Inhibition of viral replication by interferon-gamma-induced nitric oxide synthase.

Interferons (IFNs) induce antiviral activity in many cell types. The ability of IFN-gamma to inhibit replication of ectromelia, vaccinia, and herpes simplex-1 viruses in mouse macrophages correlated with the cells' production of nitric oxide (NO). Viral replication was restored in IFN-gamma-treated macrophages exposed to inhibitors of NO synthase. Conversely, epithelial cells with no detectable NO synthesis restricted viral replication when transfected with a complementary DNA encoding inducible NO synthase or treated with organic compounds that generate NO. In mice, an inhibitor of NO synthase converted resolving ectromelia virus infection into fulminant mousepox. Thus, induction of NO synthase can be necessary and sufficient for a substantial antiviral effect of IFN-gamma.

Ectromelia virus replication in major target organs of innately resistant and susceptible mice after intravenous infection.

The kinetics of ectromelia virus replication in the spleen and liver and of alpha/beta interferon production in the spleen were determined during the first 3 days after intravenous infection with the virulent Moscow strain in resistant C57 BL/6 and susceptible DBA/2 mice. Virus replication in the spleen as measured by assays for virus DNA and infectious centers was suppressed in C57BL/6 mice relative to DBA/2 mice within the first 1 or 2 days of infection. Infectious centers increased in DBA/2 mice but not in C57 BL/6 mice. Differences in virus replication between strains were less discrete when spleens were assayed for infectious virus than when they were assayed for infectious centers because infectious centers of most C57 BL/6 mice had more infectious virus than infectious centers of DBA/2 mice. Virus replication in the liver, the major target organ, as measured by virus DNA and infectious virus assays, was suppressed in C57 BL/6 mice relative to DBA/2 mice 3 days after infection but not before that interval. The results indicate that genetic control of ectromelia virus replication begins within the first 1 or 2 days of infection in the spleen but is delayed in the liver and that genetic control is directed at the prevention of virus spread more than at virus replication.

Electron microscopy, plaque assay and preliminary serological characterization of three ectromelia virus strains isolated in Poland in the period 1986-1988.

Three strains of viruses have been identified as ectromedia virus (EV) based on their origin, clinical features, morphology, size of virions (140 x 220 nm), replicative ability and specific cytoplasmatic fluorescence. The mean diameter of plaques produced by EV strains was 0.76 mm (range 0.5-1.0 mm). The neutralizing properties of the tested sera were evaluated by seroneutralization (SN) and hemagglutination inhibition (HAI) tests.

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. IV. Studies with the Moscow strain.

The pathogenesis and transmission of infection with the Moscow strain of ectromelia virus were studied in inbred mice. BALB/cAnNcr had high morbidity and mortality and C57BL/6Ncr (B6) mice had high morbidity and low mortality. Virus was detected in B6 mice for 2 weeks after subcutaneous (s.c.) inoculation and infected mice developed lesions compatible with acute mousepox. B6 inoculated by footpad transmitted infection to cagemates for up to five weeks and soiled cages that had housed infected mice were infectious for three weeks. S.c.-inoculated B6 mice also transmitted by contact for 2 weeks. Transmission was attributed to oronasal excretion of virus. Airborne transmission of infection between adjacent cages occurred at a low rate. Ectromelia virus-free progeny were derived from previously infected dams. These studies indicate that the highly virulent and infectious Moscow strain of ectromelia virus caused self-limiting infection in inbred mice and that direct contact is the most efficient means of transmission. These findings support the concept that mousepox can be contained by husbandry practices that minimize or eliminate the spread of infection by direct contact or fomites.

Thioglycollate elicited macrophages demonstrate enhanced virus replication and depressed bacterial killing.

Thioglycollate elicited peritoneal macrophages of Balb/c mice exhibited minimal antibacterial activity against Listeria monocytogenes but were fully permissive for the replication of ectromelia virus. By comparison, resident and LPS elicited macrophages did not exhibited depressed antibacterial activity nor did they support viral replication. The thioglycollate effects were demonstrated in macrophages cultured in vitro and also in intact Balb/c mice. Mice given thioglycollate intraperitoneally and challenged by the same route suffered overwhelming virus and bacterial infections as a result of early local proliferation within peritoneal macrophages with subsequent spread to the liver. Balb/c mice challenged intravenously with similar doses of the virus of bacterial pathogen after administration of thioglycollate by the i.p. route did not succumb to either infection. Thus the ability of thioglycollate to compromise cellular host defenses against the infectious agents appears to be site specific; i.e. restricted to the peritoneal cavity where exudate macrophages and challenge inocula first come into contact.

Ectromelia virus-induced changes in primary cultures of mouse hepatocytes.

Mouse hepatocytes were isolated by collagenase perfusion, maintained in non-proliferating monolayer culture and shown to retain liver cell function as judged by gluconeogenesis for 15 to 18 h. Such cells could be infected with and support the replication of a virulent strain of ectromelia virus. Virus antigen and characteristic cytoplasmic 'B'-type poxvirus inclusion bodies were demonstrated by immunofluorescence in virtually all cells. By electron microscopy it was shown that 'B'-type inclusions were the site of virus replication, and that the biogenesis of ectromelia virus and ultrastructural changes in hepatocytes were similar to those observed in infected mouse livers. Early cell rounding effects, a normal characteristic of poxvirus infections in tissue culture cells, were not seen in ectromelia-infected hepatocytes, although late degenerative changes did occur. Pulse-labelling of hepatocyte cultures with [35S]methionine showed that ectromelia virus inhibited the rise in protein synthesis seen in controls and imposed a gradual decline in host protein synthesis to an extent and at a rate significantly different from that in mouse L929 cells. Gluconeogenesis was inhibited by ectromelia virus infection of hepatocytes.

Kinetics of ectromelia virus (mousepox) transmission and clinical response in C57BL/6j, BALB/cByj and AKR/J inbred mice.

Research was undertaken to answer basic questions on susceptibility, clinical response and transmission of ectromelia virus in selected strains of inbred mice. C57BL/6J and AKR/J were found to be markedly more resistant to a virulent strain of ectromelia virus (isolated during the 1979-80 outbreak at the National Institutes of Health) than C57LJ, BALB/cByJ, DBA/2J, A.By/SNJ and C3H/HeJ when infected by footpad inoculation. In C57BL/6J and AKR/J the LD50 was about 7 logs higher than the ID50. With one exception, C57LJ, the LD50 and ID50 titers in the other strains were about equal. In C57LJ the LD50 titer was intermediate. Following intragastric inoculation, virus was isolated from feces of C57BL/6J mice for as long as 46 days and up to 29 days from BALB/cByJ mice. Transmission to cage mates from intragastrically infected C57BL/6J and BALB/cByJ occurred up to 36 and 30 days respectively after infection. Virus was isolated from the spleen in 2 of 5 BALB/cByJ mice and 1 of 7 C57BL/6J mice tested 95 days after gastric inoculation. Following footpad inoculation, BALB/cByJ mice consistently transmitted virus to cage mates before death at 10-12 days. C57BL/6J mice transmitted between days 8 and 17, but not beyond. Virus was maintained in C57BL/6J mice by exposure to infected cage mates for seven passages, which was the most attempted. Clinical signs in infected C57BL/6J mice were usually subtle or inapparent.

Mechanisms of enhanced resistance of Mycobacterium bovis BCG-treated mice to ectromelia virus infection.

The mechanism of enhanced resistance of Mycobacterium bovis BCG-treated mice to ectromelia virus infection was investigated by determining the effect of splenectomy, antithymocyte serum, and antimacrophage serum on resistance. It was greatly reduced by these treatments, not only in normal mice, but also in mice treated with live or heat-inactivated BCG. Production of circulating interferon by ectromelia virus and Newcastle disease virus was augmented in BCG-treated mice and was markedly depressed by splenectomy and antithymocyte and antimacrophage serum treatments in both BCG-treated and normal mice. Carbon clearance activity was activated in BCG-treated mice, but splenectomy did not influence phagocytic activity. These results suggest that augmented interferon production in the spleens of BCG-treated mice plays a major role in enhanced resistance. Other possible mechanisms are discussed.

Mechanism of protection during the early phase of a generalized viral infection. I. Contribution of phagocytes to protection against ectromelia virus.

Effects of carrageenan and gamma-irradiation on virus titre in the liver were observed after intravenous inoculation of 8 X 10(3) p.f.u. of ectromelia virus which was not lethal for untreated mice. Trapping of virus by the liver within 30 min and an initial transient reduction in titre by day 1 were not affected by gamma-irradiation but were inhibited by pretreatment with carrageenan. An increase from day 1 to day 3 was not affected by gamma-irradiation but was augmented by pretreatment with carrageenan. Therefore, protection within 3 days may depend principally upon carrageenan-sensitive and irradiation-resistant cells, namely, fixed macrophages. Elimination of virus from day 4 to day 7 depended upon cell-mediated immunity. When carrageenan was given 3 days after virus inoculation, the titre of virus increased progressively from day 4 ultimately to kill the hosts. The cytotoxic activity of spleen cells against infected target cells was raised in carrageenan-treated mice as well as in untreated mice. Immune elimination of virus may be mediated by a mechanism requiring the cooperation of sensitized T lymphocytes and blood monocytes.

Pathology and diagnosis of mousepox.

The pathologic changes of mousepox were studied during an outbreak at the National Institutes of Health in 1979. The most consistent lesions were necrosis of lymphatic tissues, especially the spleen, lymph nodes, and Peyer's patches. Hepatic necrosis and jejunal hemorrhage also were found. In two transmission studies, the disease was experimentally induced in BALB/cAnN and C3H/HeN-nu mice. Athymic mice were found to be highly susceptible, and they developed fulminant disease. The diagnosis was confirmed by demonstration of pox virions in infected tissues by electron microscopy, staining of viral antigen by immunoperoxidase methods, and by isolation of the virus in chorioallantoic membranes of hen's eggs and in cultures of chick embryonic cells.

Studies on poxvirus infections in irradiated animals.

If rabbits were given total body irradiation and infected wih vaccinia virus (strain Elstree) a severe disease developed with a viraemia lasting up to 12 days. The clearance of the virus from the peripheral blood was severely impaired by x-ray doses above 800 R. The attenuated vaccinia virus strain MV did not turn virulent, if it was injected to irradiated rabbits. With caution it can be assumed that live vaccines, containing attenuated viruses, may be given to immunosuppressed persons. Rats are not susceptible to ectromelia-virus (mouse-poxvirus); overt clinical sympatoms, however, with a mortality of 30 per cent developed in irradiated rats. This proofs that specific poxviruses can be transferred to another species. As the experimental conditions are unnatural, this may occur only rarely in immunosuppressed persons. After intracerebral infection of Balb-C-mice with low doses of vaccinia virus two types of infection were seen: 1. a severe cytocidal infection of leptomeninges, chorioid plexus and vessels; 2. a noncytocidal, latent infection of glial cells and neurons. Several animals developed a picture resembling experimental allergic encephalomyelitis. It seems that irradiation altered the antigenic conditions of the cytoplasmic membranes in non-cytocidally infected cells. The model might explain some processes in the pathogenesis of demylinating diseases.