Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. II. Pathogenesis

The pathogenesis of mousepox due to infection with ectromelia virus strain NIH-79 was characterized in genetically susceptible (BALB/cAnNCr) and genetically resistant (C57BL/6NCr) mice. BALB/c mice inoculated subcutaneous (s.c.) or intranasally (i.n.) had high mortality. Most mice died within 7 days from severe necrosis of the spleen and liver. Necrotic foci in livers of BALB/c mice that survived beyond 7 days often were accompanied by mononuclear cell infiltrates and by hyperplasia of lymphoid tissues. C57BL/6 mice inoculated by either route remained asymptomatic and necrotic lesions were mild or absent, whereas focal non-suppurative hepatitis and lymphoid hyperplasia were prominent. Infectious virus and viral antigen were distributed widely in tissues of BALB/c mice, but had limited distribution in C57BL/6 mice. Both mouse strains had infection of the respiratory tract, genital tract, oral tissues and bone marrow, and BALB/c mice also had infection of the intestines. Both strains also developed serum antibody to vaccinia virus antigen after infection. The results show that ectromelia virus occurs in tissues conducive to mouse to mouse transmission and that the severity and character of mousepox lesions correlate directly with resistance and susceptibility to infection. They also support the concept that cellular immunity contributes to survival from infection.     

Murine viruses in an island population of introduced house mice and endemic short-tailed mice in Western Australia

House mice (Mus domesticus) were recently introduced to Thevenard Island, off the northwest coast of Western Australia. This island is also habitat for an endangered native rodent, the short-tailed mouse (Leggadina lakedownensis). Concerns have been raised that house mice may pose a threat to L. lakedownensis both through competition and as a source of infection. To assess the threat to L. lakedownensis posed by viral pathogens from M. domesticus, a serological survey was conducted from 1994 to 1996 of both species for evidence of infection with 14 common murine viruses (mouse hepatitis virus, murine cytomegalovirus, lymphocytic choriomeningitis virus, ectromelia virus, mouse adenovirus strains FL and K87, minute virus of mice, mouse parvovirus, reovirus type 3, Sendai virus, Theiler's mouse encephalomyelitis virus, polyoma virus, pneumonia virus of mice, and encephalomyocarditis virus) and Mycoplasma pulmonis. Despite previous evidence that populations of free-living M. domesticus from various locations on the Australian mainland were infected with up to eight viruses, M. domesticus on Thevenard Island were seropositive only to murine cytomegalovirus (MCMV). Antibodies to MCMV were detected in this species at all times of sampling, although seroprevalence varied. Infectious MCMV could be isolated in culture of salivary gland homogenates from seropositive mice. In contrast, L. lakedownensis on Thevenard Island showed no serological evidence of infection with MCMV, any of the other murine viruses, or M. Pulmonis, and no virus could be isolated in culture from salivary gland homogenates. Although MCMV replicated to high titers in experimentally infected inbred BALB/c laboratory mice as expected, it did not replicate in the target organs of experimentally inoculated L. lakedownensis, indicating that the strict host specificity of MCMV may prevent its infection of L. lakedownensis. These results suggest that native mice on Thevenard Island are not at risk of MCMV infection from introduced house mice, and raise interesting questions about the possible selective survival of MCMV in small isolated populations of M. domesticus.     

Abrogation of resistance to severe mousepox in C57BL/6 mice infected with LP-BM5 murine leukemia viruses.

Strain C57BL/6 (B6) mice infected with LP-BM5 murine leukemia virus (MuLV) develop a disease which combines abnormal lymphoproliferation with profound immunosuppression and has many features in common with human acquired immunodeficiency syndrome induced by HTLV-III/LAV retroviruses. To determine whether this LP-BM5 MuLV infection would affect the innate resistance of B6 mice to a naturally occurring, highly virulent murine pathogen, mice were exposed to ectromelia virus at various times after treatment with LP-BM5 viruses. At week 4 after infection with LP-BM5, mice challenged with ectromelia virus were unable to generate a humoral immune response to this virus, and between weeks 8 and 10 after infection, challenged mice lost the ability to generate an ectromelia virus-specific cytotoxic-T-cell response. Loss of the cellular immune responses to ectromelia virus was associated with an increased susceptibility to the lethal effects of the virus.     

Mousepox in the Netherlands

Two independent outbreaks of ectromelia in mice occurred in The Netherlands. In both cases, the causative virus was isolated and identified as ectromelia virus on the basis of serology, demonstration of antigen by indirect immunofluorescence, negative contrast electron microscopy, morphology of lesions on chorioallantoic membranes of embryonated chicken eggs, and cytopathogenicity for mouse cells. Inoculation of the virus into the dermis of rabbits demonstrated a low virulence for this species.     

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. III. Experimental transmission of infection and derivation of virus-free progeny from previously infected dams

The incidence and duration of transmission of infection with ectromelia virus strain NIH-79 was tested in innately resistant (C57BL/6) and innately susceptible (BALB/c) inbred mice. Transmission by C57BL/6 index mice occurred through 3 weeks and by BALB/c index mice through 4 weeks, although the duration of infection in individual index mice was often shorter. Soiled caging that previously housed infected mice was inconsistently infectious. Transmission was high in cages where infected mice died and were cannibalized by cagemates, but was low to moderate in cages where there was no cannibalism. Infected mice that were bred 6 weeks after they were infected, delivered virus-free progeny and did not transmit infection to their non-immune breeding partners. Sentinel mice housed in the room with experimentally infected mice were seronegative for antibody to ectromelia virus and to other murine viruses. These results support the view that infection with NIH-79 virus is typically short-lived. They also indicate that breeding of recovered mice can save valuable colonies that have been exposed to ectromelia virus.     

Comparison of mucosal and systemic humoral immune responses and subsequent protection in mice orally inoculated with a homologous or a heterologous rotavirus.

Rotaviruses are the single most important cause of severe diarrhea in young children worldwide, and vaccination is probably the most effective way to control the disease. Most current live virus vaccine candidates are based on the host range-restricted attenuation of heterologous animal rotaviruses in humans. The protective efficacy of these vaccine candidates has been variable. To better understand the nature of the heterologous rotavirus-induced active immune response, we compared the differences in the mucosal and systemic immune responses generated by heterologous (nonmurine) and homologous (murine) rotaviruses as well as the ability of these infections to produce subsequent protective immunity in a mouse model. Sucking mice were orally inoculated with a heterologous simian or bovine rotavirus (strain RRV or NCDV) or a homologous murine rotavirus (wild-type or tissue culture-adapted) strain EHP at various doses. Six weeks later, mice were challenged with a virulent murine rotavirus (wild-type strain ECW) and the shedding of viral antigen in feces was quantitated. Levels of rotavirus-specific serum immunoglobulin G (IgG) and fecal IgA prior to challenge were measured and correlated with subsequent viral shedding or protection. Heterologous rotavirus-induced active protection was highly dependent on the strain and dose of the virus tested. Mice inoculated with a high dose (10(7) PFU per mouse) of RRV were completely protected, while the protection was diminished in animals inoculated with NCDV or lower doses of RRV. The ability of a heterologous rotavirus to stimulate a detectable intestinal IgA response correlated with the ability of the virus to generate protective immunity. Serum IgG titer did not correlate with protection. Homologous rotavirus infection, on the other hand, was much more efficient at inducing both mucosal and systemic immune responses as well as protection regardless of the virulence of the virus strain or the size of the immunizing dose.     

Hygienic monitoring of Mongolian gerbils: which mouse viruses should be included?

The Mongolian gerbil (Meriones unguiculatus) serves as an animal model for a wide range of diseases. A practical limitation in its use is the definition of the hygienic status, as not much is known about viruses that potentially infect gerbils and might be transmitted to other rodents. As successful re-derivation was recently described for gerbils, we now aimed at investigating which mouse viruses induce seroconversion in gerbils and might be transmitted to mice. Gerbils were inoculated with viral agents of mice and co-housed with mouse contact sentinels. Seroconversion in gerbils was observed after oronasal inoculation with Sendai virus (SeV), mammalian orthoreovirus serotype 3 (Reo-3) and rotavirus A (RV-A, EDIM), seroconversion to RV-A also in sentinel mice. Pneumonia virus of mice (PVM) was not detected by serology but by polymerase chain reaction in gerbils and respective sentinel mice. No seroconversion towards or transmission of murine hepatitis virus, murine norovirus, minute virus of mice or mouse cytomegalovirus was detected. Anti-gerbil IgG antibodies did not increase sensitivity of indirect immunofluorescence (IFA) compared with anti-mouse IgG. In conclusion, seroconversion to SeV, Reo-3 and RV-A as well as transmission of RV-A and PVM indicate that these agents should be included in health monitoring of gerbils. Furthermore, anti-mouse IgG is suitable as a secondary antibody for IFA with gerbil serum.     

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.     

Administration of vaccinia virus to mice may cause contact or bedding sentinel mice to test positive for orthopoxvirus antibodies: case report and follow-up investigation

Routine testing of bedding sentinels from a barrier room revealed one mouse seropositive to ectromelia virus (EV). Results of hemagglutination-inhibition testing and western blot analysis were confirmatory for orthopoxvirus antibodies. Additional seropositive animals were not identified. Interviews indicated that replication-competent vaccinia virus (VV), Western Reserve strain (VV-WR), recently had been given to mice. Although VV-WR was not expected to spread by contact or via fomites, the case evidence suggested transmission of vaccinia via soiled bedding. In a follow-up experiment, 15 index mice were inoculated with 10(7) plaque-forming units of VV by either subcutaneous or intrarectal instillation. A dedicated contact sentinel and a bedding sentinel were provided for each index mouse. All 15 index mice were positive for antibodies when tested 22 days after inoculation. One mouse, inoculated by the subcutaneous route, appeared ill and developed lesions on the proximal portion of the tail. The contact sentinel mouse housed with this index mouse was the only sentinel to seroconvert. We conclude that VV-WR can spread to contact sentinels and potentially to bedding sentinels. The ability of other VV strains to be transmitted horizontally and the susceptibility of different mouse strains to infection merit further investigation. The use of VV in animal facilities must be managed carefully since the available serologic tests do not distinguish between VV and EV, an exotic agent of major concern to laboratory animal facilities.     

<Emphasis Type="Italic">Ly49h</Emphasis> is necessary for genetic resistance to murine cytomegalovirus

Natural killer (NK) cells play critical roles in antiviral immunity. While the importance of effector mechanisms such as interferons has been demonstrated through knockout mice, specific mechanisms of how viruses are recognized and controlled by NK cells are less well defined. Previous genetic studies have mapped the resistance genes for murine cytomegalovirus (MCMV), herpes simplex virus-1 (HSV-1), and ectromelia virus to the NK gene complex on murine chromosome 6, a region containing the polymorphic Ly49 and Nkrp1 families. Genetic resistance to MCMV in C57BL/6 has been attributed to Ly49H, an activation receptor, through susceptibility of the recombinant inbred strain BXD-8 that lacks Ly49h (also known as Klra8) but derived about half of its genome from its DBA/2 progenitor. However, it remained possible that epigenetic effects could account for the MCMV phenotype in BXD-8 mice. Herein, we report the generation of a novel congenic murine strain, B6.BXD8-Klra8 ( Cmv1-del )/Wum, on the C57BL/6 genetic background to evaluate the effect of deletion of a single NK activation receptor, Ly49H. Deletion of Ly49H rendered mice much more susceptible to MCMV infection. This increase in susceptibility did not appear to be a result of a difference in NK cell expansion or interferon-gamma (IFN-gamma) production between the C57BL/6 and the B6.BXD8 strains. On the other hand, the deletion of Ly49h did not otherwise affect NK cell maturation or Ly49D expression and had no effect on susceptibility to HSV-1 or ectromelia virus. In conclusion, Ly49h is necessary for genetic resistance to MCMV, but not HSV-1 or ectromelia virus.     

Reexamination of the efficacy of vaccination against mousepox

Experiments were conducted to evaluate the efficacy of three strains of vaccinia virus, IHD-T, Lister and Wyeth, to immunize the BALB/cByJ mouse against infection with ectromelia virus. Mice vaccinated with any of the strains were protected for at least 12 weeks against clinically apparent disease when challenged with cage-mates infected with a virulent stain (NIH-79) of ectromelia virus. However, 4 to 8 weeks after vaccination mice were capable of transmitting virus to non-vaccinated cage-mates. The results are discussed within the context of the current practices for preventing and controlling ectromelia epizootics.     

A Changing Pattern of the Cell Cycle during the First Two Cleavage Divisions of the Mouse

The cell cycles of the early cleavage stages of the mouse were analyzed by examining Feulgen-stained ova. The period from ovulation to the completion of second cleavage division was investigated. The ova donors were C57BL/6 × DBA/2 female mice, which were hormonally superovulated. To estimate the durations of DNA synthesis and mitotic phases of the cleavage divisions, the ova were pooled into culture medium, and as a function of time, aliquots were removed from the batch of pooled ova. The ova specimens were Feulgen-stained and classified as the ova nuclei in G₁, S, G₂ or mitosis by use of a cytophotometric technique and then the durations of these phases were determined by probit analysis.
The pronuclear stage had a generation time of 11 hr, with a G₁ phase of 6 hr and a short S phase of 1.7 hr. In contrast the two-cell stage had a generation time of 18 hr, with a G₁ phase of 2 hr and an S phase of 3 hr. The duration of cleavage division also changed; the first cleavage division spanned 3 hr while the second spanned 1 hr.     

Examination of latent infection in germfree mice for some murine viruses.

Sera from some groups of germfree mice were examined for reactivity against Sendai, reo 3, Theiler's GD VII, ectromelia, and mouse hepatitis virus antigens. Of 51 sera collected in September and October 1976 and in August and September 1977 from one germfree mouse breeding colony, there was no positive reaction against all the antigens tested. Moreover, the attempt of virus isolation from homogenates of various organs including lungs, liver, kidneys, and intestines of 6 germfree mice was unsuccessful.     

Identification of a spleen focus-forming virus in erythroleukemic mice infected with a wild-mouse ecotropic murine leukemia virus

An NFS/N mouse inoculated at birth with an ecotropic murine leukemia virus (MuLV) obtained from wild mice (Cas-Br-M MuLV) developed a lymphoma after 18 weeks. An extract prepared from the lymphomatous spleen was inoculated into newborn NFS/N mice, and these mice developed erythroleukemia within 9 weeks. Spleens from the erythroleukemic mice contained ecotropic and mink cell focus-inducing (MCF) MuLVs; however, when these viruses were biologically cloned and reinoculated into newborn NFS/N mice, no erythroleukemia was induced. In contrast, cell-free extracts prepared from the erythroleukemic spleens induced erythroleukemia within 5 weeks. Analysis of cell-free extracts prepared from the erythroleukemic spleens showed that they contained a viral species that induced splenomegaly and spleen focus formation in adult mice, with susceptibility controlled by alleles at the Fv-2 locus. The spleen focus-forming virus coded for a 50,000-dalton protein precipitated by antibodies specific to MCF virus gp70. RNA blot hybridization studies showed the genomic viral RNA to be 7.5 kilobases and to hybridize strongly to a xenotropic or MCF envelope-specific probe but not to hybridize with an ecotropic virus envelope-specific probe. The virus described here appears to be the fourth independent isolate of a MuLV with spleen focus-forming activity.     

Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox

Genetic resistance to clinical mousepox (ectromelia virus) varies among inbred laboratory mice and is characterized by an effective natural killer (NK) response and the early onset of a strong CD8(+) cytotoxic T-lymphocyte (CTL) response in resistant mice. We have investigated the influence of virus-expressed mouse interleukin-4 (IL-4) on the cell-mediated response during infection. It was observed that expression of IL-4 by a thymidine kinase-positive ectromelia virus suppressed cytolytic responses of NK and CTL and the expression of gamma interferon by the latter. Genetically resistant mice infected with the IL-4-expressing virus developed symptoms of acute mousepox accompanied by high mortality, similar to the disease seen when genetically sensitive mice are infected with the virulent Moscow strain. Strikingly, infection of recently immunized genetically resistant mice with the virus expressing IL-4 also resulted in significant mortality due to fulminant mousepox. These data therefore suggest that virus-encoded IL-4 not only suppresses primary antiviral cell-mediated immune responses but also can inhibit the expression of immune memory responses.     

Observations on the replication of ectromelia virus in mouse-derived cell lines: implications for epidemiology of mousepox

Ectromelia virus was shown to replicate in vitro in all lymphoma cell lines and in a small proportion of hybridoma lines tested. It was demonstrated that certain hybridoma cell lines, which were passed in ectromelia virus-infected mice, yielded ectromelia virus infectivity on explantation into tissue culture. This finding further substantiated the belief that ascitic fluid and hybridoma cell lines exposed to virus during mouse-passage could be important in the epidemiology of mousepox.     

Physical mapping of the paralysis-inducing determinant of a wild mouse ecotropic neurotropic retrovirus.

We have recently shown that a molecularly cloned ecotropic retrovirus, initially isolated from the brain of a paralyzed wild mouse, retained the ability to induce hind limb paralysis when inoculated into susceptible mice (Jolicoeur et al., J. Virol. 45:1159-1163, 1983). To map the viral DNA sequences encoding the determinant of paralysis, we constructed chimeric viral DNA genomes in vitro between parental cloned infectious viral DNA genomes from this neurotropic murine leukemia virus (MuLV) and from nonneurotropic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered after microinjection of NIH 3T3 cells with these recombinant DNAs, were inoculated into newborn SIM.S and SWR/J mice to test the paralysis-inducing potential. We found that the 3.9-kilobase-pair SalI-ClaI fragment of the neurotropic MuLV comprising the 3' end of pol and all env sequences was sufficient to confer the paralysis-inducing potential to chimeric viruses. Therefore, this region of the neurotropic MuLV genome most likely harbors the primary determinant of paralysis.     

IL-12 delivery from recombinant vaccinia virus attenuates the vector and enhances the cellular immune response against HIV-1 Env in a dose-dependent manner.

To develop vaccination strategies against HIV-1 infection aimed to specifically enhance the cell-mediated immunity (CMI), we have engineered vaccinia virus (VV) recombinants expressing HIV-1 Env (rVVenv) and murine IL-12 (rVVlucIL-12) genes or coexpressing both genes (rVVenvIL-12). In mice inoculated with rVVlucIL-12 there is a rapid clearance of the virus, and this correlates with the induction of high levels of IL-12 and IFN-gamma in serum and spleen early after infection. Enzyme-linked immunospot analysis of mice inoculated with rVVlucIL-12, revealed a nearly 2-fold increase in the number of specific anti-VV CD8+ T cells compared with that in mice given control rVV, and the serum Ab response was biased in favor of a Th1 response. An enhancement of about 2-fold in the number of anti-gp160 IFN-gamma-secreting CD8+ T cells was observed in mice inoculated with rVVenvIL-12, when a dose of 1 x 107 PFU/mouse was used, but this enhancement was not observed when mice were given 5 x 107 PFU. This variation with virus dosage was confirmed in mice immunized simultaneously with different multiplicities of rVV expressing singly the env or IL-12 genes. The highest specific CMI was obtained in mice coadministered a low dose (2 x 104 PFU) of rVVlucIL-12 and 1 x 107 PFU of rVVenv. Our findings provide evidence for specific enhancement of the CMI to HIV-1 Env by the differential expression of IL-12 and env genes delivered from VV recombinants. This approach can be of wide vaccination interest as a means to improve immune responses to other Ags.     

Genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues.

The molecular mechanism of genetic resistance of inbred mouse strains to mouse hepatitis virus, a murine coronavirus, was studied by comparing virus binding to plasma membranes of intestinal epithelium or liver from susceptible BALB/c and resistant SJL/J mice with a new solid-phase assay for virus-binding activity. Virus bound to isolated membranes from susceptible mice, but not to membranes from resistant mice. F1 progeny of SJL/J X BALB/c mice had an intermediate level of virus-binding activity on their enterocyte and hepatocyte membranes. This correlated well with previous studies showing that susceptibility to mouse hepatitis virus strain A59 is controlled by a single autosomal dominant gene (M. S. Smith, R. E. Click, and P. G. W. Plagemann, J. Immunol. 133:428-432). Because virus binding was not prevented by treating membranes with sodium dodecyl sulfate, the virus-binding molecule could be identified by a virus overlay protein blot assay. Virus bound to a single broad band of Mr 100,000 to 110,000 in membranes from hepatocytes or enterocytes of susceptible BALB/c and semisusceptible C3H mice, but no virus-binding band was detected in comparable preparations of resistant SJL/J mouse membranes. Therefore, SJL/J mice may be resistant to mouse hepatitis virus A59 infection because they lack a specific virus receptor which is present on the plasma membranes of target cells from genetically susceptible BALB/c and semisusceptible C3H mice.