- Volume 5, Issue 3, 1969

The BSC/SV 40 virus carrier state was established by infecting monolayers of BSC1 cells with high concentrations of SV40. In these carrier cultures, only 4% of the cells contained intranuclear tumour (T) antigen as determined by the immunofluorescent method, but every cell produced infectious virus. During passage of the BSC/SV40 cultures, a cell line was selected in which all the cells exhibited T antigen and only 0.2−1% of the cells produced infectious virus (transformed state). The BSC1 transformed cells were like the BSC1 parent cells sensitive to challenge with heterologous viruses (attenuated polio 1, herpes simplex and vaccinia), but were resistant to superinfection by the homologous SV40. Cytosine arabinoside, actinomycin D and antiserum to SV40 inhibited virus and T antigens in the BSC/SV40 carrier cultures. In the transformed cells, infectious virus and virus antigen only were inhibited but synthesis of the T antigen remained unaffected. By cell cloning, two virus-free clones were obtained.

A total of 212 arbovirus serotypes, representing 29 arbovirus serogroups, were screened for their plaque-forming ability and titrated in two continuous simian kidney cell lines (VERO and LLC-MK2). Plaquing data were obtained with 205 viruses in either one or both of these cell lines; seven viruses failed to plaque in either line. Both cell lines were susceptible to 169 arbovirus serotypes. Seventeen viruses failed to plaque in VERO and 18 in LLC-MK2. Plaques appeared as early as 2 days, and as late as 18 days (Triniti virus in VERO) Plaque diameters ranged from 1 to 28 mm. during a 24-day observation period.

By pulse-labelling cells infected by adenovirus type 5 with [3H]thymidine, extraction of the DNA and equilibrium density gradient centrifugation in caesium chloride, viral and cellular DNA synthesis could be readily differentiated. In the experimental system used, cellular DNA synthesis was inhibited early in infection and virus DNA synthesis began about 2 to 3 hr later. These events were related chronologically to the production of the virus antigens. When arginine was withdrawn from the cell culture medium, the production of virus DNA and the inhibition of cellular DNA synthesis was still apparent.

By labelling at later times when only virus DNA was being synthesized, the fate of the label could be followed during virus maturation. The results suggested that virus DNA was associated with heterogeneous membrane-containing structures before being assembled into virus particles.

Suspensions of human leucocytes purified by NH4Cl produced interferon in the same time after induction by Sendai virus whether the medium contained serum or not. In the absence of serum the interferon titre levelled off after as little as 5 hr, and the yield was less than 10% of that obtained when serum was present. The production of interferon was completely inhibited by 2 μg./ml. of actinomycin D added simultaneously with the virus. Interferon synthesis gradually became insensitive to actinomycin during the first 4 to 5 hr after induction in both the presence and absence of serum. Large amounts of interferon rapidly appeared after addition of serum to cultures induced and incubated in serum-free medium. The cells responded to added serum by interferon production up to 16 to 20 hr after induction. The rate of interferon production after addition of serum was not affected by actinomycin. Removal of serum from the medium resulted in a reduced yield of interferon; which would not be prevented by addition of actinomycin. The low yields of interferon in a medium devoid of serum were not due to reduced synthesis of the DNA-dependent RNA needed for interferon production.

The effects of the synthetic polyanionic interferon inducers maleic acid/divinyl ether copolymer (MA/DVE) and polyinosinic/polycytidylic acid (polyrI/polyrC) were studied in mice challenged intranasally with vesicular stomatitis virus. Young (14-day or 24-day) animals were used. Either local (intranasal) or systemic (intraperitoneal) treatment protected significantly against virus infection. Optimal local protection was given by 40 μg. of polyrI/polyrC instilled 4 hr before virus challenge. Intraperitoneal MA/DVE exerted a more durable effect lasting for at least 10 days, and polyrI/polyrC proved to be active when applied several days after inoculation of virus. PolyrI/polyrC treatment started after virus in the brain had reached maximum concentration retarded progression of the disease, but when delayed until clinical signs of illness occurred, polyrI/polyrC treatment was not effective. The protective activity of MA/DVE and polyrI/polyrC injected intraperitoneally against virus infection at a distant site suggested the mediation by a systemic antivirus state, presumably due to interferon production.

Three kinds of polyanionic polysaccharides, chick allantoic mucopolysaccharide, capsular polysaccharide of Klebsiella pneumoniae and heparin depressed interferon synthesis induced by ultraviolet-irradiated Newcastle disease virus in chick embryo cells. The minimum concentrations causing complete inhibition of interferon synthesis were respectively 20, 0.5 and 50 μg./ml. The polysaccharides did not affect the action of exogenous interferon at concentrations sufficient to depress interferon synthesis. It seems unlikely that these polysaccharides were toxic for the cells, and that the depression of interferon synthesis was due to an inhibition of the early steps of virus-cell interaction.

Correlations between the amino acid and nucleotide compositions of the particles of 41 plant viruses suggest that plant viruses, and hence presumably plants, use the same genetic code as bacteria, and that the gene for the protein in the particles of each of these viruses has a nucleotide composition similar to that of the whole nucleic acid molecule of the virus.

A microelectrophoretic technique was used to study the behaviour of a number of different pox virus preparations from chick embryos. The viruses were suspended in buffered media made molar to sucrose to reduce Brownian movement. The mobility/pH curves obtained were consistent with the view that in all preparations the virus surface was protein or lipo-protein. Cowpox, vaccinia and neurovaccinia viruses gave similar results with an iso-electric point close to 4. The white variant of cowpox virus differed from the parent strain with an iso-electric point at approx. 2.8. Rabbit pox virus had the lowest iso-electric point at approx. 2.3. Variola, alastrim and monkey pox viruses all had iso-electric points of approx. 3.4 but at greater pH than this monkey pox showed somewhat higher mobility than the other two viruses.

Respiratory syncytial virus in purified suspensions was inactivated exponentially at temperatures from 50° to 0° and at -78°. The rate constants (K) decreased from 11 log. p.f.u./hr at 50° to 0.2 log. p.f.u./hr at 25°. The constants then increased again to 0.7 log. p.f.u./hr at 0°. An Arrhenius plot showed a linear relationship between ln K and 1/T in the range from 50° to 37°, with thermodynamic constants corresponding with those of protein denaturation. As has been reported for other viruses (reviewed by Woese, 1960), a linear relationship was not observed below 37°.

In molar solutions of NaCl, MgSO4, Na2SO4, sodium-phosphate and glucose the virus was almost completely protected against thermal inactivation at all temperatures greater than 0°. The extent of protection decreased with molarity and 0.1 m-solution of these salts failed to protect. At 0° the virus was protected by glucose, but only partially by salts. The protection against thermal inactivation was apparently not an ion effect and the influence of glucose and of molarity suggests an osmotic effect. The unexpectedly high inactivation rate at 0°, as well as the different effects of NaCl and MgSO4, suggest a unique mechanism at this temperature.

Aerosols of respiratory syncytial virus were kept at different relative humidities (RH). Viability was studied by a plaque method. Virus recoveries 1 min. after aerosolization were highest at high RH. The stability of the aerosol was maximal at 60% RH. The total decay after 61 min. varied from 1.1 log. p.f.u. to 2.5 log. p.f.u., with peak values at 30% RH (2.3 log. p.f.u.) and 80% RH (2.5 log. p.f.u.), suggesting two processes with different kinetics. Oxidation was excluded as a cause of inactivation by experiments in pure nitrogen.

A soluble fraction containing transplantation antigens was isolated from cells infected with adenoviruses and from the membrane fraction from cells of adenovirus-induced hamster tumours. It was not identified either as a part of the virus structure or as a T antigen. This material regularly protected young adult hamsters against the development of tumours resulting from the transplantation of adenovirus tumour cells. Protection was given by soluble antigens isolated from more than one cell type; the antigens could be induced by at least two groups of adenovirus, with probable cross-protection within groups. The antigens did not protect animals challenged with tumour cells produced by a different DNA virus, SV40.

When neutral red is added to the liquid medium in tissue cultures, more dye is taken up by healthy cells than by damaged cells. This forms the basis of a method for assessing the relative extent of cell damage in different cultures, the amounts of bound dye being determined directly by inspection of the stained cell sheets, or indirectly by estimating extracted dye colorimetrically. This procedure can be used to measure the effects of a cytocidal virus and can also be applied to the assay of interferons. Here it provides a precise and reproducible method which is unusually sensitive for the assay of mouse interferon, and has also been used to assay rat, chick, rabbit and human interferons.

There are many reports in the literature indicating that the administration of exogenous interferon or induction of endogenous interferon can protect experimental animals against certain virus infections (reviewed by Finter, 1966, and Vilcek, 1969). In most of these studies, challenge was by parenteral inoculation of relatively large quantities of virus and the protective effects of interferon were assessed in terms of decreased mortality or less severe disease. However, naturally acquired virus infection is generally the consequence of exposure to much smaller quantities of virus, and with respiratory viruses, infection is initiated by deposition of virus in appropriate areas of the respiratory tract and not by parenteral inoculation. Accordingly, experiments were designed to determine whether interferon induced by Newcastle disease virus (NDV) could protect mice from infection by influenza virus transmitted by other mice. This experimental system was described in detail by Schulman & Kilbourne (1963) and was used to study the effects on transmission of infection of other modifications of the host such as immunization (Schulman, 1967).

Infection of human diploid cells with Simian virus 40 (SV 40) leads to an abortive virus cycle characterized by little virus production, little or no cell destruction, no inhibition of cell replicating capacity, low efficiency of induction of tumour antigen (T-antigen) and eventual transformation of the morphological, chromosomal, and growth characteristics of the cells (Koprowski et al. 1962; Shein & Enders, 1962; Rabson et al. 1962; Weinstein & Moorhead, 1965). In marked contrast, in stationary-phase primary African green monkey kidney (GMK) cells, SV 40 infection leads to high yields of virus, highly efficient induction of T-antigen and extensive cell destruction (Carp & Gilden, 1966). The determination of the cause of these marked differences might clarify the mechanism of the transformation process. Previous results indicated that the virus adsorbed and entered into eclipse phase with equal efficiency for the two cell types (Carp & Gilden, 1966).

Infection of protoplasts and spheroplasts by isolated bacteriophage DNA with production of whole phage has been reported for a number of organisms (Riggs & Rosenblum, 1969). This phenomenon has not previously been described in Proteus strains.

DNA was prepared from phage 13 vir (Prozesky, de Klerk & Coetzee, 1965) by the technique of Gierer & Schramm (1956). The concentration of DNA, determined by optical extinction at 260 nm. (1 μg. DNA/ml. = 0.0205 absorbency units), ranged from 200 to 600 μg./ml. Sedimentation coefficients of the DNA at concentrations of 10 to 50 μg./ml. were determined in a Spinco model E ultracentrifuge (Rosenblum & Schumaker, 1963). The DNA was homogeneous and an average molecular weight of 26.3 × 106 was calculated. Sedimentation of the DNA through an alkaline sucrose gradient revealed no single-strand breaks.

A cell line (HT-1) derived from a hamster tumour induced by the Moloney sarcoma virus has been extensively used for preparation of murine pseudotype sarcoma viruses (Huebner et al. 1966; Hartley et al. 1969). Infectious virus could not be isolated from these cells nor was the group specific (gs) antigen of the murine C-type RNA viruses detectable in complement-fixation tests (Huebner et al. 1966). In further studies with this cell line, clonal sublines were derived and tested for infectious virus and virus particle production, gs antigen using both complement-fixation and precipitation-ingel assays, and the presence of virus genome by a highly reproducible rescue technique.

HT-1 cells in the 80th in vitro passage were seeded into ten 60 × 15 mm. plastic Petri dishes (50 cells/dish). Each culture yielded 5 to 10 individual colonies, of which five were isolated by trypsinization within glass cloning rings. Each of the 50 clones was maintained separately in Eagle’s basal medium with 10% foetal bovine serum.

Bee chronic paralysis virus (cryptogram R/1:*/*:X/X:I/O.) is common in honey bees (Apis mellifera L.) throughout the world (Bailey, 1967). Its ellipsoidal particles contain ribonucleic acid and are of three sizes; 22 nm. wide and about 41, 54 and 64 nm. long, and have sedimentation coefficients (S 20, w) of 97S, 110S and 125S respectively (Bailey, Gibbs & Woods, 1968). The particles of bee chronic paralysis virus (CBPV) seem unlike those of any other described virus, except perhaps mouse lactic dehydrogenase virus ((R)/*:*/*:X/*:V/O), (Notkins, 1965). The particles of lactic dehydrogenase virus (LDHV) may contain RNA, and are elliptical in outline, 36 to 42 × 45 to 75 nm. when sectioned but 60 to 65 × 70 to 85 nm. in suspension (De The & Notkins, 1965). In this note some properties of the particles of LDHV are compared with those previously reported for CBPV.

Gel filtration on agarose (agar) has been used to separate viruses differing in size (Bengtsson & Philipson, 1964; Čech, 1962; Fridborg et al. 1965; Steere & Ackers, 1962). We therefore tried it for the separation of adeno-associated viruses (AAV) from adenoviruses, including the structural components of the latter. The results presented here indicate that chromatography on Sepharose 2B (Pharmacia, Uppsala, Sweden) represents a useful step for such separation.

The source of AAV and adenovirus was similar to that used in previous studies (Hoggan, Blacklow & Rowe, 1966) with adenovirus type 7, strain LLE46+, containing the adenovirus SV40 hybrid and AAV type 1. The viruses were grown in African green monkey kidney cells. The preparation of [3H]thymidine labelled adenovirus type 7 and the measurement of radioactivity are described in a subsequent paper (A. R. Neurath, B. A. Rubin & R. W. Hartzell, 1969, in preparation).

We have exposed suspensions containing the infective agent of scrapie to doses of ultraviolet light (u.v.) up to 12 times greater than those used in the experiments reported by Alper et al. (1967). Suspensions were prepared and assayed by the methods described by those authors and by Alper, Haig & Clarke (1966). Two different suspensions were irradiated on 2 days separated by a week and were exposed to nearly monochromatic u.v. at 254 nm. from a 15 w ‘germicidal’ lamp. All of the samples were assayed in duplicate and about the same level of activity remained after similar doses in both experiments (Fig. 1). Ultraviolet absorption spectra taken on several preparations made at various times, were constant within fairly narrow limits. Transmittances of the total samples (thickness 2 mm.) irradiated were 0.93. Samples were stirred during irradiation.