Restriction by Polycations of Infection with Myxoma Virus in Rabbits

THE JOURNAL OF INFECTIOUS DISEASES VOL. 125, NO. 2. FEBRUARY 1972 1972 by the University of Chicago. All rights reserved. Restriction by Polycations of Infection with Myxoma Virus in Rabbits Dennis L. Wegner and Harry C. Hinze From the Department of Medical Microbiology, University of Wisconsin, Madison, Wisconsin Nine mg of DEAE-dextran, polyornithine, and polylysine were mixed with a low dose of myxoma virus (50 plaque-forming units) and injected intradermally into rabbits, resulting in both restriction of viral replication at the site of inoculation and prevention of generalized myxomatosis. These effects were not seen with the neutral polymer, dextran, or with the negatively charged polymer, dextran sulfate. No restriction was observed with DEAE-dextran when this compound was given at a different time or at a different site from that of virus. The restrictive effect could be overcome by injection into adult rabbits of a mixture of DEAE-dextran and a high dose of virus (3 X 10 5 pfu), or by injection into weanling rabbits of a low dose of virus mixed with DEAE-dextran. DEAE-dextran, when mixed with the related Shope rabbit-fibroma virus and injected intradermally into rabbits, resulted in reduced size of tumors induced by the virus. DEAE-dextran had no effect on an unrelated virus, pseudorabies. Previous studies have shown that DEAE-dextran may act to enhance virus-induced plaque formation in cultured cells. Of particular interest to our study was the demonstration by Woodroofe and Fenner that addition of DEAE-dextran to the overlay facilitated plaque formation by rabbit myxoma virus on chick-embryo fibroblasts [1]. DEAE-dextran also increases the in-vitro infectivity of preparations of viral nucleic acid, e.g., polyoma DNA [2] and polio RNA [3]. On the other hand, Dianzani et a1. used a mixture of DEAE-dextran and polyriboinosinic:polyribocytidylic acid (poly I:C) to increase the resistance of mice to challenge with Columbia SK virus through the induction of high levels of interferon [4]. We designed the present study to examine both the effects in rabbits of DEAE-dextran on Shope rabbit fibroma virus, a virus that produces a local, self-limiting infection, and the effects on two viruses, rabbit myxoma virus and pseudorabies vi- Received for publication June 15, 1971, and in revised form September 14, 1971. This investigation was supported by Public Health Service grant no. CA-I0395 from the National Cancer Institute, and was submitted by Mr. Wegner in partial fulfillment of the requirements for the degree of Master of Science. Please address requests for reprints to Mr. Dennis Wegner, Department of Medical Microbiology, University of Wisconsin, Madison, Wisconsin 53706. rus, that produce systemic, lethal disease. The most marked results were seen with myxoma virus; therefore, most of the investigations reported in this paper are concerned with this virus. Materials and Methods Rabbits. Animals used for this study were adult (weight, 6 Ib or more) New Zealand white rabbits purchased from a local rabbitry. Viruses. The Moses strain of rabbit myxoma virus, the Patuxent strain of Shope rabbit fibroma virus, and pseudorabies virus were available in this laboratory as mycoplasma-free stocks, which were originally obtained from the American Type Culture Collection. The viruses were grown in serially cultured, domestic rabbit-kidney cells. Chemicals. DEAE-dextran (mol wt, 2 X 10 6 ), dextran (mol wt, 5 X 10 5 ), and dextran sulfate (mol wt, 5 X 10 5 ) were purchased from Pharmacia Fine Chemicals, Inc., Piscataway, N.J. We prepared sterile stock solutions (20 mg/ml) by mixing the powder with phosphate-buffered saline (PBS) and autoclaving. The solutions were stored at 4 C. Poly-L-ornithine HBr (mol wt, 12,000-50,000) and poly-dl-iysine HBr (mol wt, 15,000-20,000) were purchased from Nutritional Biochemicals Corporation, Cleveland, Ohio, and dissolved in PBS (20 mg/ml). The solutions were sterilized by filtration and used immediately. 141

142 Wegner and Hinze Assays of virus. In-vivo assays of myxoma virus were performed in adult domestic rabbits by intradermal inoculation of serial lo-fold dilutions of each suspension. On the sixth day after inoculation, regularly observed to be the last day before the onset of secondary lesions, each site was examined for induration; the titer was recorded as the highest dilution that produced a detectable lesion. Results Inhibition of myxoma virus by intradermal DEAE-dextran. In the initial experiment, a relatively low dose of virus (50 pfu of myxoma mixed with 9 mg of DEAE-dextran in PBS), was injected intradermally into each of four sites (0.5 ml per site) on the backs of eight adult rabbits. Six controls received the same amount of virus in PBS. Slightly raised, circular lesions, 1-2 em in diameter, developed five days after inoculation at the sites of injection on test animals. These lesions attained maximum size of 2-5 em at seven days and persisted unchanged throughout the 20-day period of observation. No secondary lesions or other symptoms of systemic disease were observed (figure 1). Infection in controls followed the classic clinical progression of myxomatosis [5]. Tumors 1-2 em in diameter developed on the skin at the sites of inoculation four to five days after infection and gradually enlarged throughout the course of the disease. The first sign of systemic disease was reddening of the conjunctivae at seven days. Secondary nodules were seen on the ears and skin at eight days and continued to enlarge until the death of the animals at nine to 13 days (figure 2). In contrast, weanlings receiving the same mixture of virus and DEAE-dextran and adults receiving a larger dose of virus (3 X 10 5 pfu mixed with 9 mg of DEAE-dextran) were not protected and exhibited the same progression of disease and time of death as controls. It was also found that administration of 9 mg of DEAE-dextran into the same skin sites as virus one, three, or six days before infection or one day after infection had no effect on the outcome of the disease. Effect of DEAE-dextran given by systemic routes. To determine whether DEAE-dextran given systemically would have an effect on virus inoculated intradermally, we injected 50 pfu of myxoma into each of four sites on the backs of 15 adult rabbits. On the day of infection, two of the animals were started on a daily regimen of 50 mg of DEAE-dextran (2.5 ml of stock solution) given in the marginal ear vein, and two infected controls received equal volumes of PBS. Nine of the rabbits received higher intraperitoneal doses of DEAE-dextran in amounts approaching the maximal tolerable level. These regimens included 200 mg of DEAE-dextran given on days 0, 3, 6, and 9; 150 mg given on days 0, 3, 6, and 9; and 100 mg given on days 0, 3, 6, 8, and 10. Two infected controls received PBS intraperitoneally on days 0, 3, 6, 8, and 10. A one- to twoday lag in the appearance of secondary lesions Figure 1. Test rabbit nine days after four O.5-ml intradermal injections of 50 pfu of myxoma virus mixed with 9 mg of DEAE-dextran. This animal did not develop systemic myxomatosis during its 20-day period of observation. Figure 2. Control rabbit nine days after four intradermal injections of 50 pfu of myxoma virus alone. This animal shows typical symptoms of generalized myxomatosis. Death occurred 24 hr later.

Polycationic Restriction of Myxoma Virus 143 was observed in rabbits receiving DEAE-dextran by both systemic routes, but survival time of these animals was not affected. Effect of intradermal DEAE-dextran on fibroma and pseudorabies viruses. The effect of DEAE-dextran on a closely related poxvirus was examined. Shope rabbit fibroma virus (2 X 10 6 pfu) was mixed with 9 mg of DEAE-dextran in PBS and injected intradermally into each of four sites on the backs of two adult rabbits. Control sites on the same animals received an equal volume of virus and PBS. At eight days, fibromata 17 mm in diameter developed at control sites, while macules 3 mm or less in diameter were seen at test sites. From this point on, the lesions at both control and test sites regressed until the animals were asymptomatic at one month. To determine the effect of DEAE-dextran on an unrelated DNA virus, two adult rabbits each received four intradermal injections of 50 pfu of pseudorabies virus mixed with 9 mg of DEAEdextran in PBS. Two controls received the same amount of virus in PBS. DEAE-dextran had no effect on the outcome of this viral disease, as both the controls and the test animals died on the third day. Effect of other polymers on myxoma virus. To determine whether the observed inhibition of myxoma virus was related to the molecular backbone of DEAE-dextran or to its polycationic nature, we tested the effects of the uncharged compound, dextran, negatively charged dextran sulfate, and the polycations polyornithine and polylysine. Nine mg of each compound in PBS was mixed with 50 pfu of myxoma virus and injected intradermally into each of four sites on the backs of rabbits; two rabbits were used in the tests of each compound. Those rabbits receiving polyornithine and polylysine exhibited modified circular lesions similar to those seen in animals protected by DEAEdextran. No secondary lesions or other symptoms of systemic disease were seen during a 20-day period of observation. Rabbits that received dextran and dextran sulfate were not protected and had symptoms of progressive myxomatosis identical to those of controls. Thus, it appears that molecular charge, rather than the nature of the molecular backbone, is important in this inhibition. Viral multiplication at the site of intradermal inoculation. Since preliminary experiments in this laboratory had shown that DEAE-dextran did not interfere with the replication in vitro of myxoma virus, and did not inactivate extracellular virus in vitro, it was of interest to determine the effect of DEAE-dextran on the in-vivo replication of virus. Three or four sites on each of five rabbits were inoculated with a mixture of 9 mg of DEAE-dextran and 50 pfu of virus. Controls received the same amount of myxoma in PBS. On days 1, 3, and 5 after inoculation, one animal treated with DEAE-dextran and one control were sacrificed. Three sites of inoculation were excised from each animal and stored at -20 C. On day 7, two test animals and their controls were sacrificed, and four skin sites were taken from each animal. All samples of skin were prepared for assay by submergence in liquid nitrogen for 5 min, followed by pulverization. The skin powders were placed into tared vials and weighed. Five rnl of PBS was added to each vial, and the samples were subjected to four cycles of freezing and thawing at -70 C. Following sonication for 2 min, the samples were assayed in vivo for infectious virus. The results (table 1) indicated that myxoma virus multiplied Table 1. Multiplication of myxoma virus at the site of inoculation in the presence of DEAE-dextran. Titer of virus (infectious doses* of virus per g of skin) Day Treated Control It 9 X 10 4 10 4 <10 2 X 10 4 <10 10 4 3t 10 8 X 10 7 10 107 10 2 X 10 7 5t 10 4 2 X 10 8 107 3 X 10 7 9 X 10 7 6 X 10 6 7* 9 X 10 5 5 X 10 8 6 X 10 6 10 9 10 8 3 X 108 2 X 10 5 6 X 10 7 4 X 10 6 10 9 7 X 10 7 3 X 10 8 2 X 10 6 9 X 10 7 2 X 10 5 7 X 10 8 *The smallest unit of virus that can produce a detectable lesion. t Values given are from three sites each on one control rabbit. *Values given are from four sites each on two treated rabbits and two control rabbits.

144 Wegner and Hinze more slowly in the presence of DEAE-dextran and reached a lower maximal titer. However, the observed inhibition of systemic myxomatosis could not be explained by local elimination of the virus. Effect of DEAE-dextran on myxoma viremia. Fenner and Woodroofe have shown that multiplication of myxoma in the skin of rabbits is followed by invasion of the lymph nodes and subsequent viremia [6]. Since DEAE-dextran did not eliminate virus from the site of inoculation, it was of interest to examine the blood of test animals and controls for virus. Two rabbits each received four intradermal injections of 50 pfu of myxoma virus mixed with 9 mg of DEAE-dextran, and two controls received the same amount of virus in PBS. Each rabbit was bled from the marginal ear vein on days 1, 3, 8, and 9, and the blood was immediately assayed in vivo. No measurable viremia was detected during this time in treated animals. In contrast, controls had titers in blood of 10 infectious doses of virus per m1 on day 3 and a peak titer of 10 4 infectious doses per m1 on day 8. Importance of the immune response. A possible explanation for the inhibition of systemic disease and the absence of viremia was that DEAE-dextran might restrict myxoma virus at the site of inoculation long enough to allow the rabbit to initiate a protective immune response. To test this hypothesis, we gave four intradermal injections of 50 pfu of myxoma virus mixed with 9 mg of DEAE-dextran to each of two rabbits. Eleven days later, when the animals had no systemic symptoms, they were each challenged with two intradermal injections at distal sites of 50 pfu of myxoma virus alone. No protection had developed during the interval between the first and second series of injections. The treated animals exhibited the same progression of disease as controls and died 10 and 11 days after the second inoculation. Sera from these rabbits were drawn on the day of reinoculation and examined for antibody by plaque-reduction neutralization. No detectable antibody was formed. Cell-mediated immunity was not studied. Discussion All of the commonly known compounds possessing in-vivo antiviral activity function by blocking some stage of the cycle of viral replication. In contrast, DEAE-dextran allows myxoma virus to mul- tiply at the site of intradermal inoculation but, under the conditions described in this study, prevents it from producing a generalized infection. An inhibitory effect is demonstrable on a closely related poxvirus, Shope fibroma virus, but not on an unrelated herpesvirus, pseudorabies. It appears that molecular charge is important in this inhibition. Dextran sulfate, dextran, and DEAE-dextran have identical molecular backbones, similar physical properties, and comparable molecular weights; but only the positively charged DEAE-dextran can limit the spread of myxoma virus. On the other hand, the only properties shared by DEAE-dextran, polyornithine, and polylysine are a polycationic nature and the ability to prevent generalized myxomatosis. Polyanions have been shown to have an inhibitory effect in vitro on certain viruses. Hochberg and Becker [7] and Nahmias and Kibrick [8] have shown that heparin interferes with the adsorption of herpes simplex. Takemoto and Liebhaber demonstrated that addition of dextran sulfate to a viral inoculum inhibited plaque formation by encephalomyocarditis, ECHO, Coxsackie A9, and influenza B viruses [9]. Previous studies on the effect of polycations on poxviruses have been limited to the enhancement of plaque formation, but inhibition of these viruses in vivo by such compounds has not been demonstrated. Kim and Sharp [10] used DEAE-dextran to increase plaquing efficiency of vaccinia and rabbitpox on mouse L cells and showed by electron microscopy that this effect was due neither to an aggregation of viral particles nor to a modification of the process of adsorption. Several workers have shown that polycations will bind to the negatively charged surface of mammalian cells both in vivo and in vitro and, in some instances, cause a direct cytotoxicity [11, 12]. The lack of localized destruction of cells or necrosis of tissues at the site of injection of DEAE-dextran makes it unlikely that the inhibition of generalized myxomatosis is due to overt destruction of cells in the area of initial viral infection. Since poxviruses are known to be negatively charged at physiologic ph [13, p. 324], it seems most likely that myxoma virus, injected together with the polycations, is electrostatically bound at the site of inoculation. This concept is strengthened by the finding that DEAE-dextran, injected

Polycationic Restriction of Myxoma Virus 145 intradermally before or after inoculation with virus, has no inhibitory effect. The failure of DEAE-dextran to inhibit a large dose of virus may indicate that this binding force is easily overwhelmed. References 1. Woodroofe, G. M., Fenner, F. Viruses of the myxoma-fibroma subgroup of the poxviruses. I. Plaque production in cultured cells, plaque-reduction tests, and cross-protection tests in rabbits. Aust. J. Exp, BioI. Med. Sci. 43: 123-142, 1965. 2. Warden, D., Thorne, H. V. The infectivity of polyoma virus DNA for mouse embryo cells in the presence of diethylaminoethyl-dextran. J. Gen. Virol. 3:371-377, 1968. 3. Pagano, J. S., McCutchan, J. H., Vaheri, A. Factors influencing the enhancement of the infectivity of polio virus ribonucleic acid by diethylaminoethyldextran. J. Virol. 1:891-897, 1967. 4. Dianzani, F., Rita, G., Cantagalli, P., Gagnoni, S. Effect of DEAE-dextran on interferon production and protective effect in mice treated with the double-stranded polynucleotide complex polyinosinic:polycytidylic acid. J. Immun. 102:24-27, 1969. 5. Findlay, G. M. Notes on infectious myxomatosis of rabbits. Brit. J. Exp. Path. 10:214-219, 1929. 6. Fenner, F., Woodroofe, G. M. The pathogenesis of infectious myxomatosis: the mechanism of infection and the immunological response in the European rabbit (Oryctolagus cuniculus). Brit. J. Exp, Path. 34:400-411, 1953. 7. Hochberg, E., Becker, Y. Adsorption, penetration, and uncoating of herpes simplex virus. J. Gen. Virol. 2:231-241, 1968. 8. Nahmias, A. J., Kibrick, S. Inhibitory effect of heparin on herpes simplex virus. J. Bact. 87: 1060 1066, 1964. 9. Takemoto, K K, Liebhaber, H. Virus-polysaccharide interactions. II. Enhancement of plaque formation and the detection of variants of poliovirus with dextran sulfate. Virology 17:499-501, 1962. 10. Kim, K S., Sharp, D. S. The influence of DEAEdextran on plain and synergistic plaque formation by two poxviruses. 1. Gen. Virol. 4:505-512, 1969. 11. Katchalsky, A. Polyelectrolytes and their biological interactions. Biophys. J. 4(Suppl.) :9-41, 1964. 12. Moroson, H. Polycation-treated tumor cells in vivo and in vitro. Cane. Res. 31:373-380,1971. 13. Andrewes, C. H., Pereira, H. G. Viruses of vertebrates. 2nd ed. Williams and Wilkins, Baltimore, 1967. 432 p.