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.     

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.     

Evidence for Persistence of Ectromelia Virus in Inbred Mice,Recrudescence Following Immunosuppression and Transmission to Na?ve Mice

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.     

Genetic variability of immunomodulatory genes in ectromelia virus isolates detected by denaturing high-performance liquid chromatography

The genetic variability of nine genes in 12 isolates and strains of ectromelia virus, which causes a smallpox-like disease (mousepox) in mice, was determined and allows for classification of ectromelia viruses. The low genetic variability suggests that evolutionary pressure maintains the activity of immunomodulatory genes in natural poxvirus infections.     

Chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality.

Four genetic loci were tested for linkage with loci that control genetic resistance to lethal ectromelia virus infection in mice. Three of the loci were selected because of concordance with genotypes assigned to recombinant inbred (RI) strains of mice derived from resistant C57BL/6 and susceptible DBA/2 (BXD) mice on the basis of their responses to challenge infection. Thirty-six of 167 male (C57BL/6 x DBA/2)F1 x DBA/2 backcross (BC) mice died (22%), of which 27 (75%) were homozygous for DBA/2 alleles at Hc and H-2D. Twenty-eight percent of sham-castrated and 6% of sham-ovariectomized BC mice were susceptible to lethal mousepox, whereas 50% of gonadectomized mice were susceptible. There was no linkage evident between Hc or H-2D and loci that controlled resistance to lethal ectromelia virus infection in 44 castrated BC mice. Mortality among female mice of BXD RI strains with susceptible or intermediate male phenotypes was strongly correlated (r = 0.834) with male mortality. Gonadectomized C57BL/6 mice were as resistant as intact mice to lethal ectromelia virus infection. These results indicate that two gonad-dependent genes on chromosomes 2 and 17 and one gonad-independent gene control resistance to mousepox virus infection, that males and females share gonad-dependent genes, and that the gonad-independent gene is fully protective.     

Comparison of protective properties of the smallpox DNA-vaccine based on the variola virus A30L gene and its variant with modified codon usage

Efficacy of candidate DNA-vaccines based on the variola virus natural gene A30L and artificial gene A30Lopt with modified codon usage, optimized for expression in mammalian cells, was tested. The groups of mice were intracutaneously immunized three times with three-week intervals with candidate DNA-vaccines: pcDNA_A30L or pcDNA_A30Lopt, and in three weeks after the last immunization all mice in the groups were intraperitoneally infected by the ectromelia virus K1 strain in 10 LD50 dose for the estimation of protection. It was shown that the DNA-vaccines based on natural gene A30L and codon-optimized gene A30Lopt elicited virus, thereby neutralizing the antibody response and protected mice from lethal intraperitoneal challenge with the ectromelia virus with lack of statistically significant difference.     

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.     

Innate resistance to lethal mousepox is genetically linked to the NK gene complex on chromosome 6 and correlates with early restriction of virus replication by cells with an NK phenotype.

Most inbred strains of mice, including DBA/2 (D2), are highly susceptible to the lethal effects of ectromelia virus, but C57BL/6 (B6) mice are innately resistant. Resistance is controlled by multiple, unlinked, autosomal dominant genes. Of 101 male (B6 x D2)F1 x D2 backcrossed (N2) mice, 18 died after ectromelia virus challenge and all were homozygous for the D2 allele at the proline-rich protein (Prp) locus on distal chromosome 6 (P < 0.001). This association was suggested by the patterns of susceptibility to lethal mousepox in recombinant inbred strains derived from B6 and D2 mice (D. G. Brownstein, P. N. Bhatt, L. Gras, and R. O. Jacoby, J. Virol. 65:1946-1951, 1991). The association between the Prp locus and susceptibility to lethal mousepox also held for N2 male mice that were castrated as neonates, which increased the percentage that were susceptible to 40. Spleen virus titers were significantly augmented in B6 (NK1.1+) mice depleted of asialo GM1+ or NK1.1+ cells, whereas spleen virus titers were unaffected in D2 (NK1.1-) mice depleted of asialo GM1+ cells. These results suggest that a gene or genes within the natural killer gene complex, adjacent to the Prp locus, determine strain variations in resistance to lethal ectromelia virus infection.     

Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer

Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.     

Protective immunity against secondary poxvirus infection is dependent on antibody but not on CD4 or CD8 T-cell function

Renewed interest in smallpox and the need for safer vaccines have highlighted our lack of understanding of the requirements for protective immunity. Since smallpox has been eradicated, surrogate animal models of closely related orthopoxviruses, such as ectromelia virus, have been used to establish critical roles for CD8 T cells in the control of primary infection. To study the requirements for protection against secondary infection, we have used a prime-challenge regime, in which avirulent ectromelia virus was used to prime mice that were then challenged with virulent ectromelia virus. In contrast to primary infection, T cells are not required for recovery from secondary infection, since gene knockout mice deficient in CD8 T-cell function and wild-type mice acutely depleted of CD4, CD8, or both subsets were fully protected. Protection correlated with effective virus control and generation of neutralizing antibody. Notably, primed mice that lacked B cells, major histocompatibility complex class II, or CD40 succumbed to secondary infection. Thus, antibody is essential, but CD4 or CD8 T cells are not required for recovery from secondary poxvirus infection.     

Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene.

With the aid of three monoclonal antibodies (MAbs), a glycoprotein specifically localized to the outer envelope of vaccinia virus was shown to be encoded by the A33R gene. These MAbs reacted with a glycosylated protein that migrated as 23- to 28-kDa and 55-kDa species under reducing and nonreducing conditions, respectively. The protein recognized by the three MAbs was synthesized by all 11 orthopoxviruses tested: eight strains of vaccinia virus (including modified vaccinia virus Ankara) and one strain each of cowpox, rabbitpox, and ectromelia viruses. The observation that the protein synthesized by ectromelia virus-infected cells reacted with only one of the three MAbs provided a means of mapping the gene encoding the glycoprotein. By transfecting vaccinia virus DNA into cells infected with ectromelia virus and assaying for MAb reactivity, we mapped the glycoprotein to the A33R open reading frame. The amino acid sequence and hydrophilicity plot predicted that the A33R gene product is a type II membrane protein with two asparagine-linked glycosylation sites. Triton X-114 partitioning experiments indicated that the A33R gene product is an integral membrane protein. The ectromelia virus homolog of the vaccinia virus A33R gene was sequenced, revealing 90% predicted amino acid identity. The vaccinia and variola virus homolog sequences predict 94% identical amino acids, the latter having one fewer internal amino acid. Electron microscopy revealed that the A33R gene product is expressed on the surface of extracellular enveloped virions but not on the intracellular mature form of virus. The conservation of this protein and its specific incorporation into viral envelopes suggest that it is important for virus dissemination.     

Obligatory requirement for antibody in recovery from a primary poxvirus infection

To understand the correlates of protective immunity against primary variola virus infection in humans, we have used the well-characterized mousepox model. This is an excellent surrogate small-animal model for smallpox in which the disease is caused by infection with the closely related orthopoxvirus, ectromelia virus. Similarities between the two infections include virus replication and transmission, aspects of pathology, and development of pock lesions. Previous studies using ectromelia virus have established critical roles for cytokines and effector functions of CD8 T cells in the control of acute stages of poxvirus infection. Here, we have used mice deficient in B cells to demonstrate that B-cell function is also obligatory for complete virus clearance and recovery of the host. In the absence of B cells, virus persists and the host succumbs to infection, despite the generation of CD8 T-cell responses. Intriguingly, transfer of naive B cells or ectromelia virus-immune serum to B-cell-deficient mice with established infection allowed these animals to clear virus and fully recover. In contrast, transfer of ectromelia virus-immune CD8 T cells was ineffective. Our data show that mice deficient in CD8 T-cell function die early in infection, whereas those deficient in B cells or antibody production die much later, indicating that B-cell function becomes critical after the effector phase of the CD8 T-cell response to infection subsides. Strikingly, our results show that antibody prevents virus from seeding the skin and forming pock lesions, which are important for virus transmission between hosts.     

The cell-mediated immune response to ectromelia virus infection. Secondary response in vitro: specificity, nature of effector and responder cells and requirements for induction of antigenic changes in stimulator cells.

An in vitro culture method was used to study secondary cell-mediated responses to ectromelia virus infection in mice. Infected, syngeneic spleen cells or peritoneal cells were efficient "stimulator" cells when cultured with "responder" cells obtained from mice infected with ectromelia 4-6 weeks previously. The kinetics of generation of cytotoxic cells in cultures were determined; a peak occurred on days 4-5. A separation procedure performed on the cytotoxic cells showed that activity was associated mainly with the Ig-negative subpopulation (T cell-rich) and that H-2 compatibility between cytotoxic cells and target cells was required. The secondary response was virus-specific, at the level of both induction and target cell lysis, at least so far as ectromelia and lymphocytic choriomeningitis (LCM) viruses are concerned. Seperation of responder cells prior to culture showed that a potent secondary response was generated with the Ig-negative (T cell-rich) subpopulation and only a weak response was observed when the responder cells were Ig-positive (rich in B cells). Infected stimulator cells did not appear to secrete significant amounts of soluble antigen into the medium over 4 days of culture. Thus, antigenic patterns effective in memory T cell stimulation may be largely associated with the surfaces of infected cells.Pretreatment of ectromelia virus with UV- or gamma-irradiation did not impair its ability to induce antigenic changes in stimulator cells. Stimulator cells treated with UV-or gamma-irradiated virus for 1 h and then immediately with pactamycin to inhibit further viral protein synthesis and replication were efficient stimulators, thus indicating that antigenic changes are induced very rapidly on the surface of stimulator cells after uptake of virus. These treatments are being used to further characterize the cellular requirements in the stimulator population.     

Detection of mouse ectromelia virus using the heterologous rabbit antiserum conjugated with fluorescein isothiocyanate

A technique for direct determination of cells infected with mouse ectromelia virus was elaborated using the indirect immunofluorescence method and heterologous rabbit antiserum against vaccinia virus. The proposed method can be performed in laboratories with simple equipment and does not require special virological laboratory.     

Preparation of monoclonal antibodies cross-reactive with orthopoxviruses and their application for direct immunofluorescence test

Variola virus (smallpox virus), vaccinia virus (VV), cowpox virus (CPV) and ectromelia virus (EV) belong to the genus Orthopoxvirus of the family Poxviridae. To establish the possible diagnosis for smallpox infection, monoclonal antibodies (MAbs) against VV and CPV were produced. The cross-reactivity of seven MAbs with cells infected with various strains of the orthopoxviruses (CPV, VV and EV) was confirmed by an immunofluorescence (IF) test and other immunological analyses. Four and three MAbs reacted with the common antigen of all poxviruses (probably NP antigen) and the antigen involved in neutralization, respectively. We developed the IF test using these MAbs. The direct IF test required only 45 min to perform. Smallpox infection is now eradicated, but it is important to prepare for the diagnosis of smallpox in an emergency. The direct IF assay using MAbs cross-reactive with orthopoxviruses is rapid, simple, specific, applicable for multiple samples, and will make it possible to screen for and detect orthopoxviruses that include variola virus with tissue impression smears from skin lesions in most laboratories or institutes.     

Chromosome mapping of Rmp-4, a gonad-dependent gene encoding host resistance to mousepox.

DBA/2 (D2) mice are susceptible and C57BL/6 (B6) mice are resistant to lethal mousepox. A congenic resistant strain, D2.B6-Rmp-4r (D2.R4), was developed by serially backcrossing male mice that survived ectromelia virus infection with D2 mice, beginning with (B6 x D2)F1 mice. Male D2.R4 mice were at least 300-fold more resistant to lethal mousepox than male D2 mice. Female D2.R4 mice were 100-fold more resistant than male D2.R4 mice and 500-fold more resistant than female D2 mice. Neonatal gonadectomy prevented development of resistance in D2.R4 mice of both sexes. Differences in resistance between strains and between sexes correlated with restriction of virus replication in spleen and liver, but gender differences were less evident in liver than in spleen. High-resolution interval mapping of the 19 autosomes of D2.R4 mice using dispersed informative microsatellites as marker loci revealed a segment of distal chromosome 1 to be of B6 origin. Haplotypes for a marker locus, D1Mit57, from the differential segment were determined in (D2.R4 x D2)F1 x D2 backcross mice, which were then infected with ectromelia virus. Significantly more heterozygotes than homozygotes survived ectromelia virus infection in both sexes. Whereas nearly all surviving males were heterozygotes, 44% of surviving females were homozygotes. These results indicate that resistance in D2.R4 mice is determined by a gonad-dependent gene on distal chromosome 1, provisionally named Rmp-4, and by an ovary-dependent factor that is not genetically linked to Rmp-4.     

Vaccinia and ectromelia recombinant viruses, causing an infection, characteristic for ectromelia, in mice

Ten recombinants between the viruses of vaccinia and ectromelia were isolated that cause the ectromelia virus specific lesions in mice. The structure of recombinant viral genomes, the efficiency of viral propagation in mice, the nature of lesions induced by viruses have been studied. Eight of obtained recombinants have a DNA insertion originating from the right end of ectromelia viral genome, nine recombinants have an insertion originating from the left end, seven recombinants possess both insertions. The latter recombinants have more pronounced pathogenicity for mice. Both revealed regions are supposed to define the specific pathogenicity of ectromelia virus for mice.     

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.     

Epithelial Immunization Induces Polyfunctional CD8+ T Cells and Optimal Mousepox Protection

We assessed several routes of immunization with vaccinia virus (VACV) in protecting mice against ectromelia virus (ECTV). By a wide margin, skin scarification provided the greatest protection. Humoral immunity and resident-memory T cells notwithstanding, several approaches revealed that circulating, memory CD8⁺ T cells primed via scarification were functionally superior and conferred enhanced virus control. Immunization via the epithelial route warrants further investigation, as it may also provide enhanced defense against other infectious agents.     

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.