, strain armstrong, produced infectious progeny; this was unaccompanied by visible signs of cell damage. Serial cultivation of infected cells in a medium free of antibodies was easily accomplished. Initially, retardation of cell multiplication and of first subculture cytopathic effects were observed. Later, a state of equilibrium was attained in which the cells multiplied at a normal rate, while continually producing and releasing virus. Although almost every cell of these carrier cultures could be shown by immunofluorescence to be infected, morphologically and functionally pathological alterations were not found.

After prolonged cultivation the carried virus was found to be incomplete; its ability to spread from cell to cell was greatly reduced; its ability to kill mice was abolished. These losses were not regained by passages in various hosts. Otherwise the carried virus resembled the original armstrong virus in conferring solid immunity to mice, reacting specifically with antibodies and sedimenting in the ultracentrifuge like lymphocytic choriomeningitis virus.

To characterize more fully the virus-host relationship, the following facts have been established. No antigens, alien for L cells, were found on the cell surface. Most clones from single cells carried lymphocytic choriomeningitis-specific antigen in every cell. A few clones were evidently completely free of the virus. Some cultures derived from single cells consisted of a mixed population of infected and antigen-free cells.

Homologous interference was absolute; heterologous interference against vesicular stomatitis, encephalomyocarditis and vaccinia viruses could not be demonstrated.

Treatment of L (Arm) cultures with lymphocytic choriomeningitis-neutralizing antibodies slowly decreased the proportion of antigen-containing cells. This ‘cure’ was real, and, furthermore, not caused by the inhibition of multiplication of L (Arm) cells reacting with antibodies. This observation indicated that the persistent infection of L cells with the lymphocytic choriomeningitis virus, strain armstrong, was not only maintained by a vertical transfer of virus from parent to daughter cells but that horizontal spread played a role.

The antigenic structure of a recombinant virus, FPV-A2 (r4), obtained by the interaction of an avian and a human influenza A virus was investigated by means of haemagglutination-inhibition, neuraminidase-inhibition and immunoprecipitation tests. The neuraminidase of the recombinant was found to be antigenically identical to that of its A2/singapore/1/57 parent whilst its haemagglutinin was identical to that of fowl plague virus, the other parent. Similar studies on a recombinant obtained by the interaction of two avian influenza A virus strains, fowl plague virus and A/turkey/massachusetts/65 virus, indicated that it contained neuraminidase of the antigenic type found in the turkey virus and haemagglutinin like that of fowl plague virus. A/turkey/massachusetts/65 was selected for study since its neuraminidase is antigenically closely related to that of human A2 virus strains.

Studies on the electrophoretic mobilities of the structural proteins of FPV-A2 (r4) indicated that it contained structural components derived from each parent and thus supported the immunological findings.

The significance of these findings in relation to possible genetic interactions between human and avian influenza viruses in nature is discussed.

When preparations of a tobacco necrosis virus were negatively stained with 2% sodium or potassium phosphotungstate, pH 6 or 7, immediately before viewing in an electron microscope, many of the virus particles were degraded or were penetrated by the stain. However, when the potassium phosphotungstate was at pH 3, 4 or 5 few of the particles were degraded or were penetrated by the stain. A correlation between the pH of stability and the isoelectric point of the virus is suggested. Fixation for 20 min. in 5, 10 or 20% formaldehyde effectively stabilized most of the particles of tobacco necrosis virus when stained with neutral potassium phosphotungstate, whereas fixation in 1.25 or 2.5% formaldehyde did not.

Several types of mutants variously affected by the concentration of calcium ions in the medium may be isolated from phage P2 (host: Escherichia coli). As compared with the wild type, the mutants may be more or less dependent on calcium for adsorption, more rapidly inactivated in the presence of calcium, or less prone to establish lysogeny at low calcium concentrations.

Magnesium pyrophosphate gel (Mg-PPi), previously employed for the purification of animal viruses and bacteriophages, was used for the purification of plant viruses with filamentous and isometric particles. The viruses studied were turnip yellow mosaic, two strains of tomato bushy stunt and tobacco mosaic virus. Ultracentrifugal analysis of the purified products obtained indicated a high degree of purification. The infectivity of the preparation of the viruses was not altered by the chromatographic method. Intact were separated from empty isometric virus particles. Tobacco mosaic virus particles were separated according to their length and molecular weight.

The coliphage α 3, which is morphologically similar to φX174, was shown by acridine orange staining to contain single-stranded DNA. Its infective process was studied by the electron microscopy of thin sections of Escherichia coli infected at high multiplicity. The latent period was found to be about 13 min. but no obvious structural changes were visible in host cells until 10 min. after infection, when the plasma membrane began to retract from the poles. At 14 min. intracellular phage was detected and bulges appeared on the cells at their midpoint. At 21 min. many cells were in the form of spheroplasts and others were beginning to lyse. Spheroplasts formed by phage infection lysed differently from those produced by penicillin.

Purified suspensions of Sericesthis iridescent virus were treated with a nasal decongestant, negatively stained and examined in the electron microscope. The outer icosahedral surface of the virus particles showed morphological subunits apparently in close-packed hexagonal array and 70 Å apart. The orientation of untreated particles was determined from their outline. This made it possible to measure precisely their icosahedral edge-length (860 ± 27 Å).

Prolonged storage of purified virus in distilled water at 4° resulted in disintegration of the particles into triangular, pentagonal and linear fragments. The triangles of side 700 Å were clearly composed of 55 hexagonally arrayed subunits 70 Å apart. Edges of the pentagons appeared to consist of three subunits also about 70 Å apart, while the linear fragments had a broad length distribution about a mean of 438 Å.

All these observations, interpreted by a new approach to the ‘Goldberg diagram’, suggested that the virus surface is composed of 1562 morphological subunits, though alternatives of 1292 and 1472 subunits cannot be excluded.

During our investigation of the structure of arboviruses (Bergold, Graf & Munz, 1966; Bastardo, Bergold & Munz, 1968) 9 more viruses have been studied (Table 1). Serological tests kindly done by the Trinidad Regional Virus Laboratory (TRVL) and the Middle American Research Unit (Maru) in Panama helped to confirm the identity of the viruses. Viruses were grown in roller flasks in BHK 21 cells and titrated by plaquing in the same cells, using a serum-free maintenance medium (Bergold & Mazzali, 1968). The viruses were concentrated (400 ×) and purified by centrifugation (Bergold et al. 1966; Bastardo et al. 1968). Only two high-speed centrifugations were done with most viruses (Table 2) because the additional cycle caused considerable loss of infectivity. For electron-microscopy the virus samples were mixed (1 + 1) with sodium phosphotungstate (pH 7) and glycerol (1 + 2) was added (Horne, 1965).

Since the partial degradation of influenza virus particles by trypsin was first reported (Mayron & Rafelson, 1960), the virus has been shown to be susceptible to the action of many other proteases. Degradation by these means is known to cause the release of low molecular weight neuraminidase from the particles, but the other activities associated with the viral surface, that is haemagglutination and V-antigen complement fixation, are apparently destroyed. Degraded virus particles obtained after such treatment show markedly altered physical properties which may be attributed to the loss of about 50% of the viral protein; the bulk of nucleic acid however is still particle-associated (Biddle, 1968; Kendal, Biddle & Belyavin, 1968). This suggests that there is a membrane resistant to protein digestion beneath the characteristic protein outer coat of the virus. To investigate this structural feature, protease digested virus was examined electron microscopically, using both negative staining and thin-sectioning techniques.

The infectivity of polyoma virus may be assayed by measuring haemagglutinin induced in a single cycle of infection in mouse kidney cells by a method similar to that used for quantification of influenza virus (Cairns et al. 1952). Although less sensitive than plaque assay (Dulbecco & Freeman, 1959) or end-point titration (Rowe et al. 1959) the method is more rapid.

Primary mouse kidney cells (Nordenskjöld & Krakoff, 1968) were grown in Eagle's minimum essential medium in Earle's solution + 10% calf serum. For virus production the cells were grown in Roux bottles and for plaque assays in 60 mm. plastic Petri dishes (Falcon Plastics Inc.). The medium was changed daily for the first 3 days. The Glasgow variant of polyoma virus (Crawford, Crawford & Watson, 1962) was kindly provided by Dr G. di Mayorca, Naples.

Because of the unusual properties of scrapie agent and doubt as to its being truly a virus, experiments were undertaken to find out if an interferon stimulating agent might influence the development or course of the disease following inoculation of infective material into mice. A group of 12 three-week-old male mice was inoculated intraperitoneally with 0.1 ml. of 5% Statolon made up in sterile 1% sodium bicarbonate solution one week before challenge with 0.1 ml. 10-1 scrapie mouse brain suspension injected intraperitoneally. A second Statolon injection was given the next day and at weekly intervals until the end of the experiment. At the same time six similar mice were inoculated intraperitoneally with the same scrapie material to serve as controls.

Experimental and control animals both developed clinical signs after 7 months, with no difference between the groups as to incubation period or the way in which signs evolved. All animals were examined histologically for confirmation of disease.

The flexible elongated plant virus, potato virus X, has been studied by a number of physical and physical-chemical methods. Here we wish to compare the results of these studies and to make some observations on the structure of the virus which these studies suggest.

X-ray diffraction studies of potato virus X were first made by Bernal & Fankuchen (1941), who obtained diffraction patterns suggesting a periodicity along the virus particle of 33 Å. Later X-ray studies (Tollin et al. 1967) showed that this periodicity varied from 33 to 36 Å depending on the amount of water in the specimen. A dry specimen of potato virus X with improved orientation has been obtained in an identical fashion to that for narcissus mosaic virus (Tollin, Wilson & Young, 1968). The X-ray diffraction pattern (Pl. 1) can be interpreted as being due to a helical arrangement of subunits with a pitch of 33 to 36 Å, depending on water content.

Investigation of a Zygocactus X Schlumbergera hybrid (Christmas Cactus), which showed no symptoms, led to the isolation of a strain of cactus virus X (CVX) and another, new virus for which the common name zygocactus virus (ZV) is suggested.

Crude sap from a cladophyll of this cactus hybrid inoculated to Chenopodium quinoa Willd. caused many local lesions after about 6 days, and some days later slight distortion and curling of young leaves. While the local lesions were typical of CVX, the systemic symptoms supported the existence of a second virus (ZV). Mechanical transmission of sap from systemically infected C. quinoa to Nicotiana glutinosa L., which is not a host for CVX, caused systemic infection and resulted in light green spotting of the younger leaves. The virus infected but caused no symptoms in Nicotiana clevelandii Gray and Solanum demissum Lindl. The thermal inactivation point of ZV in crude sap of these four herbaceous hosts was at 10 min. exposure 72 to 74°.