The mature virion of ectromelia virus, a pathogenic poxvirus, is capable of intrahepatic spread and can serve as a target for delayed therapy.

Orthopoxviruses (OPVs), which include the agent of smallpox (variola virus), the zoonotic monkeypox virus, the vaccine and zoonotic species vaccinia virus, and the mouse pathogen ectromelia virus (ECTV), form two types of infectious viral particles: the mature virus (MV), which is cytosolic, and the enveloped virus (EV), which is extracellular. It is believed that MVs are required for viral entry into the host, while EVs are responsible for spread within the host. Following footpad infection of susceptible mice, ECTV spreads lymphohematogenously, entering the liver at 3 to 4 days postinfection (dpi). Afterwards, ECTV spreads intrahepatically, killing the host. We found that antibodies to an MV protein were highly effective at curing mice from ECTV infection when administered after the virus reached the liver. Moreover, a mutant ECTV that does not make EV was able to spread intrahepatically and kill immunodeficient mice. Together, these findings indicate that MVs are sufficient for the spread of ECTV within the liver and could have implications regarding the pathogenesis of other OPVs, the treatment of emerging OPV infections, as well as strategies for preparedness in case of accidental or intentional release of pathogenic OPVs.

The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses.

Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.

IL-12p40 and IL-18 play pivotal roles in orchestrating the cell-mediated immune response to a poxvirus infection.

A strong cell-mediated immune response is critical for controlling viral infections and is regulated by a number of cytokines, including IL-12 and IL-18. Indeed, some viruses have evolved to specifically target these pathways to counter the host immune response. Orthopoxviruses, including ectromelia virus, encode immune evasion molecules that specifically target IL-18 and IFN-gamma. We hypothesized that IL-12 and IL-18 are pivotal for induction of IFN-gamma production and subsequent generation of an effective host response to ectromelia virus infection. In this study, we demonstrate that absence of both IL-12p40 and IL-18 resulted in increased susceptibility to infection that was associated with skewing of the cytokine response to Th2 and a reduction in NK and CTL responses. The decrease in CTL response correlated with a defect in CD8(+) T cell proliferation and lower numbers of virus-specific CD8(+) T cells. Lack of either IL-12p40 and/or IL-18 was also associated with reduced numbers of CD8(+) T cells at sites of infection and with an increase in the numbers of splenic T regulatory cells. Taken together, our data indicate that IL-12p40 and IL-18 act in concert and play an important antiviral role through the up-regulation of IFN-gamma production and cell-mediated immune responses.

Surviving mousepox infection requires the complement system.

Poxviruses subvert the host immune response by producing immunomodulatory proteins, including a complement regulatory protein. Ectromelia virus provides a mouse model for smallpox where the virus and the host's immune response have co-evolved. Using this model, our study investigated the role of the complement system during a poxvirus infection. By multiple inoculation routes, ectromelia virus caused increased mortality by 7 to 10 days post-infection in C57BL/6 mice that lack C3, the central component of the complement cascade. In C3(-/-) mice, ectromelia virus disseminated earlier to target organs and generated higher peak titers compared to the congenic controls. Also, increased hepatic inflammation and necrosis correlated with these higher tissue titers and likely contributed to the morbidity in the C3(-/-) mice. In vitro, the complement system in naïve C57BL/6 mouse sera neutralized ectromelia virus, primarily through the recognition of the virion by natural antibody and activation of the classical and alternative pathways. Sera deficient in classical or alternative pathway components or antibody had reduced ability to neutralize viral particles, which likely contributed to increased viral dissemination and disease severity in vivo. The increased mortality of C4(-/-) or Factor B(-/-) mice also indicates that these two pathways of complement activation are required for survival. In summary, the complement system acts in the first few minutes, hours, and days to control this poxviral infection until the adaptive immune response can react, and loss of this system results in lethal infection.

The efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin-4.

Improved vaccines and therapies for virulent poxvirus infection are required, particularly in the light of recent threats of bioterrorism. Cidofovir (HPMPC) is an acyclic nucleoside analog with proven efficacy against poxviruses. Here, we evaluated HPMPC in mice given a recombinant ectromelia virus (ECTV) encoding interleukin-4 (ECTV-IL-4) that is highly immune suppressive. Mousepox-sensitive BALB/c mice given HPMPC for five consecutive days after infection were protected against the lethal effects of a control ECTV recombinant, although they suffered a chronic form of mousepox disease. High doses of the drug resulted in a milder localized disease. In contrast, HPMPC failed to protect mousepox-resistant C57BL/6 mice against ECTV-IL-4, although its lethal effects were delayed by five daily doses of 20 mg/kg or a single dose of 100 mg/kg. Higher daily doses further delayed mortality, although the majority of animals eventually succumbed to infection. It appears that HPMPC inhibited ECTV-IL-4 replication without clearance, with the virus having a lethal effect when the drug was removed. Resistance of ECTV-IL-4 to HPMPC treatment may relate to the virus's ability to inhibit antiviral cell-mediated immunity. Interestingly, ECTV-IL-4-mediated immune suppression was not accompanied by a reduction in systemic IFN-gamma expression, suggestive of an alternative or highly localized suppressive mechanism.

Deficiency in Th2 cytokine responses exacerbate orthopoxvirus infection.

Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbpΔ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbpΔ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.

[The virucidal properties of neoaquasept]. / Virulitsidnye svoistva neoakvasepta.

The paper provides the evidence suggesting that the disinfectant neoaquasept (NA) obtained by using sodium salt of dichlorisocyanuric acid has virucidal activity against ectromelia virus in BALB/c mice. Three concentrations of NA were tested. The recommended application rate and concentration of the disinfectant is 3 and 10 times higher. NA was demonstrated to decrease the degenerative changes of the liver and spleen and increases the survival of the animals infected with ectromelia virus. It is proposed to use the water disinfected with NA as a preventive agent in the spread foci of various infections.

TLR3 and TLR9 agonists improve postexposure vaccination efficacy of live smallpox vaccines.

Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.

The attenuated NYCBH vaccinia virus deleted for the immune evasion gene, E3L, completely protects mice against heterologous challenge with ectromelia virus.

The New York City Board of Health (NYCBH) vaccinia virus (VACV) vaccine strain was deleted for the immune evasion gene, E3L, and tested for its pathogenicity and ability to protect mice from heterologous challenge with ectromelia virus (ECTV). NYCBHΔE3L was found to be highly attenuated for pathogenicity in a newborn mouse model and showed a similar attenuated phenotype as the NYVAC strain of vaccinia virus. Scarification with one or two doses of the attenuated NYCBHΔE3L was able to protect mice equally as well as NYCBH from death, weight loss, and viral spread to visceral organs. A single dose of NYCBHΔE3L resulted in low poxvirus-specific antibodies, and a second dose increased levels of poxvirus-specific antibodies to a level similar to that seen in animals vaccinated with a single dose of NYCBH. However, similar neutralizing antibody titers were observed following one or two doses of NYCBHΔE3L or NYCBH. Thus, NYCBHΔE3L shows potential as a candidate for a safer human smallpox vaccine since it protects mice from challenge with a heterologous poxvirus.

Mousepox in the C57BL/6 strain provides an improved model for evaluating anti-poxvirus therapies.

The intranasal lethal mousepox model employing the A/Ncr mouse strain is used to evaluate anti-orthopoxvirus therapies. These infections mimic large droplet transmission and result in 100% mortality within 7-10 days with as little as 1 PFU of ectromelia virus. Unlike the A/Ncr model, humans are less susceptible to lethal respiratory infections with variola virus and monkeypox virus as demonstrated by their lower mortality rates. In this study we show that a low dose intranasal infection of C57BL/6 mice results in 60-80% mortality and better models smallpox. Comparing CMX001 (HDP-cidofovir) efficacy in the A/Ncr strain and the C57BL/6 strain revealed that delayed treatment with CMX001 is more efficacious at preventing severe disease in the C57BL/6 strain. The increased efficacy of CMX001 in C57BL/6 over A/Ncr following an intranasal infection with ectromelia appears to be mediated by a stronger Th1 cell mediated response. Following footpad infection we show that the C57BL/6 strain has earlier and more robust transcriptional activity, Th1 cytokine secretions, antigen presenting activity and IFNgamma splenic CD8+ T cell responses as compared to the A/Ncr strain. As a result of the enhanced immune response in the C57BL/6 strain, non-lethal intradermal ectromelia infections can therapeutically protect up to 3 days following a homologous, lethal intranasal infection - much like how smallpox vaccination can protect humans for up to 4 days following intranasal variola infection.

Synergic effects of interferon and interferon inducer against ectromelia virus infection in mice.

Intraperitoneal injection of mouse brain interferon into mice 24 hr before inoculation of ectromelia virus, significantly reduced the mortality rate and prolonged the mean day of death (MDD). Intravenous injection of Newcastle disease virus (NDV) 24 hr before virus inoculation, also had significant beneficial effects. Furthermore, combined treatment with interferon and NDV had more protective effect against virus infection than either interferon or NDV treatment alone.

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. I. Clinical responses.

Clinical responses to infection with ectromelia virus strain NIH-79 were determined in several strains of inbred mice. All mice were equally susceptible to infection, but mortality was strain dependent. BALB/c AnNCr, A/JNCr, DBA/2NCr and C3H/He/NCr MTV- mice were highly susceptible to lethal infection whereas AKR/NCr and SJL/NCr mice were moderately susceptible and C57BL/6NCr mice were highly resistant. Death rates were influenced strongly by virus dose and by route of inoculation. High doses were associated with early and high mortality. For a given dose, intraperitoneal inoculation resulted in the highest mortality and death rates were progressively reduced in mice inoculated by the footpad, subcutaneous and intranasal routes. Footpad swelling was prominent in resistant mice and in survivors among susceptible strains. Deaths among AKR and SJL mice were sporadic and often occurred late irrespective of virus dose. It is suggested that this pattern could be influenced by secondary contact infections or by immunologic injury associated with host responses to ectromelia virus.

Combined protective effects on interferon and interferon induction on herpes simplex and ectromelia virus infections in mice.

Mouse interferon or the induction of mouse interferon with polyriboinosinic acid-polyribocytidylic acid significantly protected mice against herpes simplex and ectromelia viral infections. When polyriboinosinic acid-polyribocytidylic acid was administered 24 h before herpes simplex or ectromelia viral infection and mouse interferon was administered shortly before and 24 h after infection, a combined protective effect against either herpes simplex or ectromelia viral infection in mice was evident. There was a significant decrease in the mortality rate with the combined treatment as compared either with the rate in group treated with interferon or polyriboinosinic acid-polyribocytidylic acid.

Effect of polychlorinated biphenyl on viral infections in mice.

Mice that were fed a diet containing 400, 200 or 100 microgram of polychlorinated biphenyls (PCB) per g were significantly more susceptible to Herpes simplex virus than mice that were fed a PCB-free diet. The mortality of mice that were fed a diet containing 400 or 200 microgram of PCB per g and infected with ectromelia virus was higher than that of normal control mice infected with virus. There was no significant difference in inducibility of interferon by polyinosinic acid-polycytoidylic acid between PCB-fed mice and control mice.

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. V. Genetics of resistance to the Moscow strain.

The genetics of resistance to the Moscow strain of ectromelia virus was examined in crosses derived from resistant C57BL/6 (B6) and susceptible DBA/2 (D2) mice. Infection with 10(1) to 10(5) PFU of virus resulted in mortalities of 90 to 100% of D2, 0% of B6 and 0 to 3% of (B6 x D2) F1 mice by day 21. Among F1 x D2 backcross progeny, 49% of male and 18% of female mice died. Reciprocal backcrossing did not alter male or female mortality rates. These data are consistent with a single autosomal dominant gene controlling resistance to ectromelia in male mice and at least one additional dominant sex-limited gene controlling resistance in female mice. Fewer male F2 mice died than were predicted based on single-locus control and 32% of recombinant inbred (RI) strains derived from B6 and D2 progenitors expressed non-parental phenotypes. Therefore, additional resistance genes, not expressed in backcross mice, were apparently expressed in F2 mice and RI strains.

Differential pathogenesis of lethal mousepox in congenic DBA/2 mice implicates natural killer cell receptor NKR-P1 in necrotizing hepatitis and the fifth component of complement in recruitment of circulating leukocytes to spleen.

Innate resistance of C57BL/6 (B6) mice to lethal mousepox is controlled by multiple genes. Previously, four resistance genes were localized to specific subchromosomal regions and transferred onto a susceptible DBA/2 (D2) background by serial backcrossing and intercrossing to produce congenic strains. Intraperitoneally inoculated ectromelia virus was uniformly lethal and achieved similar titers in B6 and D2 mice but elicited differential responses in liver, spleen, and circulating blood leukocytes. The distribution of these response phenotypes in congenic strains linked control of phenotypes with specific subchromosomal regions. D2.R1 mice, which carried a differential segment of chromosome 6, exhibited a B6 liver response and intermediate spleen and circulating leukocyte responses. D2.R2 and D2.R4 mice, which carried differential segments of chromosomes 2 and 1, respectively, exhibited a D2 liver response, a B6 spleen response, and an intermediate circulating leukocyte response. The localization of control of liver response phenotypes to chromosome 6 implicates cells that express natural killer (NK) cell receptor NKR-P1 alloantigens. The localization of control of spleen and circulating leukocyte responses to chromosomes 1, 2, and 6 implicates NK cells, the fifth component of complement, and a gene near the selectin gene complex in recruitment of circulating leukocytes to spleen.