Bioluminescence imaging of vaccinia virus: effects of interferon on viral replication and spread

Whole animal imaging allows viral replication and localization to be monitored in intact animals, which provides significant advantages for determining viral and host factors that determine pathogenesis. To investigate effects of interferons on spatial and temporal progression of vaccinia infection, we generated recombinant viruses that express firefly luciferase or a monomeric orange fluorescent protein. These viruses allow vaccinia infection to be monitored with bioluminescence or fluorescence imaging, respectively. The recombinant viruses were not attenuated in vitro or in vivo relative to a control WR virus. In cell culture, reporters could be detected readily by 4 h post-infection, showing that these viruses can be used as early markers of infection. The magnitude of firefly luciferase activity measured with bioluminescence imaging in vitro and in vivo correlated directly with increasing titers of vaccinia virus, validating imaging data as a marker of viral infection. Replication of vaccinia was significantly greater in mice lacking receptors for type I interferons (IFN I R-/-) compared with wild-type mice, although both genotypes of mice developed focal infections in lungs and brain after intranasal inoculation. IFN I R-/- mice had greater dissemination of virus to liver and spleen than wild-type animals even when mortality occurred at the same time point after infection. Protective effects of type I interferons were mediated primarily through parenchymal cells rather than hematopoietic cells as analyzed by bone marrow transplant experiments. Collectively, our data define a new function for type I interferon signaling in systemic dissemination of vaccinia and validate these reporter viruses for studies of pathogenesis.     

Role of macrophages,interferon gamma and procoagulant activity in the resistance of genetic heterogeneous mouse populations to mouse hepatitis virus infection

Summary Genetic heterogeneous mouse populations selected for high (H_III) and low (L_III) antibody response were used to study some aspects of mouse hepatitis virus 3 (MHV3) infection, such as the resistance pattern, virus replication in the liver and peritoneal exudate or in cultured peritoneal macrophages, the interferon (IFN) synthesis in the serum and peritoneal exudate and the procoagulant activity (PCA) of the peritoneal exudate (PEC) and spleen cells (SC). The H_III mice, when compared to their L_III mice counterparts, were susceptible to MHV3 infection showing higher virus titres in the liver and peritoneal exudate, comparable IFN alpha/beta or IFN gamma titres in the peritoneal exudate or in the serum, and higher levels of PCA of PEC and SC. A higher virus titre was detected in the supernatants of H_III mouse macrophages infected with MHV3. The activation of H_III mouse macrophages with LPS, IFN alpha/beta or IFN gamma, in contrast to that of L_III mouse macrophages, did not induce an antiviral effect with partial restriction of the MHV3 replication. The LPS antiviral activity was shown to be partially exerted by IFN alpha/beta synthesis. The IFN gamma was shown to be more effective in inducing an antiviral state in L_III macrophages, when compared to IFN alpha/beta. The data obtained are consistent with the notion that the resistance mechanisms to the MHV3 infection involve the PCA and the sensitivity of macrophages to IFN.     

Sex hormone modulation of interferon (IFN) alpha/beta and gamma production by mouse spleen cell subsets following picornavirus infection

Replication of the diabetogenic variant of encephalomyocarditis virus (EMCV-D) in spleen cells and its association with subpopulations of spleen cells (L3T4+, Lyt-2+, Mac 1+, 33D1+ and AGM1+ cells) from both sexes of ICR Swiss mice were examined. Virus replication was limited to less than 0.5 log in suspensions of whole spleen cells, nonadherent cells or a B cell subfraction from both sexes of ICR Swiss mice following infection with EMCV-D at an MOI of 10; no virus replication was seen in adherent spleen cells from either sex. After 1 hour adsorption of EMCV-D onto spleen cells at a multiplicity of infection (MOI) of either 10 or 0.1, virus-associated cells were isolated using a monoclonal murine anti-EMCV-D and anti-mouse IgG conjugated to magnetic beads. Using an MOI of 0.1, less than 1% of spleen cells bound virus particles after 1 hour adsorption at 4 degrees C. Among the virus-positive cells, relatively higher percentages of adherent cell populations (Mac 1+ and 33D1+ cells) of both sexes bound virus particles within the first hour post-infection (PI) than did the other spleen cell subpopulations. Interferon (IFN) alpha/beta production was detected as early as 4 hours PI in female spleen cell cultures infected with EMCV-D at an MOI of 0.1 while no IFN alpha/beta activity was found in comparably infected male spleen cell cultures. Inhibiting IFN alpha/beta activity in the virus-infected spleen cell cultures during the first 20 hours of infection using polyclonal rabbit anti-mouse IFN alpha/beta serum eliminated production of IFN gamma as well as IFN alpha/beta. Spleen cell cultures depleted of adherent cells were unable to produce IFN alpha/beta or IFN gamma in the first 24 hours PI. The capacity to produce IFN gamma at 12 hours after virus infection of spleen cells from both sexes of mice was restored to adherent cell-depleted cultures by addition of mouse IFN alpha/beta at the time of infection. These results suggest that IFN alpha/beta and adherent cells play critical roles in the early production of IFN gamma (less than 16 hours PI) characteristic of the infected spleen cell cultures of females. Production of IFN alpha/beta and IFN gamma by spleen cells from both sexes of ICR Swiss mice was enhanced by administrating estrone to donor mice during the week before harvesting spleen cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of Natural Killer Cells and Interferon during Mouse Hepatitis Virus Infection of Resistant and Susceptible Inbred Mouse Strains

Following infection by mouse hepatitis virus (JHM strain), an induction of natural killer (NK) cell activity was observed in C3H mice, which are considered to be sensitive to JHM virus infection. In contrast, mice of the resistant SJL strain did not show any increase of NK cell activity after JHM virus infection. However, infection of both SJL and C3H mice with mouse hepatitis virus type 3 (MHV3) resulted in an increase of NK level, comparable to that observed with the JHM virus infection in the C3H strain. No significant differences were observed in the NK cell activity of the peritoneal exudate or spleen cells of infected mice. Low levels of interferon were detected in serum or peritoneal exudate of C3H mice infected with JHM virus 18 or 24 hours before, but no detectable early interferon production was found. Also no interferon could be detected in the resistant SJL mice. After JHM virus infection, the number of peritoneal exudate cells (PEC) was increased significantly in C3H mice but not in SJL mice. Macrophages obtained from the C3H mice supported virus replication, whereas SJL macrophages did not. Our data suggest that NK cells do not play a role in the resistance of SJL mice against JHM virus infection but may participate in the defence mechanisms against this virus in C3H mice.     

Replication of mumps virus in mouse: transient replication in lung and potential of systemic infection

Summary A mumps virus strain, which replicated in mouse lung after aerosol inhalation, was obtained by selective replication of a wild strain in L929 cells and by further passaging in mice by intraperitoneal inoculation. All of infected mice survived and rechallenge of the survived mice with the same virus resulted in no virus growth in the lung. Treatment of infected mice with antiserum against interferon (IFN) or asialo GM1 delayed virus clearance from lung. Mice at 5 weeks of age were also sensitive to the virus as well as those at 1 week. When injected intravenously, the virus could grow not only in lung but also in salivary glands, heart and spleen. Furthermore, the virus replicated in liver, spleen, pancreas and testis after intraperitoneal inoculation. Antibody response of mice infected by aerosol inhalation was slower than that of intraperitoneally infected ones in either IgG or IgM production. These results indicated that the adapted virus replicated in mouse lung by a natural route of infection and had a potential to cause systemic infection in mouse.     

Resistance of vaccinia virus to interferon is related to an interference phenomenon between the virus and the interferon system

In this investigation the sensitivity of vaccinia virus to interferon (IFN) has been examined in cultured cells. In a variety of mouse and human cells of different origins vaccinia virus functions (RNA, protein, and virus yields) were found to be relatively resistant to IFN. In these systems, the levels of the IFN-mediated enzyme activities (2-5A synthetase and protein kinase) were severely impaired by the virus. This virus-mediated inhibitory effect developed with time after infection and was dependent on viral protein synthesis. Mixed infections between vaccinia virus and viruses (VSV or polio) which are sensitive to IFN showed that both protein synthesis and virus yields were not inhibited. These findings show that vaccinia virus can overcome the antiviral action of IFN and that viral gene functions appear to be involved in this interference phenomenon.     

Protection of mice against vaccinia and herpes simplex virus infection by Propionibacterium acnes

Heat-killed cell suspension of several Propionibacterium acnes strains were prepared and studied for their protective activity in viral infection of mice and immunomodulating properties. Majority of the strains caused marked spleen enlargement in the treated mice. These changes persisted for several weeks. Only some of the tested strains enhanced significantly primary humoral immune response against sheep red blood cells. There was no increase, however, in neutralizing or hemagglutination-inhibition antibody production against vaccinia virus in mice treated with Propionibacteria. No evidence in the increase of spleen lymphocytes migration inhibition of mice infected with vaccinia virus and treated with P. acnes or in hypersensitivity reaction to oxazolone have been found. Significant resistance enhancement of mice pretreated with P. acnes against vaccinia virus or herpes simplex virus type 1 infection was observed. Activity of Propionibacteria depended on the applied strain, dose and scheme of administration.     

Interferon in experimental viral infections in mice: tissue interferon levels resulting from the virus infection and from exogenous interferon therapy.

In mice given single intraperitoneal doses of interferon, serum interferon levels peaked at 1 h postinjection and were reduced to zero at about 8 h. The interferon concentrations in spleen, liver, and lungs were about 100-fold higher than could be expected from the amount of serum contained in these organs. In the brain only low levels of antiviral activity were detected. In mice infected intraperitoneally with Mengo virus, viral replication in the brain occurred around day 4 and was accompanied by the appearance of large amounts of interferon (approximately 10(3.25) U/g). This was preceded, however, by viral replication in the spleen and by the appearance of modest amounts of interferon in spleen and serum. In these mice protection could be obtained with relatively small doses of interferon, provided protection could be obtained with relatively small doses of interferon, provided they were given before the time of maximal levels of endogenous serum interferon. In mice infected intranasally with vesicular stomatitis virus, virus replication in the brain started within 24 to 48 h and increased with time; also, small amounts of interferon (10(2) to 10(2.5) U/g) were already detectable on days 1 and 2. The major peak of virus replication in the brain occurred on days 5 to 6 and was accompanied by the appearance of large amounts of interferon (approximately 10(3.25) U/g). In this model early treatment with interferon also provided protection, but only if given in larger doses than in the Mengo virus system. Athymic (nu/nu) mice developed a chronic systemic infection when inoculated with a demotropic strain of vaccinia virus. No interferon was detected in sera, livers, spleens, or lungs of these animals; some mice had low levels of interferon-like antiviral activity in the brain, but no attempt was made to characterize this material. Daily administration of large doses of interferon failed to exert an effect on the development of this chronic disease. Yet, normal (NMRI) mice were protected against acute infection with dermotropic or neurotropic strains of vaccinia virus, and athymic mice were partially protected against acute lethal infection with neurotropic vaccinia virus.     

Toxoplasma-induced activities of peritoneal and spleen natural killer cells from beige mice against thymocytes and YAC-1 lymphoma targets.

Homozygous (bg/bg) and heterozygous (bg/+) beige mice were infected with Toxoplasma gondii, and splenic and peritoneal natural killer (NK) cell activities were assayed against YAC-1 lymphoma (NK-YAC) and thymocyte (NK-THY) targets. Although uninfected bg/bg mice were devoid of NK-YAC activity when compared with bg/+ mice, NK-THY activity was at a completely normal level. Both effector cells showed NK-1.2+ Thy-1.2 +/- asialo GM1+ asialo GM2+ phenotype. T. gondii infection induced a marked augmentation in splenic NK-YAC activity of bg/+ mice, whereas a slight increase was shown in the bg/bg mouse spleen cells. On the other hand, the infection did not change the splenic NK-THY activity of either strain of mice. An increased expression of Thy-1.2 antigen was shown on both NK-THY and NK-YAC effector cells from the infected mouse spleen. The T. gondii-induced augmentation was dramatic in the peritoneal cavity of the both mice. The activated peritoneal NK cells were of the NK-1.2- Thy-1.2+ asialo GM1 +/- asialo GM2+ phenotype and were considered to be generated from functionally inactive peritoneal cells. Splenic effector cells obtained from the infected mice were selectively depleted with target cell monolayer, whereas peritoneal cells from the infected mice were strongly absorbed by the target monolayers without selectivity. A weak but significant interferon (IFN) titer was detected in the peritoneum, but not in the spleen, of the infected mice. Most of the IFN titer was acid labile. Treatment with anti-IFN-alpha/beta resulted in partial decline of both NK and IFN activities of bg/bg mice, but not bg/+ mice. Thus, involvement of both IFN-alpha/beta and IFN-gamma in the generation of peritoneal NK cells and IFN-independent augmentation of splenic NK cells in toxoplasmosis were suggested.     

Abortive replication of vaccinia virus in activated rabbit macrophages.

During the course of infection of rabbits with vaccinia virus, macrophages obtained from the peritoneal cavity develop bactericidal activity and the replication of vaccinia virus becomes restricted in these cells. The abortive replication of vaccinia virus in the activated macrophages was characterized in the present study. The virus adsorbed to and was uncoated equally well in macrophages from both normal and infected rabbits. A burst of deoxyribonucleic acid synthesis of comparable magnitude took place 3 to 6 h after infection in both normal and activated macrophages. Although the production of viral antigens, as detected by immunodiffusion and immunofluorescence, was the same in both types of cells, very few virus particles were formed in activated as compared with normal macrophages. We conclude that a block in a late step of the virus replication cycle occurred in the activated macrophages.     

Lactate dehydrogenase-elevating virus replication, maturation, and viral RNA synthesis in primary mouse macrophage cultures.

High titers of lactate dehydrogenase-elevating virus (LDV) are obtained after infection of 1-day primary peritoneal mouse macrophage cultures. The capacity of these cells to support LDV replication, however, progressively decreases as the cells age. Almost no replication occurs in 7-day cultures, in spite of the fact that the cells are viable and metabolically active, though nondividing. Mitotically active cultures of simian virus 40 (SV40)-transformed macrophages also fail to support LDV replication. The synthesis of LDV-specific RNA was studied by labeling infected 1-day macrophage cultures with [5-³H]uridine in the presence of actinomycin D. The synthesis of single-stranded 48 S viral RNA and RNase-resistant 27 S RNA commence between 4 and 5 hr after infection, and the first mature virions are released between 5 and 6 hr. The time period between synthesis of viral RNA and its appearance in mature extracellular virions is about 1.5 hr. The induction of viral RNA synthesis is completely inhibited by treatment of cells with cycloheximide 0.5 hr after infection.Electron microscopic examination of thin sections of infected macrophages shows that LDV matures by budding from the cytoplasm into intracytoplasmic vesicles. Mature virions seem to be rapidly released into the culture fluid by an as yet unknown mechanism.     

Efficacy of low-dose oral use of type I interferon in cytomegalovirus infections in vivo.

Oral administration of type I interferons (IFNs; murine IFN-alpha and IFN-beta) reduces early replication of murine cytomegalovirus (MCMV) in both the spleen and liver of MCMV-infected BALB/c mice. Examination of a range of doses of IFN (1 to 1000 IU) showed that 10 IU administered daily for 1 week prior to virus infection was optimal for inhibition of MCMV replication. Furthermore, low-dose orally administered IFN (10 IU/day) was effective in mice challenged with lethal and sublethal virus inocula. The antiviral efficacy of low-dose orally administered IFN was not restricted by either the route of virus inoculation or the mouse genotype. Analysis by immunohistochemistry of IFN-alpha receptor-bearing cells of the gastrointestinal tract revealed predominant staining of perivascular smooth muscle and the lamina propria of the anterior tongue, small intestine and rectum. These tissues, dense in IFN-alpha receptor-bearing cells, are likely to be the sites of interaction of the orally administered IFNs with the mucosal immune system. In conclusion, we propose that low-dose oral use of type I IFN therapy may have broad applications in the treatment of CMV infections.     

Rescue of SV 40 from interferon-treated heterokaryons

Summary Simian virus 40 (SV 40)-transformed nonpermissive cells express only the early products of SV 40. Heterokaryons formed by fusion of these transformed cells with uninfected permissive cells support the activation of the resident viral genome leading to subsequent viral DNA replication, late protein synthesis and release of progeny virus. Pretreatment of heterokaryon cultures with either mouse or monkey interferon (IFN) before fusion with polyethylene glycol (PEG) produced a dose-dependent inhibition in the appearance of free viral DNA as well as production of infectious virus. The decreased yield of SV 40 in these cultures was similar to the inhibition which was observed in mouse or monkey cells incubated with homologous IFN prior to exogenous infection with SV 40. when IFN was added to the cultures at progressively later times after fusion with PEG, there was less inhibition of virus production. Although there was a comparable decrease in the production of virus by pretreatment with either mouse or monkey IFN, monkey IFN exerted the inhibition for a longer period of time when added after heterokaryon formation. These results demonstrate that IFN treatment applied even after initiation of SV 40 replication can still inhibit virus multiplication.With 1 Figure     

Replication of Langat virus in immunocompetent cells of mice subjected to immobilization stress

Immobilization stress (hypokinesis) in Balb/c mice may aggravate asymptomatic infection with Langat virus (strain TP-21) as evidenced by 4-fold increased lethality in comparison with control animals. The virus levels in the spleen and brain of stressed and infected mice and the in vitro yield of the virus in immunocompetent cells derived from stressed mice were significantly higher than in controls. Enhanced virus replication in latter cells may contribute to increased accumulation of the infectious agent in lymphatic tissues, which would facilitate virus invasion into CNS followed with acute disease and death of animals.     

The immune response to vaccinia virus infection in mice: analysis of the role of antibody

Summary Immune response to primary intraperitoneal infection with vaccinia virus (strain IHD-J) was studied in C3H/Hej mice. Antibodies reactive with virus structural proteins were detected 6 days and neutralizing antibodies 8 days after infection. Although serum antibodies from infected mice bound to vaccinia virus infected cells, these antibodies were ineffective in complement mediated lysis of infected cells and were only moderately active in experiments with antibody dependent cellular cytotoxicity (ADCC). Immunoblotting analysis showed that serum antibody reacted with a number of structural proteins of both intracellular and extracellular forms of vaccinia virus. Immunoprecipitation results showed antibody binding of nonstructural proteins and glycoproteins. Correlation of the kinetics of NK and CTL activities in infected mice with neutralizing antibodies indicated that the cellular functions clearly precede the appearance of serum neutralizing antibody. The resolution of primary infection in mice thus appears to be mediated by functions of cellular immunity while resistance to reinfection may be dependent on circulating neutralizing antibody.     

Vaccinia virus proteins on the plasma membrane of infected cells. III. Infection of peritoneal macrophages

Primary macrophage cultures were prepared from the peritoneal exudate cell population harvested from mice challenged intraperitoneally with saline, thioglycollate, or vaccinia virus. Vaccinia virus was adsorbed and penetrated into primary macrophages and L-cells with similar kinetics. As evidenced by the expression of some "early" virus-specified proteins, partial uncoating and activation of the virion-associated DNA-dependent RNA polymerase occurred in the infected macrophages. Subsequently, the viral replication cycle in macrophages was aborted; with time after infection, viral DNA and virion proteins initially associated with infected cells could be detected in an acid-soluble form in the medium harvested from infected macrophage cultures. The results suggest that at the time that the final stages of virus uncoating should have occurred, intracellular subviral particles were, instead, degraded in the infected, primary macrophages. Viral DNA synthesis could not be measured in vaccinia virus-infected macrophages, no "late" virus functions were expressed, and progeny virions were not assembled. As measured by the binding of antiviral-antibody-125I-protein A complexes to the surface of vaccinia virus-infected cells, the expression of virus-specified antigens on the surfaces of infected macrophages was significantly reduced and never exceeded that measured at 2 hr after infection on the surfaces of infected L-cells. The expression of virus-specified polypeptides with mol mass of 48-50, 45-46, 36-37, and 25 kDa on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages, rendered the infected macrophages susceptible to lysis by vaccinia virus-specific cytotoxic T-cells.     

Factors involved in the age-dependent resistance of mice infected with low-virulence mouse hepatitis virus

Summary Four-week-old weanling mice survived, whereas 1-week-old suckling mice died, after intraperitoneal inoculation of mouse hepatitis virus, MHV-S strain. The factors involved in this difference in susceptibility were studied. After virus inoculation, differences in virus growth in the liver and spleen were observed, which correlated with the susceptibility of animals to the virus. Interferon, detected at an early stage of infection, was considerably lower in suckling mice than in weanling mice. Titers of MHV-S in peritoneal cells from infected animals were at least 100 times greater in suckling than in weanling mice, and a similar, but less prominent difference in virus growth was also found in the corresponding cultured macrophages. After transfer of peritoneal cells from weanling to suckling mice, a decrease in mortality of infected suckling mice was observed. These results suggest that both interferon and macrophages may be important in the age-dependent resistance of mice to MHV-S infection.With 4 Figures