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In the present study, we show that adenosine (Ado) affects vaccinia virus (VV) replication in BSC-40 cells. In order to identify its effects on VV replicative cycle we analyzed the synthesis of virus macromolecules in cells incubated with 0.5 mM Ado. A 50% reduction in the steady-state level of virus DNA synthesis was observed. Consequently, virus post-replicative gene expression was also affected. A prolonged synthesis of the F11L early virus protein was also observed and it is likely related to a slow decline in the steady-state level of early mRNAs, as suggested by northern blot analysis of the VGF early transcript. The involvement of cAMP-signaling pathway as mediator of Ado response was also evaluated. Ado stimulated a three-fold increase in cAMP levels in BSC-40 cells and cAMP-mimetic agents reduced virus yield in a dose-dependent manner. Co-incubation of infected cells with H-89 reduced the inhibitory effects of 8-Br-cAMP and Ado on VV yields suggesting PKA involvement. A(2) receptor-mediated activation of PKA was indicated by antagonism of Ado response by theophylline and DMPX. Together, these results indicate that virus DNA replication is the main target of Ado. The mechanism involved is not related to reduction of the pyrimidine nucleotide synthesis. Furthermore, Ado-induced PKA activation modulates negatively an unidentified step of the virus replicative cycle.

Vaccinia virus (VV) is one of the largest and most complex of animal viruses, with a virion that contains about 100 different polypeptides. Assembly of the viral proteins occurs in discrete cytoplasmic sites leading to formation of two infectious forms, an abundant (>90%) intracellular mature virus (IMV) with an envelope, and a minor extracellular enveloped virus (EEV) with an extra membrane acquired from the trans-Golgi network. It has been shown that while EEV contains in the outer membrane cellular proteins probably acquired during virus release from cells, however, IMV exclude host proteins during assembly. Since VV recombinants (VVr) expressing genes of interest are candidates as potential vaccines against pathogens and cancer, it becomes important to know if VVr can acquire foreign proteins during morphogenesis. In this investigation we show that purified virions (IMVs) from VVr can incorporate foreign proteins into different sites in the virus particle. By sequential fractionation of virion compartments with detergents, we found foreign proteins in the lipid envelope (cytokine IL-12 and CS antigen of Plasmodium yoelii), as part of a protein matrix-like membrane (HIV-1 gp41 of env), or more closely associated with the core containing the DNA complexes (HIV-1 gp160; a multiepitopic protein with the V3 loop of HIV-1 Env from different isolates, and beta-galactosidase). Similar findings were observed with purified virions derived from the WR strain as well as from the highly attenuated modified vaccinia virus Ankara (MVA) strain. These observations should be taken into consideration when VVr are used in clinical trials or in other vaccination approaches.

Interferon-beta (IFN-beta) strongly inhibited the expression of the hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV), a paramyxovirus, in HeLa cells under the conditions where it did not affect the expression of the four upstream genes encoding the nucleocapsid protein, phosphoprotein, membrane protein and fusion protein. Even the downstream gene, encoding the large protein as well as the genome replication, appeared to be less susceptible to IFN-beta than the HN gene. This selective action of IFN-beta did not appear to be attributable to its well characterized antiviral mechanisms such as acceleration of RNA decay and translation inhibition. No similar down-regulation of a particular gene expression was found with another paramyxovirus, Sendai virus, or with a rhabdovirus, vesicular stomatitis virus, or seems to have been reported previously with any negative-strand RNA viruses. This new effect of IFN-beta thus suggests gene expression mechanism unique to NDV and may further lead to the discovery of a novel biochemical effect of IFN-beta.

The effect of the immune modulator, Cyclosporin A (CsA) on vaccinia virus replication has been examined in cell cultures. In the present study we report that CsA is anti-viral towards vaccinia virus. Viral yield was inhibited by more than 97% after 24 h postinfection in the presence of 16 microM to 40 microM CsA. An analysis of the infectious cycle in greater detail revealed that CsA did not effect the total level of [35S] methionine incorporation into vaccinia infected cells. However, both early and late viral gene expression were inhibited by CsA. Late viral protein synthesis appeared to be more sensitive to the drug. At least one late viral polypeptide of approximately Mr 38,000 was virtually undetected up to 8 h postinfection in the presence of 40 microM CsA. Host protein synthesis which is normally inhibited by the virus was not turned off until very late in infection. Viral DNA replication was also inhibited by the addition of CsA at levels comparable to those observed for late protein synthesis.

The development of Vaccinia-virus plaques on the chorioallantois of embryonated eggs was studied under the influence of active cyclophosphamide metabolites. The viruses were inoculated on the 9th or the 13th incubation day. Cyclophosphamide was injected in doses of 62.5--500 mug in 0.2 ml NaCl into the yolk sac either 7 hrs after the virus inoculation or 3, 6, or 9 hrs in advance. The plaque development was not influenced when cyclophosphamide was given after the virus inoculation. But in treating the embryos before the infection a decrease dependent on the dose in the number of embryos with recognizable plaques occurred. This decrease was more marked with cyclophosphamide treatment 6 or 9 hrs, than with cyclophosphamide treatment 3 hrs before the virus inoculation. When the embryos were inoculated on the 13th day and treated with cyclophosphamide 9 hrs in advance, the mean effective dose was 316 mug/egg. The range of effective doses corresponds with that of the inhibitory effective doses of cyclophosphamide on transplantation tumors grown on the chorio-allantois of embryonated eggs.

U cells (a permanent, human amnion cell line) were protected against infection with Semliki Forest Virus (SFV) by human interferon (HIF) from peripheral leukocytes. Despite the usual genus-specific action of interferons, mouse L929 cultures (a permanent mouse fibroblast cell line) were also protected by HIF. The antiviral action of HIF in six other primate cell cultures was also examined. It is of interest that two lymphoblastoid cell lines, RPMI1788 and Kaplan, were insensitive to HIF. The sensitivity of ten different viruses against HIF in primary African green monkey kidney cell cultures was compared. Among the viruses tested SFV was the most sensitive whereas two strains of vaccinia virus were barely inhibited. In contrast, type 1 herpes simplex virus was relatively sensitive to the action of HIF.

The antiviral activity and induction of interferon-like substance by mepacrine (quinacrine, Atabrine) and Acranil in mice, described previuosly, was studied in more detail and compared with tilorone. The serum substance induced by Acranil was characterized as mouse interferon. Acranil, given parenterally, proved to be as strong as tilorone regarding interferon stimulation, in spite of the presence of only one side chain in the Acrainl molecule. Mepacrine was found to be a wealer interferon inducer than either tilorone or Acranil. The mode of interferon induction by Acranil and tilorone (effect of metabolic inhibitors, hyporesponsiveness to repeated doses, correlation between acute toxicity and antiviral activity, failure of effectiveness in chicks and chicken embryo tissue culture) was found comparable. However, the body temperature reactions of mice to the drugs were different: a striking hypothermic effect of tilorone, a lower one of mepacrine and the absence of body temperature depression by Acranil was observed.

Carbopol, a polymer of acrylic acid cross-linked with allylsucrose, was found to impart resistance to virus infection in mice which received an intraperitoneal injection of the polycarboxylate 1 or 4 days before either intravenous vaccinia virus challenge or intranasal herpes simplex virus challenge. An interferon-like substance was detected in the blood stream 20-40 hours after the intraperitoneal injection of carbopol. The antiviral activity of this cross-linked polycarboxylate could be due to activation of peritoneal macrophages and/or interferon production.

Both monensin and cytochalasin D reduced the production of infectious cell associated virus and infectious extracellular virus with the latter clearly being the more sensitive. The difference in yields were even more clearly seen if the yield of virus particles was monitored instead of yield of infectious virus. Addition of 1 microgram/ml cytochalasin D or 1 microM monensin to the growth medium of vaccinia virus-infected cells inhibited the appearance of extracellular enveloped vaccinia virus (EEV) in the growth medium without affecting the production of intracellular naked vaccinia virus (INV) particles. Although EEV was not released into the medium of cytochalasin D treated cells, EEV was, nevertheless, detectable in CsCl gradients of cell associated virus. Monensin treatment did not affect the synthesis of vaccinia glycoproteins but did significantly reduce the transport of these glycoproteins to the cell surface and also reduced the secretion of proteins. Monensin had the additional effect of causing the appearance of numerous large vacuoles in the cytoplasm. Vaccinia is normally wrapped by cytoplasmic membranes in preparation for release. The monensin induced conversion of cytoplasmic membranes to large vacuoles is presumably the basis for the block in virus wrapping and subsequent release. Cytochalasin D did not alter any of the steps in protein synthesis, transport or secretion. Electronmicroscopic studies confirmed the existence of EEV on the surface of infected cells treated with cytochalasin D. This drug which specifically affects cellular microfilament organisation thus imposes a block on the final release of EEV from the cell surface.

The inhibitory effect of N,N'-bis(methylisatin-beta-thiosemicarbazone)-2-methylpiperazine (compound TSKI-VI) and methisazone (Marboran) on the growth of vaccinia virus (IHD strain) was studied in vitro and in vivo. The therapeutic indices of both compounds determined in vivo were similar, but TSKI-VI was found more efficient in vitro.