African swine fever virus transmembrane protein pEP84R guides core assembly.

African swine fever virus (ASFV) causes a devastating hemorrhagic disease with worldwide circulation and no widely available therapeutic prevention. The infectious particle has a multilayered architecture that is articulated upon an endoplasmic reticulum (ER)-derived inner envelope. This membrane acts as docking platform for the assembly of the outer icosahedral capsid and the underlying core shell, a bridging layer required for the formation of the central genome-containing nucleoid. While the details of outer capsid assembly are relatively well understood, those of core formation remain unclear. Here we report the functional characterization of pEP84R, a transmembrane polypeptide embedded in the inner envelope that surrounds the viral core. Using an ASFV recombinant inducibly expressing the EP84R gene, we show that absence of pEP84R results in the formation of non-infectious core-less icosahedral particles displaying a significant DNA-packaging defect. Concomitantly, aberrant core shell-like structures formed by co-assembly of viral polyproteins pp220 and pp62 are mistargeted to non-ER membranes, as also occurs when these are co-expressed in the absence of other viral proteins. Interestingly, co-expression of both polyproteins with pEP84R led to the formation of ER-targeted core shell-like assemblies and co-immunoprecipitation assays showed that pEP84R binds to the N-terminal region of pp220. Altogether, these results indicate that pEP84R plays a crucial role in core assembly by targeting the core shell polyproteins to the inner viral envelope, which enables subsequent genome packaging and nucleoid formation. These findings unveil a key regulatory mechanism for ASFV morphogenesis and identify a relevant novel target for the development of therapeutic tools against this re-emerging threat.

An Adenovirus Vector Expressing FMDV RNA Polymerase Combined with a Chimeric VLP Harboring a Neutralizing Epitope as a Prime Boost Strategy to Induce FMDV-Specific Humoral and Cellular Responses.

Foot and mouth disease is a highly contagious disease affecting cattle, sheep, and swine among other cloven-hoofed animals that imposes serious economic burden by its direct effects on farm productivity as well as on commerce of farmed produce. Vaccination using inactivated viral strains of the different serotypes is an effective protective measure, but has several drawbacks including a lack of cross protection and the perils associated with the large-scale growth of infectious virus. We have previously developed chimeric virus-like particles (VLPs) bearing an FMDV epitope which induced strong specific humoral responses in vaccinated pigs but conferred only partial protection against homologous challenge. While this and other FMD vaccines under development mostly rely on the induction of neutralizing responses, it is thought that induction of specific T-cell responses might improve both cross protective efficacy as well as duration of immunity. Therefore, we here describe the development of a recombinant adenovirus expressing the highly conserved nonstructural FMDV 3D protein as well as its capacity to induce specific T-cell responses in a murine model. We further describe the generation of an FMDV serotype C-specific chimeric VLP and analyze the immunogenicity of two different prime-boost strategies combining both elements in mice. This combination can effectively induce both humoral and cellular FMDV-specific responses eliciting high titers of ELISA and neutralizing antibodies anti-FMDV as well as a high frequency of IFNγ-secreting cells. These results provide the basis for further testing of this anti FMD vaccination strategy in cattle or pig, two of the most relevant natural host of this pathogen.

Poxvirus-encoded TNF receptor homolog dampens inflammation and protects from uncontrolled lung pathology during respiratory infection.

Ectromelia virus (ECTV) causes mousepox, a surrogate mouse model for smallpox caused by variola virus in humans. Both orthopoxviruses encode tumor necrosis factor receptor (TNFR) homologs or viral TNFR (vTNFR). These homologs are termed cytokine response modifier (Crm) proteins, containing a TNF-binding domain and a chemokine-binding domain called smallpox virus-encoded chemokine receptor (SECRET) domain. ECTV encodes one vTNFR known as CrmD. Infection of ECTV-resistant C57BL/6 mice with a CrmD deletion mutant virus resulted in uniform mortality due to excessive TNF secretion and dysregulated inflammatory cytokine production. CrmD dampened pathology, leukocyte recruitment, and inflammatory cytokine production in lungs including TNF, IL-6, IL-10, and IFN-γ. Blockade of TNF, IL-6, or IL-10R function with monoclonal antibodies reduced lung pathology and provided 60 to 100% protection from otherwise lethal infection. IFN-γ caused lung pathology only when both the TNF-binding and SECRET domains were absent. Presence of the SECRET domain alone induced significantly higher levels of IL-1ß, IL-6, and IL-10, likely overcoming any protective effects that might have been afforded by anti-IFN-γ treatment. The use of TNF-deficient mice and those that express only membrane-associated but not secreted TNF revealed that CrmD is critically dependent on host TNF for its function. In vitro, recombinant Crm proteins from different orthopoxviruses bound to membrane-associated TNF and dampened inflammatory gene expression through reverse signaling. CrmD does not affect virus replication; however, it provides the host advantage by enabling survival. Host survival would facilitate virus spread, which would also provide an advantage to the virus.

African Swine Fever Virus Protein pE199L Mediates Virus Entry by Enabling Membrane Fusion and Core Penetration.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) causing a lethal hemorrhagic disease that currently threatens the global pig industry. Despite its relevance in the infectious cycle, very little is known about the internalization of ASFV in the host cell. Here, we report the characterization of ASFV protein pE199L, a cysteine-rich structural polypeptide with similarity to proteins A16, G9, and J5 of the entry fusion complex (EFC) of poxviruses. Using biochemical and immunomicroscopic approaches, we found that, like the corresponding poxviral proteins, pE199L localizes to the inner viral envelope and behaves as an integral transmembrane polypeptide with cytosolic intramolecular disulfide bonds. Using an ASFV recombinant that inducibly expresses the E199L gene, we found that protein pE199L is not required for virus assembly and egress or for virus-cell binding and endocytosis but is required for membrane fusion and core penetration. Interestingly, similar results have been previously reported for ASFV protein pE248R, an inner membrane virion component related to the poxviral L1 and F9 EFC proteins. Taken together, these findings indicate that ASFV entry relies on a form of fusion machinery comprising proteins pE248R and pE199L that displays some similarities to the unconventional fusion apparatus of poxviruses. Also, these results provide novel targets for the development of strategies that block the first stages of ASFV replication.IMPORTANCE African swine fever virus (ASFV) causes a highly lethal swine disease that is currently present in many countries of Eastern Europe, the Russian Federation, and Southeast Asia, severely affecting the pig industry. Despite extensive research, effective vaccines or antiviral strategies are still lacking and relevant gaps in knowledge of the fundamental biology of the viral infection cycle exist. In this study, we identified pE199L, a protein of the inner viral membrane that is required for virus entry. More specifically, pE199L is necessary for the fusion event that leads to the penetration of the genome-containing core in the host cell. Our results significantly increase our knowledge of the process of internalization of African swine fever virus, which may instruct future research on antiviral strategies.

Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands.

Members of the tumour necrosis factor (TNF) superfamily OX40L and CD70 and their receptors are costimulating signalling axes critical for adequate T cell activation in humans and mice but characterisation of these molecules in other species including ruminants is lacking. Here we cloned and expressed the predicted ovine orthologues of the receptors OX40 and CD27, as well as soluble recombinant forms of their potential ovine ligands, OaOX40L and OaCD70. Using biochemical and immunofluorescence analyses, we show that both signalling axes are functional in sheep. We show that oligomeric recombinant ligand constructs are able to induce signalling through their receptors on transfected cells. Recombinant defective human adenoviruses were constructed to express the soluble forms of OaOX40L and OaCD70. Both proteins were detected in the supernatant of adenovirus-infected cells and shown to activate NF-κB signalling pathway through their cognate receptor. These adenovirus-secreted OaOX40L and OaCD70 forms could also activate ovine T cell proliferation and enhance IFN-γ production in CD4+ and CD8+ T cells. Altogether, this study provides the first characterisation of the ovine costimulatory OX40L-OX40 and CD70-CD27 signalling axes, and indicates that their activation in vivo may be useful to enhance vaccination-induced immune responses in sheep and other ruminants.

Addition of a Viral Immunomodulatory Domain to Etanercept Generates a Bifunctional Chemokine and TNF Inhibitor.

The inhibition of tumor necrosis factor (TNF) through the use of either antibodies or soluble receptors is a highly effective strategy for the clinical control of chronic inflammatory conditions such as rheumatoid arthritis. Different viruses have similarly exploited this concept by expressing a set of specifically tailored secreted TNF decoy receptors to block host inflammatory responses. Poxviruses have been shown to encode at least two distinct molecules, termed Cytokine response modifier D (CrmD) and CrmB, in which a TNF inhibitor is combined with a chemokine inhibitor on the same molecule. The ectromelia virus CrmD protein was found to be a critical determinant of virulence in vivo, being able to control local inflammation to allow further viral spread and the establishment of a lethal infection. Strikingly, both the TNF and the chemokine inhibitory domains are required for the full activity of CrmD, suggesting a model in which inhibition of TNF is supported by the concomitant blockade of a reduced set of chemokines. Inspired by this model, we reasoned that a similar strategy could be applied to modify the clinically used human TNF receptor (etanercept), producing a generation of novel, more effective therapeutic agents. Here we show the analysis of a set of fusion proteins derived from etanercept by addition of a viral chemokine-binding protein. A bifunctional inhibitor capable of binding to and blocking the activity of TNF as well as a set of chemokines is generated that is active in the prevention of arthritis in a murine disease model.

Lymphocystis disease virus (LCDV-Sa), polyomavirus 1 (SaPyV1) and papillomavirus 1 (SaPV1) in samples of Mediterranean gilthead seabream.

Lymphocystis disease, caused by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of tumour-like lesions on the skin of affected animals associated with several environmental factors and/or with stress due to the intensive culture conditions of fish farms. In a previous study, the genomes of a new LCDV species, LCDV-Sa, were detected, together with 2 previously unknown viruses, Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1). Gilthead seabream from 17 fish farms in Spain, Italy and Turkey were sampled between 2009 and 2015 to investigate the role of the newly described SaPV1 and SaPyV1 viruses in lymphocystis disease development. Our results show that in diseased fish, either or both of the new viruses are almost invariably detected together with LCDV (98%). In asymptomatic fish, these viruses were detected in a much lower percentage (28%) and mostly in concurrence with LCDV (24%). These data confirm the suspected association among the 3 different viruses during lymphocystis disease development in gilthead seabream and warrant future studies to establish their respective contributions.

A Proteomic Atlas of the African Swine Fever Virus Particle.

African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal swine disease for which there is no vaccine available. The ASFV particle, with an icosahedral multilayered structure, contains multiple polypeptides whose identity is largely unknown. Here, we analyzed by mass spectroscopy the protein composition of highly purified extracellular ASFV particles and performed immunoelectron microscopy to localize several of the detected proteins. The proteomic analysis identified 68 viral proteins, which account for 39% of the genome coding capacity. The ASFV proteome includes essentially all the previously described virion proteins and, interestingly, 44 newly identified virus-packaged polypeptides, half of which have an unknown function. A great proportion of the virion proteins are committed to the virus architecture, including two newly identified structural proteins, p5 and p8, which are derived from the core polyproteins pp220 and pp62, respectively. In addition, the virion contains a full complement of enzymes and factors involved in viral transcription, various enzymes implicated in DNA repair and protein modification, and some proteins concerned with virus entry and host defense evasion. Finally, 21 host proteins, many of them localized at the cell surface and related to the cortical actin cytoskeleton, were reproducibly detected in the ASFV particle. Immunoelectron microscopy strongly supports the suggestion that these host membrane-associated proteins are recruited during virus budding at actin-dependent membrane protrusions. Altogether, the results of this study provide a comprehensive model of the ASFV architecture that integrates both compositional and structural information.IMPORTANCE African swine fever virus causes a highly contagious and lethal disease of swine that currently affects many countries of sub-Saharan Africa, the Caucasus, the Russian Federation, and Eastern Europe and has very recently spread to China. Despite extensive research, effective vaccines or antiviral strategies are still lacking, and many basic questions on the molecular mechanisms underlying the infective cycle remain. One such gap regards the composition and structure of the infectious virus particle. In the study described in this report, we identified the set of viral and host proteins that compose the virion and determined or inferred the localization of many of them. This information significantly increases our understanding of the biological and structural features of an infectious African swine fever virus particle and will help direct future research efforts.

Chemokines cooperate with TNF to provide protective anti-viral immunity and to enhance inflammation.

The role of cytokines and chemokines in anti-viral defense has been demonstrated, but their relative contribution to protective anti-viral responses in vivo is not fully understood. Cytokine response modifier D (CrmD) is a secreted receptor for TNF and lymphotoxin containing the smallpox virus-encoded chemokine receptor (SECRET) domain and is expressed by ectromelia virus, the causative agent of the smallpox-like disease mousepox. Here we show that CrmD is an essential virulence factor that controls natural killer cell activation and allows progression of fatal mousepox, and demonstrate that both SECRET and TNF binding domains are required for full CrmD activity. Vaccination with recombinant CrmD protects animals from lethal mousepox. These results indicate that a specific set of chemokines enhance the inflammatory and protective anti-viral responses mediated by TNF and lymphotoxin, and illustrate how viruses optimize anti-TNF strategies with the addition of a chemokine binding domain as soluble decoy receptors.

Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream.

UNLABELLED: Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought. IMPORTANCE: Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.

Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF.

Poxviruses encode up to four different soluble TNF receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific pro-inflammatory and immunological activities. Here, we report that viral TNF receptors bound and inhibited tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus, CrmE, which does not inhibit mouse soluble TNF, could block murine tmTNF-induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors.

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin ß. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin ß. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs.

An orphan viral TNF receptor superfamily member identified in lymphocystis disease virus.

BACKGROUND: Lymphocystis disease virus (LCDV) is a large icosahedral dsDNA-containing virus of the Lymphocystivirus genus within the Iridoviridae family that can cause disease in more than 140 marine and freshwater fish species. While several isolates have been charcaterized and classified into distinct genotypes the complete genomic sequence is currently only available from two species, the LCDV-1, isolated from flounder (Platichtys flesus) in Europe and the LCDV-C, isolated from Japanese cultured flounder (Paralichthys olivaceus) in China. Analysis of the genome of LCDV-C showed it to encode a protein named LDVICp016 with similarities to the Tumour necrosis factor receptor (TNFR) superfamily with immunomodulatory potential. FINDINGS: We have expressed and purified the recombinant protein LDVICp016 and screened for potential interaction partners using surface plasmon resonance. Commercially available human and mouse members of the TNF superfamily (TNFSF), along with a representative set of fish-derived TNFSF were tested.We have found the LDVICp016 protein to be secreted and we have identified a second viral TNFR encoded by ORF 095 of the same virus. None of the 42 tested proteins were found to interact with LDVICp016. CONCLUSIONS: We show that LDVICp016 is a secreted protein belonging to the TNF receptor family that may be part of a larger gene family in Lymphocystiviruses. While the ligand of this protein remains unknown, possibly due to the species specific nature of this interaction, further investigations into the potential role of this protein in the blockade of immune responses in its fish host are required.

Complete genome sequence of the European sheatfish virus.

Viral diseases are an increasing threat to the thriving aquaculture industry worldwide. An emerging group of fish pathogens is formed by several ranaviruses, which have been isolated at different locations from freshwater and seawater fish species since 1985. We report the complete genome sequence of European sheatfish ranavirus (ESV), the first ranavirus isolated in Europe, which causes high mortality rates in infected sheatfish (Silurus glanis) and in other species. Analysis of the genome sequence shows that ESV belongs to the amphibian-like ranaviruses and is closely related to the epizootic hematopoietic necrosis virus (EHNV), a disease agent geographically confined to the Australian continent and notifiable to the World Organization for Animal Health.

Chemokines in teleost fish species.

Chemokines are chemoattractant cytokines defined by the presence of four conserved cysteine residues which in mammals can be divided into four subfamilies depending on the arrangement of the first two conserved cysteines in their sequence: CXC (α), CC (ß), C and CX(3)C classes. Evolutionarily, fish can be considered as an intermediate step between species which possess only innate immunity (invertebrates) and species with a fully developed acquired immune network such as mammals. Therefore, the functionality of their different immune cell types and molecules is sometimes also intermediate between innate and acquired responses. The first chemokine gene identified in a teleost was a rainbow trout (Oncorhynchus mykiss) chemokine designated as CK1 in 1998. Since then, many different chemokine genes have been identified in several fish species, but their role in homeostasis and immune response remains largely unknown. Extensive genomic duplication events and the fact that chemokines evolve more quickly than other immune genes, make it very difficult to establish true orthologues between fish and mammalian chemokines that would help us with the ascription of immune roles. In this review, we describe the current state of knowledge of chemokine biology in teleost fish, focusing mainly on which genes have been identified so far and highlighting the most important aspects of their expression regulation, due to the great lack of functional information available for them. As the number of chemokine genes begins to close down for some teleost species, there is an important need for functional assays that may elucidate the role of each of these molecules within the fish immune response.

The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon.

Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.

A method for the generation of ectromelia virus (ECTV) recombinants: in vivo analysis of ECTV vCD30 deletion mutants.

BACKGROUND: Ectromelia virus (ECTV) is the causative agent of mousepox, a lethal disease of mice with similarities to human smallpox. Mousepox progression involves replication at the initial site of infection, usually the skin, followed by a rapid spread to the secondary replicative organs, spleen and liver, and finally a dissemination to the skin, where the typical rash associated with this and other orthopoxviral induced diseases appears. Case fatality rate is genetically determined and reaches up to 100% in susceptible mice strains. Like other poxviruses, ECTV encodes a number of proteins with immunomodulatory potential, whose role in mousepox progression remains largely undescribed. Amongst these is a secreted homologue of the cellular tumour necrosis factor receptor superfamily member CD30 which has been proposed to modulate a Th1 immune response in vivo. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the contribution of viral CD30 (vCD30) to virus pathogenesis in the infected host, we have adapted a novel transient dominant method for the selection of recombinant ECTVs. Using this method, we have generated an ECTV vCD30 deletion mutant, its corresponding revertant control virus as well as a virus encoding the extracellular domain of murine CD30. These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date. CONCLUSIONS/SIGNIFICANCE: We show that the vCD30 is expressed as a secreted disulfide linked trimer and that the absence of vCD30 does not impair mousepox induced fatality in vivo. Replacement of vCD30 by a secreted version of mouse CD30 caused limited attenuation of ECTV. The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response. The method developed might be useful for the construction of ECTV mutants for the study of additional genes.

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus.

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.

African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.