Rabbitpox Research Articles

Effect of Isoleucine Deprivation on Rabbitpox Virus DNA Synthesis in Mouse L Cells

Synthesis of rabbitpox DNA was inhibited in mouse L cells deprived of isoleucine. Time-course patterns of incorporation of radiolabelled precursors into viral DNA revealed that synthesis of viral DNA began about 6 h after reversal of the isoleucine-deficient state.

A quantitative estimation of poxvirus genome fraction transcribed as “early” and “late” mRNA

The annealing of labeled rabbitpox virus DNA with an excess of RNA extracted from the infected cells at different stages of reproduction cycle has been performed. In chick embryo, cultured cells treated with cytosine arabinoside in order to prevent DNA replication or in the untreated cells prior to the onset of viral DNA synthesis the presence of virus-specific RNA complementary to 17–20% of virus DNA has been revealed. At later stages of the reproduction cycle the hybridization has revealed RNA sequences complementary to 40–42% of viral DNA. If the replication of viral DNA has been prevented by the inhibition of protein synthesis with cycloheximide, RNA sequences complementary to 28–33% of DNA can be detected. The results of hybridization experiments with the use of mixed RNA preparations suggest that RNA extracted from cycloheximide-treated cells comprise the predominant part of the sequences synthesized in the presence of cytosine arabinoside. Virus-specific RNA present in the cell at late stages of reproduction comprises the bulk of RNA sequences synthesized in the presence of cycloheximide. Hybridization data close to the ones obtained with chick embryo cells have also been obtained with RNA extracted from rabbitpox virus-infected HeLa cells.

Neutralizing activities of antisera to poxvirus soluble antigens.

SUMMARY Sera from rabbits that had been immunized with soluble antigens from rabbit-pox or vaccinia viruses showed high neutralizing activity against the intracellular form of rabbitpox virus, but little neutralizing activity against the extracellular form. They were almost ineffective in preventing the distant dissemination of rabbitpox virus through infected HeLa cell cultures. These results account for the disappointingly incomplete protection that was previously observed in rabbits immunized with vaccinia virus soluble antigens.

Enhanced utilization of citrulline in rabbitpox virus-infected mouse sarcoma 180 cells.

SUMMARY The replication of rabbitpox virus has been studied in mouse sarcoma 180 cells in media containing either arginine or citrulline. The yield of infective virus depended on the concentration of each amino acid, and maximum yield was obtained with 0.1 mm-arginine or 0.5 mm-citrulline. In the presence of this concentration of citrulline, cell multiplication and protein synthesis in uninfected cells was suppressed markedly compared with cell cultures in medium containing 0.5 mm-arginine. Growth of a human citrullinaemia cell line was inhibited also when citrulline substituted for arginine yet the replication of vaccinia virus continued. The incorporation of radioactivity available as [14C]-carbamoyl-citrulline into mouse sarcoma 180 cells maintained in citrulline-containing medium increased significantly following infection by rabbitpox virus. A similar, increased incorporation was observed with vaccinia virus-infected HeLa cells. Increased incorporation into rabbitpox virus-infected mouse sarcoma 180 cells occurred at 3 and 5 h post-infection and coincided with increased incorporation of [14C]-guanido-arginine into cell cultures infected in the presence of arginine. This enhanced utilization of citrulline was inhibited by actinomycin D, and rabbitpox virus replication in the presence of citrulline was inhibited by canavanine. It is concluded that virus-directed mechanisms determine arginine biosynthesis in mouse sarcoma 180 cells infected with rabbitpox virus in the presence of citrulline.

Virus-Associated Nucleases: Evidence for Endonuclease and Exonuclease Activity in Rabbitpox and Vaccinia Viruses

The endonucleolytic action of a deoxyribonuclease activity in rabbitpox and vaccinia virus was established by change in sedimentation rate of denatured (3)H-lambda deoxyribonucleic acid substrate. The presence of two deoxyribonuclease activities in pox-virus is confirmed. Exo- and endonuclease activities are unmasked by treatment of purified virus with the detergent Nonidet P-40 and further enhanced by treatment of viral "cores" with trypsin.

Mechanism of antiviral action of acetone on rabbitpox virus replication.

Acetone added to the maintenance medium in a 1% concentration reduces the yield of infectious rabbitpox virus in L-cell monolayer cultures by 90 to 97%. This concentration of the inhibitor is not toxic for cells. It was established that there is an inhibitor-sensitive stage late in the infectious cycle. Acetone exerted no significant influence on production of early viral messenger ribonucleic acid, formation of polyribosomes, deoxyribonucleic acid (DNA) replication, or protein synthesis. In the presence of acetone, the assembly of so-called "acetone particles" occurred. These particles are similar to normal virions by morphological and sedimentation properties but are slightly different from them in buoyant density. The amount of virus-specific DNA and the optical density of the "acetone particles" are the same as those of normal virions despite a 10- to 25-fold difference in the infectivity of the preparations.

Nirogen dioxide effects on alveolar macrophages.

Rabbits were exposed to 5, 15, 25, or 50 ppm of nitrogen dioxide for three hours. Their alveolar cells were harvested and tested for phagocytic activity, rate of oxygen uptake, and hexosephosphate shunt activity. Other groups of animals were given parainfluenza 3 virus intrafracheally prior to NO2 exposure in order to measure the effect of this gas on virus-induced resistance to rabbitpox virus. Virus-induced resistance and phagocytic activity were suppressed by 15 ppm of NO2. In contrast, 50 ppm stimulated oxygen uptake and hexosephosphate shunt activity. Suppression of phagocytic activity and virus-induced resistance were observed to be the most sensitive indicators of NO2 alterations of alveolar cells.

An antigenic difference between intracellular and extracellular rabbitpox virus.

SUMMARY Extracellular rabbitpox virus released naturally from infected cells differed antigenically from intracellular virus released by artificial disruption of cells. Intracellular virus was neutralized by antiserum prepared against live rabbitpox virus and by antiserum against inactivated vaccinia virus. In contrast, extracellular virus was neutralized only by rabbitpox antiserum. The antibodies responsible for the neutralization of intracellular and extracellular virus could be absorbed separately from rabbitpox antiserum. Morphologically, extracellular virus differed from intracellular virus in possessing an outer envelope. This envelope was probably the site of the virus antigen characteristic of extracellular virus, and fluorescent antibody staining of infected cells suggested that it was derived from the modified host cell membrane. Antibody directed against extracellular virus was responsible for the ability of rabbitpox antiserum to control the spread of rabbitpox virus in tissue culture and probably for its ability to protect rabbits from rabbitpox infection. Extracellular virus should therefore be used as the test virus in titrations of neutralizing antibody if these are to assess the protective activity of an antiserum.

Samplig rabbit pox aerosols of natural origin.

Natural aerosols of rabbit pox virus produced by groups of infected rabbits were sampled with a slit and Andersen sampler using an adhesive surface sampling technique (Thomas, 1970). The higher rate of successful recoveries of airborne virus compared with a previous report is believed to be due to the much larger volume of air sampled by this technique and to the processing of the whole of the sample collected by the direct addition of a cell suspension to demonstrate the presence of viable virus.

Research Note: Detection of Fowlpox Virus Antigen in Tissue Culture by Fluorescent Antibody Technique

The direct fluorescent antibody (FA) technique has been used to detect vaccinia antigens (6,8,9), small pox virus (2,4,5,7), rabbit pox virus (1), fowlpox virus (9), and Yaba Tumor Pox virus (10). In the present investigation, fowlpox viral antigens were detected in whole chicken embryo or chicken kidney cell cultures by a direct fluorescent antibody technique. Hyperimmune fowlpox virus antiserum was prepared in the chickens by cutaneous and intravenous inoculation of fowlpox virus strain ORT-101. This virus strain had been maintained by serial passages in CAM of developing chicken embryos. On cutaneous inoculation, the virus produced typical lesions of fowlpox in chickens. Specific antibodies against the fowlpox viral antigen were demonstrated by passive hemagglutination test and agar gel precipitation test. The serum was conjugated with fluorescein isothiocynate (General Biochemicals, Ohio) according to methods of Cherry and Goodman (3). The conjugated serum was adsorbed three times with acetone-extracted duck liver powder and stored at -60 C. In preliminary studies, both whole chicken embryo cell culture from nine-day-old embryos and kidney cell cultures from 19-dayold chicken embryos were grown in 10 X 150-mm rubber-stoppered sterile tubes containing 11 X 22-mm glass coverslips. Later, only chicken kidney cells were used. Confluent cell sheets were usually obtained on the coverslips in two to three days. A ten per cent suspension of fowlpox-virus-infected chorioallantoic membranes was made in Eagle's medium (Grand Island

Viremia and virus measurements of rabbit pox in CV-1 cells.

Rabbit pox virus (RPV) produced cytopathic effect (CPE) in five types of cells grown in tissue cultures. The CPE on CV-1 cells was characterized by cell fusion and lysis. The CV-1 line is a useful and sensitive cell culture for measuring concentrations of RPV in blood and tissues of infected rabbits. Viremia was detected between the 2nd and 4th days after parenteral infection. By the 6th and 7th days, the concentration of RPV in various tissues ranged from 10(5-3) to 10(8-5) TCID(50)/g. Cross-reactivity was demonstrated by the fluorescent-antibody technique between rabbit pox, vaccinia, and monkey pox viruses.

Nitrogen Dioxide Inhibition of Viral-Induced Resistance in Alveolar Monocytes

Rabbit alveolar monocytes harvested from rabbits injected intratracheally with a 5 × 105 tissue culture infective dose (TCID00) of para-influenza-3 virus are resistant to an in vitro challenge with rabbit pox virus. However, if the rabbits are exposed to 25 ppm nitrogen dioxide (NO2) for three hours immediately after the para-influenza-3 virus inoculation or at 0, 3, 6, 12, or 24 hours before the inoculation of virus, the previously observed resistance does not develop. This refractory state lasts at least 96 hours in that the alveolar macrophages from animals exposed to NO2 are unable to produce interferon when inoculated with para-influenza-3 virus in vitro. Exposure to NO2 also appears to increase the adsorption rate of para-influenza-3 virus in the lungs of rabbits, but it does not inactivate or enhance the infectivity of the virus employed.

Viremia and Virus Measurements of Rabbit Pox in CV-1 Cells

Rabbit pox virus (RPV) produced cytopathic effect (CPE) in five types of cells grown in tissue cultures. The CPE on CV-1 cells was characterized by cell fusion and lysis. The CV-1 line is a useful and sensitive cell culture for measuring concentrations of RPV in blood and tissues of infected rabbits. Viremia was detected between the 2nd and 4th days after parenteral infection. By the 6th and 7th days, the concentration of RPV in various tissues ranged from 10(5-3) to 10(8-5) TCID(50)/g. Cross-reactivity was demonstrated by the fluorescent-antibody technique between rabbit pox, vaccinia, and monkey pox viruses.

Micro-electrophoresis of Pox Viruses in Molar Sucrose

SUMMARY 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.

Effect of some proteolytic enzymes on the plaque titer of vaccinia and rabbitpox viruses

The effect of proteolytic enzymes on the plaque titer of a suspension of poxvirus particles is examined here in two parts. Most enzymes exert a dispersive effect, breaking up virion aggregates which are usually present in substantial numbers. They may raise or lower the plaque titer depending on the nature of the virus preparation. In addition, there is the direct effect of the enzyme upon the virion. Dispersive effects in this work have been measured by electron microscopy in order that the direct effect of the enzymes may be revealed. Rabbitpox and early L-cell passage vaccinia virus, both of low plaquing efficiency, are enhanced 5- to 100-fold in this respect by brief treatment with any of several proteolytic enzymes. Extended enzyme treatment reduces the titer. High-passage vaccinia virus preparations, those with maximum plaquing efficiency, are not enhanced. They are reduced in titer. High-passage vaccinia virus, degraded by pancreatin, may be partially “reactivated” by inducing aggregation among the particles of the plaquing inoculum. This synergistic response is similar to that shown by heated virus but less than that following treatment of the virus with any of several agents including UV, X-ray, and formaldehyde.

The influence of DEAE-dextran on plain and synergistic plaque formation by two poxviruses.

Summary The plaque titre of rabbitpox and early passage vaccinia viruses was enhanced by diethylaminoethyl-dextran. Both viruses have low initial plaquing efficiency. Later passage vaccinia virus of high plaquing efficiency was little enhanced either before or after exposure to u.v., X or γ rays or to heat at 56°. The dextran made an insignificant improvement in adsorption of the virus particles to the cells. It formed flat, round bodies visible in the electron microscope but these were not found to form complexes with the virus particles nor to induce them to aggregate. This eliminated synergistic infection by aggregation of virus particles as a mechanism for plaque enhancement by dextran but suggested a protective action of the dextran particles upon freshly uncoated DNA within the cell vesicle.

Conditional lethal mutants of rabbitpox virus: III. Temperature-sensitive ( ts) mutants; physiological properties, complementation and recombination

Temperature-sensitive ( ts) mutants can be readily obtained from rabbitpox virus grown in the presence of bromodeoxyuridine. Eighteen mutants were selected for study; all gave a plaque count at the restrictive temperature (39.5°) which was <10 −5 the plaque count at the permissive temperature (34.5°). A number of physiological properties of these mutants were examined. All mutants except one produced u + pocks on the chorioallantoic membrane. All the ts showed the same inactivation kinetics as wild-type rabbitpox virus, all synthesized DNA at the restrictive temperature and all except three produced the same range of soluble antigens at the restrictive temperature as they produced at the permissive temperature. Mixed infection experiments showed that most mutants complemented each other and all recombined with each other.

Antiviral effect of acetone.

We present evidence that 1% (v/v) acetone is capable of inhibiting multiplication of vaccinia and rabbitpox viruses in chick embryo fibroblast cell cultures; and when infections with these viruses were treated with both acetone and N-methyl-isatin-β-thiosemicarbazone the extent of inhibition of virus multiplication was much greater than in experiments using each preparation separately. Addition to agar overlays of acetone in concentration of 1% reduced by 1.3 to 1.5 log. the capacity of vaccinia and rabbitpox viruses to produce plaques in chick embryo fibroblast cultures. ‘Aceton zur Analyse’, DDR, VEB Laborchemie Apolda was used. The virus was adsorbed at 36° for 60 min., then agar overlay containing 1% acetone was added and the vials were immediately stoppered tightly with rubber stoppers. About the same degree of plaque formation by these viruses was observed upon addition to the agar of 1 µg./ml. N-methyl-isatin-β-thiosemicarbazone, which has a known inhibiting effect on the variola group of viruses (Bauer &amp; Sadler, 1961; Appleyard &amp; Way, 1966).

The enhancement of virus infectivity by antibody

The immunological enhancement of virus infectivity was observed only when avian antisera to the test viruses (rabbitpox virus and Murray Valley encephalitis virus) were assayed in a cell system of avian origin. The enhancing activity was found to reside in the IgG fraction of the antiserum. The enhancement phenomenon is of a transient nature, and a prolonged interaction between the virus and antibody results in the neutralization of infectivity. Studies of the dose response relationship for neutralization indicate that the combination of a single antibody molecule with the virus may be sufficient to neutralize the infectivity of the virus. The enhancing activity of chicken antisera could not be accounted for by the greater net negative charge carried by chicken IgG than the IgG of mammalian species. Enhancement could not be accounted for by the presence in chicken antisera of an antibody molecule that had an affinity for chicken embryo fibroblasts.

The photosensitivity of Semliki Forest and other viruses.

Summary Semliki Forest virus was inactivated by exposure to daylight or artificial light. The active wavelengths were in the region 3300 to 4700 Å. Inactivation was reduced by calf serum and completely prevented by cysteine or anaerobic conditions. Light destroyed the infectivity of the virus-associated RNA at the same rate as that of whole virus; it did not affect the reaction of the virus in immunodiffusion tests nor the infectivity of previously extracted RNA. Inactivation was probably the result of photo-oxidation of the viral nucleic acid which was sensitized by some naturally occurring pigment. Although riboflavin and vitamin A were able to increase the photosensitivity of Semliki Forest virus, the evidence presented indicates that neither of these substances was the natural photosensitizer. Sindbis, Murray Valley encephalitis, influenza and rabbitpox viruses also lost infectivity when exposed to light, but poliovirus was photoresistant.