Polyinosinic-Cytidylic Acid Complex (Poly I:G) and Viral Infections in Mice
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Poly I:C, the double-stranded complex of polyinosinic and polycytidylic acids, was tested for activity in mice against infections produced by Columbia SK virus and viruses of clinical importance. Poly I:C had high activity against the SK virus. It had marginal activity, and only when given intranasally, against strains of influenza virus belonging to types A, A1, A2 and B. It was active against infections produced by parainfluenza 1 virus, vaccinia virus, and herpes simplex virus. It was inactive against Coxsackie A21 and B1 virus.The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included.
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The effect of vaccinia virus on Carcinoma 755 (Ca-755) has been investigated, and it has been shown that tumor-inhibitory activity depends on the strain and concentration of the virus used. The most significant degree of oncolysis has been produced with the IHD-T and IHD-E strains of vaccinia virus. Almost complete suppression of tumor growth was obtained when 107.0 pock-forming units of virus were incubated with tumor fragments. Increasingly less tumor-inhibitory activity was demonstrable upon dilution of virus. Inhibition of tumor growth was accompanied by extensive multiplication of virus, but virus multiplication per se did not insure an oncolytic effect. The concentration of virus initially incubated with tumor fragments was found to be more critical in this respect than the titer of virus eventually obtained in the established tumor. Regardless of the extent of virus multiplication in Ca-755, hemagglutinin-inhibiting antibodies to vaccinia virus have not been detected in the sera of tumor-bearing mice. Virus shown to persist in Ca-755 tumors for periods up to 1 month has successfully been transferred along with tumor tissue through eleven serial transplants with but minimal effect on tumor growth. The multiplication and oncolytic activity of vaccinia virus were abolished upon implantation of virus-treated tumors into vaccinia-immunized mice.
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Viral Interference
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N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH) is a selective inhibitor of vaccinia virus multiplication. In concentrations up to 50 µg/ml, IMCBH causes neither toxic morphologic changes, nor does it inhibit the multiplication of cells. Viruses other than vaccinia are not affected by IMCBH. The virus-inhibitory effect of IMCBH is dependent on the type of host cell used, i.e., the compound is effective in chick embryo fibroblasts and monkey kidney cells but not in L cells. IMCBH does not exhibit any protecting effect on vaccinia virus-infected mice or rabbits. IMCBH interferes with virus release: in single cycle experiments in chick embryo fibroblasts, IMCBH strongly blocks the release of vaccinia virus at concentrations as low as 3 µg/ml, while intracellular virus synthesis is hardly affected. Viral cytopathic changes are completely suppressed by IMCBH within the span of a single cycle infection, although extensive changes eventually occur. By inhibiting virus release from initially infected cells, IMCBH markedly inhibits the multiplication of vaccinia virus in cell cultures infected at low virus/ cell multiplicities. IMCBH does not inhibit the early toxic cytopathic changes induced by large inocula of vaccinia virus in BHK21 cells.
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The capacity of RKV virus to interfere with vaccinia virus infection in cultures of rabbit kidney cells provides an additional parameter for the characterization of this relatively new member of the papova-virus group. The titration of RKV virus based on its capacity to interfere with vaccinia virus is not only 100 times more sensitive but is also more rapid than direct titration by CPE.
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Heated vaccinia virus has been applied to cultures of L cells followed by active virus. Multiplicities, determined by electron microscope particle count, were 10 and 100 for heated virus and 10 for challenge, active virus. Delay times ranged from 0 to 21 1/2 hours. Substantial inhibition was observed with inoculation of heated virus at M = 10. At M = 100 the growth of a challenge inoculum applied 21 1/2 hours later was 94% suppressed. The consequences of these findings are discussed in connection with titration of residual virus in heat-treated virus preparations.
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Infectivity
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Orthopoxvirus
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Recombinant virus
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The growth of vaccinia virus has been investigated extensively as a unique DNA virus having a large and complicated structure and inducing the synthesis of viral DNA in the cytoplasm of infected cells. It has already been reported that heat inactivated vaccinia virus could be reactivated after superinfection with another poxvirus by both Fenner et al. (1959) and ourselves (Hanafusa et al., 1959a). Several studies on the mechanism of the reactivation (Hanafusa, 1960a; Joklik et al., 1960a, b) have revealed that the reactivated virus could appear after rescue of some damaged functions of heated virus by another active virus through non-genetic interaction. Since then, our efforts have been directed to determine which step of virus growth is blocked in the infection with heated virus and what process is most crucial in the reactivation. As the first approach to these problems, the interaction of heat inactivated virus with host cells...
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Modified vaccinia Ankara
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Summary The growth of four strains of vaccinia virus in the epithelial cells of the chorioallantois of the chick embryo was found to occur in a step-wise fashion. Immediately following the initiation of infection there was a sharp reduction in the amount of virus in the membrane, and no increase was detectable for approximately 8 hours. At this time virus could be released from the cells, and it did not increase further in concentration until 16 hours following the initiation of infection. After the completion of the second infectious cycle, virus was found in higher concentration in the chorioallantoic membrane. Vaccinia hemagglutinin was detected for the first time at about 16 hours following the inoculation of virus. At this time its presence was correlated with a second increase in viral concentration. It has been assumed that vaccinia hemagglutinin was released along with virus at 8 hours, but in such minute amounts that it could not be measured by the technics employed.
Chorioallantoic membrane
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