Live Variola Virus: Considerations for Continuing Research (2009)

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3 Comparative Poxvirology A comprehensive discussion of variola virus must include comparison with other orthopoxviruses, as the similarities and differences between variola and members of its family help elucidate how variola causes disease and how it is modulated by subsequent host responses. In particular, variola virus’s unique adaptation to a single host (while other orthopoxviruses readily infect multiple mammalian species) and its ability to induce unusu- ally severe disease hint at a complex relationship between host and pathogen that may not be as easily explored using other orthopoxviruses. Variola virus also is not a single virus whose strains are identical, and the implications of this diversity could yield further information not just about the host, but also about mechanisms of antiviral therapeutic action, vaccine efficacy, and rapid diagnostic capabilities. Taking advantage of insights from comparative virology is particularly important because experiments with live variola virus must focus on critical questions. Knowledge of the related poxviruses can inform the design and refinement of experiments for which live variola virus is necessary. In the last decade, technological advances and the development of molecular techniques have made it possible to gain a deeper understanding of the general mechanisms involved in poxvirus replication, the host response, and the ways in which these pathogens have adapted to their hosts that is pertinent as background for considering the scientific needs for variola virus. POXVIRUS Taxonomy Viruses with shared characteristics are grouped into taxonomic cat- egories, including those of the Poxviridae family. Variola and the other 27 

28 LIVE VARIOLA VIRUS members of Poxviridae are among the largest and most complex known viruses. Their genome is a single linear, double-stranded DNA molecule between 130 and 360 kilobase pairs (kbp) in size, and encodes on aver- age approximately 150 proteins. The family Poxviridae is subdivided into two subfamilies based on the restriction of their host range to vertebrates ( ­ Chordopoxvirinae) or invertebrates (Entomopoxvirinae), and these sub- families are further subdivided into genera of viruses that are genetically related and share aspects of nucleotide composition, host range, and morphology. Chordopoxvirinae consists of eight genera: Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, and Yatapoxvirus (Moss, 2007). The Orthopoxvirus genus includes many of the known poxviruses that naturally infect mam- mals, including vaccinia, the prototypical poxvirus, as well as two of the three poxviruses that have most commonly infected humans, variola and monkeypox virus; the third, molluscum contagiosum virus, is a member of Molluscipoxvirus. Other members of Chordopoxvirinae, including cow- pox virus (genus Orthopoxvirus) and orf virus (genus Parapoxvirus, most commonly found in sheep and goats) are less common disease agents of humans. Table 3-1 lists the poxviruses that affect humans, along with their reservoir hosts, other infected hosts, and geographic distribution. POXVirus structure Poxviruses were first visualized by electron microscopy (EM) in 1938 (Biel and Gelderblom, 1999). The large virus particles (approximately 240 nm × 300 nm for orthopoxviruses) appear brick-shaped under standard EM, with internal structures resembling a dumbbell-shaped core and two lateral bodies (see Figure 3-1). From the late 1940s through the end of the smallpox eradication era, EM was used to diagnose smallpox and to differen- tiate between variola and varicella zoster virus (VZV), which causes chicken­ pox (Biel and Gelderblom, 1999). The distinctive morphology observed by EM can still be a first step in diagnosis of poxviruses. During the outbreak of monkeypox in the United States in 2003, the first realization that the etiologic agent was an orthopoxvirus occurred when brick-shaped virions were visual- ized in a clinical specimen by EM (Reed et al., 2004). However, it is important to recognize that the orthopoxviruses that infect humans, including variola, vaccinia, and monkeypox, cannot be differentiated by traditional EM alone because the virion structure is highly conserved among orthopoxviruses. POXVirus genomics Advances in genomic sequencing and computational molecular biology have provided new insights into the relatedness and evolutionary history 

COMPARATIVE POXVIROLOGY 29 TABLE 3-1  Poxviruses That Infect Humans Reservoir Other Infected Geographic Genus Virus Hosts Hosts Distribution Orthopoxvirus Cowpox Bank voles, Humans, cats, Europe, western long-tailed field cattle, zoo Africa mice animals Monkeypox Unknown, Humans, Western and likely rodents monkeys, zoo central Africa animals, prairie dogs Vaccinia ? Humans, ? rabbits, cattle, river buffalo Variola Humans None Eradicated (formerly worldwide) Parapoxvirus Bovine papular Cattle (beef) Humans Worldwide stomatitis Orf Sheep, goats Humans, Worldwide ruminants Pseudocowpox Cattle (dairy) Humans Worldwide Sealpox Seals Humans Worldwide Yatapoxvirus Tanapox Humans Eastern and central Africa Yabapox ? Primates Humans Western Africa Molluscipoxvirus Molluscum Humans None Worldwide contagiosum of poxviruses. This information helps place variola in its evolutionary con- text and points to significant genetic differences between variola and other orthopoxviruses. Poxvirus genes are usually nonoverlapping but closely spaced, and are arranged in blocks such that genes in the outer quadrants of the genome are transcribed toward the end of the genome in closest proximity, while genes in the central quadrants are transcribed toward the center of the genome. An analysis of 21 Poxviridae complete genome sequences in 2003 revealed a common set of 49 genes and an additional 41 genes shared by the chordopoxviruses (Upton et al., 2003). These families of shared genes encode proteins involved in basic functions such as DNA replication, transcription, and virion assembly, and are located toward the central region of the genome. In contrast, genes that are virus- or host- specific tend to be located toward the genome termini, and encode factors 

30 LIVE VARIOLA VIRUS FIGURE 3-1  Electron microscopy of orthopoxvirus structure. 

COMPARATIVE POXVIROLOGY 31 involved in subversion of host defenses and immune responses. These v ­ irulence-associated genes are believed to have been acquired more recently by the virus as it adapted to the host species (Lefkowitz et al., 2006). To date, all poxvirus genomes that have been studied have been found to have inverted terminal repeats (ITRs) at both ends of the genome (Garon et al., 1978; see Figure 3-2). Contemporary taxonomic approaches combine sequence-based phylo­ genetic and character trait analyses. This is the case for the poxviruses (Lefkowitz et al., 2006). Alignments of concatenated orthologous pro- tein sequences from the poxviruses have led to reconsideration of genus assignments for some members and genus interrelationships. Gene loss, fragmentation, and duplication all appear to have played important roles in poxvirus evolution, with subsequent restriction of virus host range. Complete genome sequences and comparative analysis have suggested the basis for differences in virulence among strains of the same orthopoxvirus species, as illustrated by monkeypox virus (MPXV). In recent years, it has become increasingly well recognized that west African strains of MPXV are less virulent than central African (Congo basin) strains, despite roughly similar degrees of host exposure in these two regions of the continent. The genome sequences of three west African MPXV strains were found to be more closely related to each other (0.01–0.07 percent difference) than to the previously sequenced strain from the Democratic Republic of the Congo (0.55–0.56 percent difference) (Chen et al., 2005). Of note, five putative virulence-associated genes contained significant deletions or fragmenta- Central conserved region (essential functions for viral replication) Variable left end Variable right end ITR ITR ~ 200,000 base pairs ~ 200 genes FIGURE 3-2  Internal terminal repeats at both ends of the poxvirus genome. Figure 3-2 

32 LIVE VARIOLA VIRUS tion in the three west African strains as compared with the central African strain, including the gene encoding the MPXV ortholog of the vaccinia complement-binding protein (VCP-MPXV). On the basis of this sequence analysis and subsequent assessment of the expressed protein, VCP-MPXV is hypothesized to play a key role in the virulence of west African MPXV strains. More genomic information about variola virus isolates, coupled with clinical data on disease severity in the cases from which the virus was recovered, has the potential to elucidate factors influencing the virulence of variola. VirAL life cycle Variola virus, like all orthopoxviruses, replicates solely in the cyto- plasm of infected cells (see Figure 3-3). Most of what is known about the orthopoxvirus life cycle has been learned from extensive study of vaccinia virus (Moss, 2007), which is closely related to variola virus. Vaccinia has been the prototypic model for experimental analysis of the orthopox viral life cycle in vitro. Not only is analysis of the vaccinia life cycle relevant for understanding variola, but because smallpox vaccines are made from vac- cinia, this information can be used to design safer alternatives. There are several key points to be kept in mind as the viral life cycle is reviewed. First, progression through this life cycle involves the action of a significant number of virally encoded products, which are largely conserved throughout the Orthopoxvirus genus but do exhibit strain-specific varia- tions. Subtle features of the variola proteome are likely to be important in this regard. Second, there is increasing evidence—particularly for the pro- cess of transcription—that host proteins participate in the viral life cycle. This participation of host proteins is likely to be a significant determinant of species specificity and may contribute to the narrow host range of variola. Third, the progression of the viral life cycle occurs within the context of the cell, and there is growing evidence that intracellular structures (e.g., cytoskeleton, membranes) are vital for efficient viral replication. How viral proteins interact with these cellular structures is likely to be strain- and species-specific, and this is a relatively understudied area. Fourth, the viral proteins that mediate the progression of the viral life cycle represent the key pool of targets for antiviral therapy. Viral Entry There is good evidence that mature virions (MV) form weak attach- ments with glycosaminoglycans and laminins on the cell surface; various proteins within the virion membrane have been shown to be responsible for these interactions ������������������������������������������������������� (Chung et al., 1998; Carter et al., 2005; Chiu et al., 

core envelope Cytoplasm DNA Attachment Entry Uncoating DNA replication enzymes Transcription Factors WV Nucleus DNA packaging Concatamer resolution EV Wrapping Maturation MV IV Crescents Golgi bodies Virus Factory 33 FIGURE 3-3  Orthopoxvirus replication. Figure 3-3 REV.eps broadside 

34 LIVE VARIOLA VIRUS 2007). It is not yet known whether other viral proteins interact strongly or ���������������������������������������������������������������������� specifically with proteinaceous receptors on the plasma membrane. If there are such receptors, however, they must be ubiquitous, since there have been no reports of cell lines that cannot support viral binding or entry. Once bound, virions can enter cells either by direct fusion of the viral and plasma membranes or by uptake of intact virus via macropinocytosis, with subse- quent release of the core from the endosomal compartment (Moss, 2006; Townsley et al., 2006; Mercer and Helenius, 2008) into the cytoplasm. The predominance of one mode of entry or the other differs with different viral strains and cell types, although the determinants of this variability have not been identified. A minority of the virions produced during poxvirus infection mature into enveloped or extracellular virions, also known as EV. EV are MV surrounded by an additional lipid bilayer carrying EV-specific surface pro- teins (Smith and Law, 2004). When EV attach to target cells, this exterior membrane is ruptured in a process known as ligand-dependent dissolution, and the MV found within then enter cells as described above (see also the discussion of morphogenesis and egress below). Gene Expression After delivery into the cytoplasm, the core of the poxvirus particle remains intact, and early gene expression begins (Broyles, 2003; Moss, 2007). Approximately 50 percent of the viral genes are expressed from the genome using the virally encoded and encapsidated transcriptional machinery. This machinery includes a multisubunit RNA polymerase, an RNA Pol accessory protein, an early transcription factor, a capping enzyme and cap modification enzyme, termination and transcript release proteins, and poly A polymerase. This phase in the viral life cycle yields a number of potential targets for antiviral drugs that may be conserved in variola and related poxviruses. The mature transcripts are released from the core and undergo translation on host cell polysomes. The initiation of two subsequent phases of poxvirus gene expression requires the prior onset of DNA replication (see below). Although the mechanism for this dependency is not known, DNA replication has been shown to have a cis-acting effect on the encapsidated viral genome that enables intermediate and late transcription to occur. Intermediate gene expression utilizes viral transcription factors that are expressed as early pro- teins, as well as some host proteins. Late gene expression also uses distinct viral transcription factors, as well as some host factors. Two features that distinguish these postreplicative phases of gene expression are the presence of 5′ polyA heads on the transcripts and imprecise termination of transcrip- tion; the latter feature leads to the presence of long overlapping transcripts, 

COMPARATIVE POXVIROLOGY 35 which leads in turn to the presence of dsRNA. The availability of distinct classes of viral mRNA at distinct times after infection is also enhanced by the expression of viral decapping enzymes, which accelerate mRNA turn- over (Parrish and Moss, 2007; Parrish et al., 2007). The cytoplasmic replication cycle of poxviruses means that both transcrip­tion and translation occur in the cytoplasm and can be both tem- porally and physically coupled. Indeed, postreplicative transcription and translation occur within distinct areas of the cytoplasm known as viral factories (Condit, 2007; Katsafanas and Moss, 2007). This compartmen- talization serves to enhance viral protein expression as well as to diminish cellular protein expression, since the translational machinery is depleted in the cytoplasmic areas outside of the viral factories. Genome Replication and Maturation After early gene expression, the core appears to disassemble and the genome is released into the cytoplasm, where it undergoes replication in the viral factories. The proteins that make up the viral replication machinery, all of which are conserved in variola, are expressed early after infection. They include a catalytic DNA polymerase, a dimeric processivity factor (one subunit of which is an enzymatically active uracil DNA glycosylase), an ssDNA binding protein, and a primase/helicase (Moss and De Silva, 2006). A virally encoded protein kinase is also essential for replication; its primary role appears to be to phosphorylate and thus overcome the inhibi- tory action of a cytoplasmic DNA sensor. Other accessory proteins that may be dispensable in tissue culture but essential in vivo are also encoded by the viral genome. The mechanism by which replication initiates is still in question, and there is as yet no clear answer as to whether replication involves only leading strand synthesis or both leading and lagging strand synthesis. Replication does lead to the synthesis of tail/tail concatemers of the genome, which undergo subsequent resolution to mature monomeric genomes. This resolution is accomplished by a virally encoded Holliday junction resolvase and leads to reformation of the unusual telomeres of the viral genome, which are incompletely base-paired hairpins with an A+T content of >95 percent. Replication is robust and occurs from ~3 to 12 hours post-infection, leading to amounts of viral DNA that are estimated to approximate one-third the amount of the cellular DNA content. Morphogenesis and Egress As structural proteins and progeny become available, morphogenesis of nascent virions commences in the cytoplasm (Condit et al., 2006). ­Electron- dense areas of proteins destined for encapsidation are among the first 

36 LIVE VARIOLA VIRUS hallmarks of morphogenesis. The appearance of membrane crescents (or cupules in three dimensions) at the periphery of these electron-dense areas is the first sign of membrane biogenesis. As these crescents enlarge and sur- round proteins destined to form the virion core, the curvature of a protein lattice that forms on their external face helps determine the size and shape of the immature virions (IV). Genome encapsidation is associated with the appearance of a nucleoid within these IV (forming IVN). Maturation of these IVN into infectious MV is accompanied by proteolytic processing of the major core proteins. This maturation is also accompanied by the transition of the oval IV to brick-shaped MV that have a characteristic dumbbell core. The majority of the MV remain within the cell as long as the cell remains intact. A minority, however, are transported to the Golgi ­apparatus (or endosomal compartment), where they become wrapped with two addi- tional lipid bilayers (forming wrapped virions, or WV). These outer enve- lopes contain a group of distinctive viral proteins that are not found in MV. WV traffic to the plasma membrane, where their outermost lipid bilayer fuses with the plasma membrane. This fusion leads to the exocytotic release of EV, which can disassociate from the plasma membrane and mediate dis- tal spread. Alternatively, some EV remain bound to the plasma membrane at the site of egress. The viral proteins that were delivered to the subjacent plasma membrane during fusion activate the formation of actin tails, which then propel the attached EV toward neighboring cells, facilitating efficient proximal spread of the virus. Host specificity and range As noted earlier, humans are the sole host for variola and molluscum contagiosum viruses, and the success of the WHO-led smallpox eradication program was achievable because variola virus has no animal reservoir. In general, poxvirus infections in vertebrate hosts show species ­ specificity; however, zoonotic infections do occasionally occur. The underlying mech- anism for host tropism, which is determined largely by host–pathogen interaction at many levels, is not well understood (McFadden, 2005). The reservoir hosts for monkeypox are rodents and squirrels, but this virus can occasionally cross the species barrier to infect monkeys and humans (Di Giulio and Eckburg, 2004). Poxviruses can bind to and enter a wide range of mammalian cells, but their success in replicating may vary. The ability of poxviruses to replicate and complete their viral life cycle in cells is dependent on many host-related factors, including cell type and species origin, cell cycle status, and intracel- lular signaling events leading to antiviral innate immunity and apoptosis. Identification of host range genes and elucidation of their interactions with 

COMPARATIVE POXVIROLOGY 37 host proteins and signaling pathways have shed light on poxvirus host tropism. For example, vaccinia E3L and K3L are host range genes target- ing interferon (IFN)-inducible dsRNA-dependent protein kinase R (PKR) (Langland and Jacobs, 2002). E3L functions by sequestering dsRNA and preventing the activation of PKR, whereas K3L mimics eIF2α and acts as a pseudosubstrate for PKR, preventing phosphorylation of eIF2α and thus inhibition of protein synthesis. Suppression of PKR expression in a non- permissive cell line for an E3L knockout virus (∆E3L) restores viral protein synthesis and viral replication. Viral-induced apoptosis is blocked in PKR- deficient cells as well (Zhang et al., 2008). E3L also blocks the activation of another IFN-inducible protein, 2′–5′ oligoadenylate synthetase (2′–5′ OAS), and the subsequent activation of ribonuclease RNaseL (Rivas et al., 1998). The C-terminal dsRNA-binding domain of E3L is required for host range as well as pathogenesis. The N-terminal Z-DNA-binding domain of E3L is not required for host range, but it is required for pathogenesis in mice (Brandt and Jacobs, 2001). Vaccinia K1L gene is an ankyrin-repeat containing host range protein that is involved in inhibiting IκBα degradation, which prevents activation of the host defense mechanisms of the NFkB pathway. The importance of the capacity to block the host cell response is demonstrated by the arrest of replication of vaccinia mutant strains lacking K1L at the stage of inter­ mediate gene transcription in Chinese hamster ovary cells. The conservation of this function, and hence its likely importance to other orthopoxviruses, was shown by the rescue of this mutant by expression of another ankyrin- repeat containing host range protein from cowpox, CP77 (Ramsey-Ewing and Moss, 1996). Another ankyrin-repeat host range protein is M-T5 of myxoma virus, whose deletion leads to an inability to replicate in rabbit T lymphocytes and reduced virulence in European rabbits (Mossman et al., 1996). M-T5 is required for myxoma replication in certain human tumor cells (Sypula et al., 2004). Induction of IFN production has been shown to contribute to maintaining the species barrier for myxoma virus (Wang et al., 2004). C7L is another host range gene, which may be functionally equiva- lent to K1L. Infection with vaccinia mutant lacking both K1L and C7L is nonpermissive in human and murine cells (Perkus et al., 1990; Oguiura et al., 1993). C7L homologues are present in the genomes of almost all mammallian poxviruses. A recent study by Meng and colleagues (2008) demonstrated that vaccinia C7L homologue, myxoma M62R, or yaba-like disease virus 67R, when reconstructed in a vaccinia mutant lacking K1L and C7L, restored the vaccinia mutant’s ability to replicate in human and murine cells, possibly by suppressing PKR activation. MVA and NYVAC are two candidates for third-generation vaccines against smallpox that have alterations in immunomodulatory and host 

38 LIVE VARIOLA VIRUS range genes. MVA is derived from more than 500 serial passages of the vac- cinia virus Ankara in chicken embryo fibroblasts (CEF); as a consequence, it lacks 31 kilobases of its genome (Antoine et al., 1998) (see Chapter 7). MVA does not replicate in most mammalian cells. The exceptions are BHK-21 cells and CEF. NYVAC is generated by deletion of 18 nonessential genes implicated in virulence or host range. It replicates in CEF, Vero cells, and BHK-21 cells (Tartaglia et al., 1992). MVA lacks K1L but retains C7L, whereas NYVAC lacks both K1L and C7L. In Hela cells, the MVA life cycle is blocked at a late stage of viral infection, probably at the assembly of immature virions (Sutter and Moss, 1992; Sancho et al., 2002). MVA with deletion of E3L still replicates in BHK-21 cells, but fails to replicate in CEF. Whereas MVA infection in Hela cells reveals a complete cascade of viral early, intermediate, and late gene transcription, MVA-∆E3L infection produces only early and intermediate transcripts. It is related to the induc- tion of 2′–5′ OAS/RNaseL and PKR (Ludwig et al., 2005, 2006). NYVAC induces apoptosis in Hela cells, which can be prevented through introduc- tion of the C7L gene (Nájera et al., 2006). Host–pathogen interactions Immune Modulation Poxviruses, including variola, encode many genes that are known or predicted to modulate host antiviral responses (Seet et al., 2003). These include secreted viral proteins that bind cytokines, chemokines, and com- plement proteins, as well as intracellular antagonists that block key signal- ing pathways leading to establishment of an antiviral state, apoptosis, or proinflammatory responses. Over the last decade, significant advances have been made in the understanding of poxvirus–host interactions and viral immune modulatory genes. Antiviral innate immunity is critical for the host to contain a viral infection initially and to activate the adaptive immune responses that result in viral clearance. Over the last several years, a number of viral-sensing pathways have been discovered in the host cell, including toll-like recep- tors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors, nucleo- tide oligomerization domain (NOD)-like receptors (NLRs), and possibly c ­ ytosolic DNA sensors to detect viral nucleic acids and other components (Akira et al., 2006). The induction of type I IFN and proinflammatory cytokines and chemokines in various cell types in response to viral patho- gens, including dendritic cells, macrophages, epithelial cells, and fibroblasts, leads to the further recruitment of other immune cells and the development of adaptive immunity. Both functional type I and type II IFN systems are required for protection against vaccinia infection. For example, mice that 

COMPARATIVE POXVIROLOGY 39 have targeted deletions in type I and type II IFN receptors show increased susceptibility to vaccinia infection (Müller et al., 1994). Although not studied for variola, IFNs can be predicted to be critical for the outcome of smallpox based on their fundamental contributions in other systemic viral infections. Through interactions with their hosts, poxviruses have evolved to p ­ ossess many mechanisms for antagonizing the production and actions of IFNs and proinflammatory cytokines and chemokines. Since the capacity of variola to block these responses is likely to be a key factor in its exceptional virulence for the human host, information about these mechanisms in other orthopoxviruses can be helpful in framing scientific questions about their contribution to the capacity of variola to overwhelm the human host. As an example of such mechanisms, many poxviruses encode IFNα/β-binding proteins and IFN-γ receptor homologs that dampen the effects of these molecules that are involved in countering viral replication and spread within the host (Upton et al., 1991; Symons et al., 1995). Specifically, the vaccinia virus (Western Reserve) B18R gene encodes for an IFNα/β-binding protein and is critical for virulence, as B18R knockout virus is attenuated in both murine intranasal and intracranial infection models (Symons et al., 1995). Similarly, ectromelia virus expresses functional homologs of IFN-γR and IFN-α/βR (Smith and Alcami, 2002). Recombinant ectromelia virus with deletion of the gene encoding type I IFN-binding protein was attenu- ated more than 107-fold compared with wild-type ectromelia virus (Xu et al., 2008). Variola virus also encodes homologs of IFN-γR and IFN-α/βR (Esposito et al., 2006; Li et al., 2007). Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates inflammation and apoptosis within the host. Many poxviruses, including variola virus, encode TNF receptor homologs (TNFRs), which block the host TNF signaling pathway. Whether they do so by binding TNF or by preventing the oligomerization of TNFR through their preligand assem- bly domains (PLADs) remains controversial (Alejo et al., 2006; Sedger et al., 2006). Myxoma virus lacking the TNF receptor homolog M-T2 has decreased virulence compared with wild-type myxoma (Upton et al., 1991). Whereas cowpox encodes four viral TNFRs, including CrmB (cytokine response modifier B), CrmC, CrmD, and CrmE, variola virus encodes one CrmB-like protein. In addition to its ability to bind to TNF, the variola CrmB-like protein is capable of binding to everal chemokines (Alejo et al., 2006). Poxviruses encode intracellular inhibitors that block caspase 1 ­activity, as well as IL-1β receptor homolog and IL-18-binding protein. IL-1β is another major cytokine mediating acute and chronic inflammation in response to infection and injury. It is produced through processing of its inactive precursor, proIL-1β, by caspase 1 to its active form, p17. Activa- 

40 LIVE VARIOLA VIRUS tion of caspase 1 is dependent on activation of a multimolecular complex termed an inflammasome, which can be triggered by various stimuli, includ- ing cytosolic bacterial infections, ATP, and alum. IL-18 is another potent inflammatory cytokine released as a result of the activation of caspase I. Vaccinia virus WR B15 encodes an IL-1β-binding protein. Infection of mice with a B15R deletion mutant induced fever, whereas wild-type vaccinia infection suppressed fever, a host response controlled by the presence of IL-1β (Alcami and Smith, 1996). Both variola virus and monkeypox encode CrmA that inhibits caspase 1, an IL-1β receptor, and an IL-18-binding pro- tein (Seet et al., 2003). Myxoma virus attenuates inflammasome activation through an early viral gene, M13L, which interacts with a critical compo- nent of the inflammasome adapter protein, ASC (Johnston et al., 2005). Poxviruses utilize multiple strategies to evade the chemokine system by encoding chemokine-binding proteins, chemokine receptor homologs, and chemokine receptor antagonists. Chemokines are a large family of small, secreted proteins that mediate the recruitment of immune cells to the sites of injury or infection. Chemokines can be divided into several categories depending on the number and position of the highly conserved cysteine residues near the N-terminus of the protein. The two major chemokine subfamilies are CC and CXC, and the two minor subfamilies are C and CX3C. Chemokines perform their functions by binding to their cognate seven transmembrane G-protein-coupled receptors on the surface of cells (Mantovani et al., 2006). Some chemokines have direct antiviral activ- ity (Nakayama et al., 2006). Myxoma virus M-T7 encodes a low-affinity c ­ hemokine-binding protein that binds not only IFN‑γ, but also a wide range of CXC, CC, and C chemokines. Myxoma M-T1 and vaccinia B29R encode high-affinity ­chemokine-binding proteins that bind to CC chemokine, but not to C, CXC, or CX3C chemokines (Graham et al., 1997; Alcami et al., 1998). Variola virus and monkeypox virus also have high-affinity c ­ hemokine-­binding proteins (Alejo et al., 2006; Jones et al., 2008). The complement system is required for successful host defense against poxviruses. Moulton and colleagues (2008) recently showed that mice deficient in complement are more susceptible to ectromelia (mousepox) infection. Variola, monkeypox, ectromelia, and vaccinia viruses encode complement-binding proteins that block both the classical and alternative pathways of complement activation (Kotwal and Moss, 1988; Rosengard et al., 2002; Liszewski et al., 2006; Parker et al., 2008). Poxviruses also produce intracellular inhibitors to block the induction of type I IFN and proinflammatory cytokines and chemokines. Vaccinia virus A46R and A52R contain a Toll/IL-1 receptor (TIR) domain that can block the recruitment of adaptor molecules to IL-1R or TLR, preventing signaling following ligand–receptor interactions (Bowie et al., 2000). A52R has been found to block multiple TLRs through association with IRAK2 

COMPARATIVE POXVIROLOGY 41 and TRAF6. Vaccinia mutant strain ∆A52R is attenuated in a murine intranasal model of infection (Harte et al., 2003). Recently, Vaccinia K7 was found to inhibit TBK1/IKKε-mediated IRF activation induced by TLR and non-TLR pathways (Schröder et al., 2008). Vaccinia E3L encodes a dsRNA-binding protein that can inhibit IRF3, IRF7, and NF-κB pathways, in addition to IFN-inducible genes (Smith et al., 2001; Xiang et al., 2002; Deng et al., 2006; Langland et al., 2006; Guerra et al., 2008) that would otherwise be activated following vaccinia infection. E3L also functions to subvert cytosolic RNA-sensing pathway mediated by MAVS and IRF3 in keratinocytes (DiPerna et al., 2004; Deng et al., 2008). Vaccinia N1L and K1L have been shown to block NF-κB signaling (Shisler and Jin, 2004). In addition, vaccinia VH1 gene encodes a phosphatase that blocks the activa- tion of IFN-induced activation of STAT-1 (Najarro et al., 2001). Apoptosis is an effective host mechanism for containing viral infections through programmed death of infected cells. Poxviruses have evolved to evade this defense mechanism through encoding of anti-apoptotic mol- ecules. The IFN-inducible PKR promotes apoptosis through inhibition of protein synthesis, which can be counteracted by vaccinia E3L and K3L (Chang et al., 1992); variola virus encodes E3L and K3L homologs. Myx- oma M11L, a virulence gene, encodes a mitochondria-targeted molecule that prevents apoptosis (Everett et al., 2000), while molluscum contagiosum virus encodes two genes, MC159 and MC160, to block the activation of initiator caspase, caspase 8 (Shisler and Moss, 2001). CrmA, a member of the serine protease inhibitor family, first identified as an inhibitor for IL-1β- converting enzyme (caspase 1), also inhibits caspase 8 and blocks apoptosis induced by various factors (Tewari and Dixit, 1995). Adaptive Immune Response Smallpox was eradicated prior to the development of modern quantita- tive cellular assays that measure virus-specific T cell numbers and function. Likewise, because of the conditions associated with smallpox outbreaks, monitoring humoral immune responses was usually difficult. Although neutralizing antibodies were measured, other more rapid and quantitative approaches, such as enzyme-linked immunosorbent assays (ELISAs), were not well established. As a result, knowledge about smallpox immunity is limited, and concepts of smallpox immunity are based on indirect information that is available about the kinetics, magnitude, and duration of ­orthopoxvirus- specific immunity derived from analysis of vaccinia-specific T cell and anti- body responses elicited by immunization with vaccinia virus. Since vaccinia replication is well controlled while variola is often life-­threatening, how well the host response to vaccinia mimics that induced by variola is not clear (see also Chapter 7). Nevertheless, these vaccinia responses define a protective 

42 LIVE VARIOLA VIRUS cross-reactive response that is relevant for vaccine design and may suggest the characteristics of those responses that protected smallpox survivors from reinfection. Following vaccinia inoculation, antiviral T cell responses are difficult to detect at 1 week, but then rapidly expand and peak within approximately 2 weeks of infection (Miller et al., 2008). Antiviral antibody responses are slightly delayed in comparison with the T cell responses and generally peak within 2–3 weeks of inoculation. After the vaccinia lesion at the site of inoculation has resolved, antiviral T cell and antibody responses decline rapidly before reaching a more long-lived plateau phase in which immunological memory is maintained for decades. During this memory phase, vaccinia-specific CD4+ and CD8+ T cell responses decline slowly, with an estimated half-life of 8–15 years (Crotty et al., 2003; Hammarlund et al., 2003), whereas antiviral antibody responses are more stable, with an estimated half-life of 92 years (Amanna et al., 2007). No specific immunological correlate predicts protection against small- pox or any of the other orthopoxviruses. Animal model experiments indi- cate that the induction of immunity to proteins present in the EV form is essential for complete protection against challenge (Kaufman et al., 2008). Results of studies in nonhuman primates indicate that vaccine-mediated immunity against lethal monkeypox challenge is due to the presence of neu- tralizing antibodies (Edghill-Smith et al., 2005). Earlier studies in humans also demonstrated that smallpox patients who developed higher antibody responses during acute smallpox infection had a lower mortality rate than those who mounted weaker antibody responses (Slifka, 2004). However, these individuals could also have had a poor virus-specific T cell response. Administration of high-dose convalescent serum to smallpox patients appeared to protect against lethal smallpox infection in uncontrolled clini- cal studies (Slifka, 2004). It is likely that preexisting antiviral antibodies provide a first line of protection against infection with orthopoxviruses, whereas antiviral T cells, along with inhibitory antibodies, are needed if the virus overcomes this barrier and gains entry into the host. During primary infection, effective induction of both T cell and B cell responses is probably necessary to prevent the virus from causing fatal complications and to clear infectious virus from the host (Slifka, 2004). References Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124:783–801. Alcami, A., and G. L. Smith. 1996. A mechanism for the inhibition of fever by a virus. Proceedings of the National Academy of Sciences of the United States of America 93:11029–11034. 

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