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Zoonotic monkeypox virus is maintained in a large number of rodent and, to a lesser extent, nonhuman primate species in West and central Africa. Although monkeypox virus was discovered in 1958, the prototypic human cases were not witnessed until the early 1970s. Before this time, it is assumed that infections were masked by smallpox, which was then widely endemic. Nevertheless, since the 1970s, reported monkeypox virus infections of humans have escalated, as have outbreaks with reported human-to-human transmission. This increase is likely due to numerous factors, such as enhanced surveillance efforts, environmental degradation and human urbanization of areas where monkeypox virus is maintained in its animal reservoir(s) and, consequently, serve as a nidus for human infection. Furthermore, viral genetic predispositions enable monkeypox virus to infect many animal species, represented in expansive geographic ranges. Monkeypox virus was once restricted to specific regions of Africa, but its environ has expanded, in one case intercontinentally – suggesting that human monkeypox infections could continue to intensify. As a zoonotic agent, monkeypox virus is far less sensitive to typical eradication measures since it is maintained in wild-animal populations. Moreover, human vaccination is becoming a less viable option to control poxvirus infections in today’s increasingly immunocompromised population, particularly with the emergence of HIV in Sub-Saharan Africa. An increased frequency of human monkeypox virus infections, especially in immunocompromised individuals, may permit monkeypox virus to evolve and maintain itself independently in human populations.

Ectromelia virus (ECTV), the causative agent of mousepox, expresses an extracellular interferon-gamma binding protein (IFN-gammaBP) with homology to the ligand binding domains of the IFN-gamma high affinity receptor (IFN-gammaR1). Unlike the cellular receptor, the IFN-gammaBP binds IFN-gamma from several species. The IFN-gammaBP is synthesized early after infection, accumulating in the extracellular milieu as dimers composed of two protein species with Mr of 34.6 or 33.0 kDa. Homodimers are covalently linked by an interchain disulphide bond at position 216. The IFN-gammaBP has complex N-linked oligosaccharides at positions 41 and 149 as determined by site-directed mutagenesis and glycosidase treatment. Glycosylation at position 41 is required for secretion from mammalian cells and may play a role in the activity of the IFN-gammaBP. Glycosylation at position 149 is not required for secretion, and the lack of glycosylation at this site does not diminish ligand binding as measured by surface plasmon resonance (SPR) and ELISA.

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-γ) receptor 1. We generated an IFN-γ binding protein (IFN-γbp) deletion mutant of ECTV to study the role of viral IFN-γbp (vIFN-γbp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-γbp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-γbp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbpΔ, produced increased levels of IFN-γ and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-γ and the virus' ability to dampen its effects.

Gamma interferon (IFN-γ) production is important in the host response to, and recovery from, infection with Ectromelia virus (ECTV) and Vaccinia virus (VACV). The orthopoxviruses have evolved several mechanisms to subvert the IFN-γ response. IFN-γ-binding protein (IFN-γBP) is a virally encoded homologue of the host IFN-γ receptor that blocks the effects of IFN-γ in the infected host. Unlike the cellular receptors, whose ligand specificity is restricted to their own species, the orthopoxvirus IFN-γBPs bind IFN-γ from several species. The reason for this relaxed specificity has yet to be explained. ECTV, a mouse pathogen, encodes an IFN-γBP that has been shown to inhibit the activity of both human and murine IFN-γ (hIFN-γ and mIFN-γ, respectively). In contrast, the IFN-γBP from VACV is unable to inhibit mIFN-γ, but retains activity against hIFN-γ. To determine which region(s) in the ECTV sequence is responsible for its ability to bind to mIFN-γ with high affinity, a series of chimeric IFN-γBPs, as well as individual point mutants in the ECTV sequence corresponding to the amino acid changes from the VACV sequence, were constructed. The affinities of the chimeric and point mutant IFN-γBPs for mIFN-γ were tested by using surface plasmon resonance and bioassay. By using this strategy, several key residues in the ligand-binding domains of the ECTV sequence have been identified that are responsible for high-affinity binding to mIFN-γ. Substitution of the ECTV residue at these positions in VACV resulted in a dramatic increase in the affinity of the VACV IFN-γBP for mIFN-γ.

Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg2+ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1–5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3′-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.

The orthopoxviruses ectromelia virus (ECTV) and vaccinia virus (VACV) express secreted gamma interferon binding proteins (IFN-gammaBPs) with homology to the ligand binding domains of the host's IFN-gamma receptor (IFN-gammaR1). Homology between these proteins is limited to the extracellular portions of the IFN-gammaR1 and the first approximately 200 amino acids of the IFN-gammaBPs. The remaining 60 amino acids at the C termini of the IFN-gammaBPs contain a single cysteine residue shown to be important in covalent dimerization of the secreted proteins. The function of the remaining C-terminal domain (CTD) has remained elusive, yet this region is conserved within all orthopoxvirus IFN-gammaBPs. Using a series of C-terminal deletion constructs, we have determined that the CTD is essential for IFN-gamma binding despite having no predicted homology to the IFN-gammaR1. Truncation of the ECTV IFN-gammaBP by more than two amino acid residues results in a complete loss of binding activity for both murine IFN-gamma and human IFN-gamma (hIFN-gamma), as measured by surface plasmon resonance (SPR) and bioassay. Equivalent truncation of the VACV IFN-gammaBP resulted in comparable loss of hIFN-gamma binding activity by SPR. Full-length IFN-gammaBPs were observed to form higher-ordered structures larger than the previously reported dimers. Mutants that were unable to bind IFN-gamma with high affinity in SPR experiments failed to assemble into these higher-ordered structures and migrated as dimers. We conclude that the unique CTD of orthopoxvirus IFN-gammaBPs is important for the assembly of covalent homodimers as well as the assembly of higher-ordered structures essential for IFN-gamma binding.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative, semi-quantitative approach described here, uses an immunological capture step and a mass spectral readout. The goal of the assay is speed rather than sensitivity or precision.

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