Bovine papular stomatitis virus as a vaccine vector for cattle.

Virus vectored vaccines are not available commercially for cattle even though compelling potential applications exist. Bovine papular stomatitis virus (BPSV), a highly prevalent parapoxvirus, causes self-limited oral lesions in cattle. Ability of virus to accommodate large amounts of foreign DNA, induce low level of antiviral immunity, and circulate and likely persist in cattle populations, make BPSV an attractive candidate viral vector. Here, recombinant BPSV were constructed expressing either Bovine herpesvirus 1 (BoHV-1) glycoprotein gD (BPSVgD), or gD and gB (BPSVgD/gB). Immunization of BPSV serologically-positive calves with BPSVgD or BPSVgD/gB induced BoHV-1 neutralization antibodies and provided protection for three of four animals following a high dose BoHV-1 challenge at day 70 pi. Results indicate BPSV suitability as a candidate virus vector for cattle vaccines.

The novel ORFV protein ORFV113 activates LPA-p38 signaling.

Viruses have evolved mechanisms to subvert critical cellular signaling pathways that regulate a wide range of cellular functions, including cell differentiation, proliferation and chemotaxis, and innate immune responses. Here, we describe a novel ORFV protein, ORFV113, that interacts with the G protein-coupled receptor Lysophosphatidic acid receptor 1 (LPA1). Consistent with its interaction with LPA1, ORFV113 enhances p38 kinase phosphorylation in ORFV infected cells in vitro and in vivo, and in cells transiently expressing ORFV113 or treated with soluble ORFV113. Infection of cells with virus lacking ORFV113 (OV-IA82Δ113) significantly decreased p38 phosphorylation and viral plaque size. Infection of cells with ORFV in the presence of a p38 kinase inhibitor markedly diminished ORFV replication, highlighting importance of p38 signaling during ORFV infection. ORFV113 enhancement of p38 activation was prevented in cells in which LPA1 expression was knocked down and in cells treated with LPA1 inhibitor. Infection of sheep with OV-IA82Δ113 led to a strikingly attenuated disease phenotype, indicating that ORFV113 is a major virulence determinant in the natural host. Notably, ORFV113 represents the first viral protein that modulates p38 signaling via interaction with LPA1 receptor.

African Swine Fever Virus CD2v Protein Induces ß-Interferon Expression and Apoptosis in Swine Peripheral Blood Mononuclear Cells.

African swine fever (ASF) is a hemorrhagic disease of swine characterized by massive lymphocyte depletion in lymphoid tissues due to the apoptosis of B and T cells, a process likely triggered by factors released or secreted by infected macrophages. ASFV CD2v (EP402R) has been implicated in viral virulence and immunomodulation in vitro; however, its actual function(s) remains unknown. We found that CD2v expression in swine PK15 cells induces NF-κB-dependent IFN-ß and ISGs transcription and an antiviral state. Similar results were observed for CD2v protein treated swine PBMCs and macrophages, the major ASFV target cell. Notably, treatment of swine PBMCs and macrophages with CD2v protein induced apoptosis. Immunoprecipitation and colocalization studies revealed that CD2v interacts with CD58, the natural host CD2 ligand. Additionally, CD58 knockdown in cells or treatment of cells with an NF-κB inhibitor significantly reduced CD2v-mediated NF-κB activation and IFN-ß induction. Further, antibodies directed against CD2v inhibited CD2v-induced NF-κB activation and IFN-ß transcription in cells. Overall, results indicate that ASFV CD2v activates NF-κB, which induces IFN signaling and apoptosis in swine lymphocytes/macrophages. We propose that CD2v released from infected macrophages may be a significant factor in lymphocyte apoptosis observed in lymphoid tissue during ASFV infection in pigs.

Thoughts on African Swine Fever Vaccines.

African swine fever (ASF) is an acute viral hemorrhagic disease of domestic swine with mortality rates approaching 100%. Devastating ASF outbreaks and continuing epidemics starting in the Caucasus region and now in the Russian Federation, Europe, China, and other parts of Southeast Asia (2007 to date) highlight its significance. ASF strain Georgia-07 and its derivatives are now endemic in extensive regions of Europe and Asia and are "out of Africa" forever, a situation that poses a grave if not an existential threat to the swine industry worldwide. While our current concern is Georgia-07, other emerging ASFV strains will threaten for the indefinite future. Economic analysis indicates that an ASF outbreak in the U.S. would result in approximately $15 billion USD in losses, assuming the disease is rapidly controlled and the U.S. is able to reenter export markets within two years. ASF's potential to spread and become endemic in new regions, its rapid and efficient transmission among pigs, and the relative stability of the causative agent ASF virus (ASFV) in the environment all provide significant challenges for disease control. Effective and robust methods, including vaccines for ASF response and recovery, are needed immediately.

Inhibition of Antiviral Innate Immunity by Foot-and-Mouth Disease Virus Lpro through Interaction with the N-Terminal Domain of Swine RNase L.

Foot-and-mouth disease virus (FMDV) is the pathogen of foot-and-mouth disease (FMD), which is a highly contagious disease in cloven-hoofed animals. To survive in the host, FMDV has evolved multiple strategies to antagonize host innate immune responses. In this study, we showed that the leader protease (Lpro) of FMDV, a papain-like proteinase, promoted viral replication by evading the antiviral interferon response through counteracting the 2',5'-oligoadenylate synthetase (OAS)/RNase L system. Specifically, we observed that the titers of Lpro deletion virus were significantly lower than those of wild-type FMDV (FMDV-WT) in cultured cells. Our mechanistic studies demonstrated that Lpro interfered with the OAS/RNase L pathway by interacting with the N-terminal domain of swine RNase L (sRNase L). Remarkably, Lpro of FMDV exhibited species-specific binding to RNase L in that the interaction was observed only in swine cells, not human, monkey, or canine cells. Lastly, we presented evidence that by interacting with sRNase L, FMDV Lpro inhibited cellular apoptosis. Taken together, these results demonstrate a novel mechanism that Lpro utilizes to escape the OAS/RNase L-mediated antiviral defense pathway. IMPORTANCE FMDV is a picornavirus that causes a significant disease in agricultural animals. FMDV has developed diverse strategies to escape the host interferon response. Here, we show that Lpro of FMDV antagonizes the OAS/RNase L pathway, an important interferon effector pathway, by interacting with the N-terminal domain of sRNase L. Interestingly, such a virus-host interaction is species-specific because the interaction is detected only in swine cells, not in human, monkey, or canine cells. Furthermore, Lpro inhibits apoptosis through interacting with sRNase L. This study demonstrates a novel mechanism by which FMDV has evolved to inhibit host innate immune responses.

Protective Immunity and New Vaccines for Lyme Disease.

Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.

Identification of T-cell epitopes in African swine fever virus CD2v and C-type lectin proteins.

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available, and progress is hindered by lack of knowledge concerning the extent of ASF virus (ASFV) strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Previously, we demonstrated that ASFV serotype-specific proteins CD2v (EP402R) and/or C-type lectin (EP153R) are important for protection against homologous ASF infection. Here, we identified six discrete T-cell epitope regions present on CD2v and C-type lectin using IFN-γ ELISpot assay and PBMCs from ASF immune animals, indicating cellular reactivity to these proteins in the context of ASFV infection and protective immunity. Notably, three of the epitope regions map to previously described serotype-specific signature regions of these proteins. Improved understanding of ASFV protective antigens, relevant epitopes and their diversity in nature will facilitate ASFV subunit vaccine design and development.

Orf Virus Encoded Protein ORFV119 Induces Cell Apoptosis Through the Extrinsic and Intrinsic Pathways.

Apoptosis, a significant form of cell death, has a leading role in the host cell defense against virus infection. Viruses have evolved a series of strategies that block apoptosis during the early stage of viral infection to enhance viral replication, and induce apoptosis in the late stages to facilitate viral particle release from the cells. Here we show that orf virus (ORFV), the causative agent of orf, encodes an apoptosis-inducing protein ORFV119. ORFV119 targets the mitochondria in host cells, inhibits cell proliferation, and induces cell apoptosis. Protein array data indicated that ORFV119 could induce apoptosis via up-regulation of Smac, Bak, and Bax and down-regulation of anti-apoptotic proteins Bcl-2 and cIAP-2. Activation of caspase-9 and caspase-3, and consequent PARP cleavage, ultimately lead to apoptosis. ORFV119 could also directly activate caspase-8 and induce Bid, involved in the extrinsic pathway, to achieve cell death. Furthermore, sequence analysis and experiments with mutants of ORFV119 introduced revealed that ORFV119 contains a key N-terminal domain that is necessary and sufficient to direct the protein to the mitochondria. Together, we report, for the first time, the identification of the novel apoptosis-inducing protein ORFV119 encoded by a parapoxvirus. This provides an important reference for the study of pathogenesis, identification of immunomodulation mechanisms of ORFV, and may lead to new strategies for orf disease control.

Complete Genome Sequencing of Two Equine Influenza A(H3N8) Virus Strains Isolated in Kazakhstan.

Here, we report the complete genome sequencing of strains A/equine/Kostanay/9/2012(H3N8) and A/equine/LKZ/9/2012(H3N8) of the equine influenza virus belonging to Florida sublineage, clade 2. The strains were isolated in 2012 in the northern and southern regions of Kazakhstan, respectively.

Identification of host cellular proteins LAGE3 and IGFBP6 that interact with orf virus protein ORFV024.

OBJECTIVE: Orf virus (ORFV) is the pathogen causing contagious pustular dermatitis in goats, sheep and herdsmen. Evidence has confirmed that ORFV can be used as a preventive and therapeutic immunomodulatory agent in several animal models. Our previous data demonstrated that ORFV024 is able to inhibit activation of the NF-κB signaling pathway and act as an important modulator for early immune responses against viral infection. However, the molecular mechanism by which ORFV024 exerting biological function remains unclear. In the present study, we explored and analyzed the function of host cellular proteins that interact with ORFV024. METHODS: The yeast two-hybrid (Y2H) assay was performed to screen proteins interacting with ORFV024 using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). Two of the screened proteins were further confirmed by confocal microscopy, His-tag pull-down assay and CO-Immunoprecipitation (CO-IP) assay. In addition, the ORFV024 interaction network was constructed using the STRING database. RESULTS: In this study, 11 ovine cellular proteins were found to interact with ORFV024. In view of the importance of LAGE3 and IGFBP6 in the ORFV024 functional analysis, we further constructed LAGE3 and IGFBP6 interaction networks. The interactions between ORFV024 and LAGE3 or IGFBP6 were confirmed by confocal microscopy, LAGE3 was further confirmed in the His-tag pull-down assay and CO-IP assay. CONCLUSIONS: Our findings indicate that ORFV024 can interact with ovine cellular proteins LAGE3 and IGFBP6.

A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling.

Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb.

Transcriptomic profiles of human foreskin fibroblast cells in response to orf virus.

Orf virus has been utilized as a safe and efficient viral vector against not only diverse infectious diseases, but also against tumors. However, the nature of the genes triggered by the vector in human cells is poorly characterized. Using RNA sequencing technology, we compared specific changes in the transcriptomic profiles in human foreskin fibroblast cells following infection by the orf virus. The results indicated that orf virus upregulates or downregulates expression of a variety of genes, including genes involved in antiviral immune response, apoptosis, cell cycle and a series of signaling pathways, such as the IFN and p53-signaling pathways. The orf virus stimulates or inhibits immune gene expression such as chemokines, chemokine receptors, cytokines, cytokine receptors, and molecules involved in antigen uptake and processing after infection. Expression of pro-apoptotic genes increased at 8 hours post-infection. The p53 signaling pathway was activated to induce apoptosis at the same time. However, the cell cycle program was promoted after infection, which may be due to the immunomodulatory genes of the orf virus. This presents the first description of transcription profile changes in human foreskin fibroblast cells after orf virus infection and provides an in-depth analysis of the interaction between the host and orf virus. These data offer new insights into the understanding of the mechanisms of infection by orf virus and identify potential targets for future studies.

Immunogenicity of ORFV-based vectors expressing the rabies virus glycoprotein in livestock species.

The parapoxvirus Orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host-innate and pro-inflammatory responses and has been proposed as a vaccine delivery vector for use in animal species. Here we describe the construction and characterization of two recombinant ORFV vectors expressing the rabies virus (RABV) glycoprotein (G). The RABV-G gene was inserted in the ORFV024 or ORFV121 gene loci, which encode for IMPs that are unique to parapoxviruses and inhibit activation of the NF-κB signaling pathway. The immunogenicity of the resultant recombinant viruses (ORFV∆024RABV-G or ORFV∆121RABV-G, respectively) was evaluated in pigs and cattle. Immunization of the target species with ORFV∆024RABV-G and ORFV∆121RABV-G elicited robust neutralizing antibody responses against RABV. Notably, neutralizing antibody titers induced in ORFV∆121RABV-G-immunized pigs and cattle were significantly higher than those detected in ORFV∆024RABV-G-immunized animals, indicating a higher immunogenicity of ORFVΔ121-based vectors in these animal species.

The biological foundation of the genetic association of TOMM40 with late-onset Alzheimer's disease.

A variable-length poly-T variant in intron 6 of the TOMM40 gene, rs10524523, is associated with risk and age-of-onset of sporadic (late-onset) Alzheimer's disease. In Caucasians, the three predominant alleles at this locus are Short (S), Long (L) or Very long (VL). On an APOE ε3/3 background, the S/VL and VL/VL genotypes are more protective than S/S. The '523 poly-T has regulatory properties, in that the VL poly-T results in higher expression than the S poly-T in luciferase expression systems. The aim of the current work was to identify effects on cellular bioenergetics of increased TOM40 protein expression. MitoTracker Green fluorescence and autophagic vesicle staining was the same in control and over-expressing cells, but TOM40 over-expression was associated with increased expression of TOM20, a preprotein receptor of the TOM complex, the mitochondrial chaperone HSPA9, and PDHE1a, and increased activities of the oxidative phosphorylation complexes I and IV and of the TCA member α-ketoglutaric acid dehydrogenase. Consistent with the complex I findings, respiration was more sensitive to inhibition by rotenone in control cells than in the TOM40 over-expressing cells. In the absence of inhibitors, total cellular ATP, the mitochondrial membrane potential, and respiration were elevated in the over-expressing cells. Spare respiratory capacity was greater in the TOM40 over-expressing cells than in the controls. TOM40 over-expression blocked Ab-elicited decreases in the mitochondrial membrane potential, cellular ATP levels, and cellular viability in the control cells. These data suggest elevated expression of TOM40 may be protective of mitochondrial function.

A parapoxviral virion protein inhibits NF-κB signaling early in infection.

Poxviruses have evolved unique proteins and mechanisms to counteract the nuclear factor κB (NF-κB) signaling pathway, which is an essential regulatory pathway of host innate immune responses. Here, we describe a NF-κB inhibitory virion protein of orf virus (ORFV), ORFV073, which functions very early in infected cells. Infection with ORFV073 gene deletion virus (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKß, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Expression of ORFV073 alone was sufficient to inhibit TNFα induced activation of the NF-κB signaling in uninfected cells. Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. Notably, ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of viral inhibition of NF-κB signaling very early in infection.

Corrigendum: Orf Virus 002 Protein Targets Ovine Protein S100A4 and Inhibits NF-κB Signaling.

[This corrects the article on p. 1389 in vol. 7, PMID: 27679610.].

Orf Virus 002 Protein Targets Ovine Protein S100A4 and Inhibits NF-κB Signaling.

Orf virus (ORFV), a member of Parapoxvirus, has evolved various strategies to modulate the immune responses of host cells. The ORFV-encoded protein ORFV002, a regulator factor, has been found to inhibit the acetylation of NF-κB-p65 by blocking phosphorylation of NF-κB-p65 at Ser(276) and also to disrupt the binding of NF-κB-p65 and p300. To explore the mechanism by which ORFV002 regulates NF-κB signaling, the understanding of ORFV002 potential binding partners in host cells is critical. In this study, ovine S100 calcium binding protein A4 (S100A4), prolyl endopeptidase-like (PREPL) and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 8 (NDUFA8) were found to interact with ORFV002 based on the yeast two-hybrid (Y2H) assay using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). GST pull-down and bidirectional co-immunoprecipitation assay results demonstrate that ORFV002 interacts with S100A4 directly. Following the pEGFP-ORFV002 (p002GFP) transfection, we found that cytoplasmic S100A4 translocates into the nucleus and co-localizes with ORFV002. Furthermore, the inhibitory effect of ORFV002 on NF-κB signaling was significantly restored by S100A4 knock-down phenotype, suggesting that ovine S100A4 participates in the ORFV002-mediated NF-κB signaling. These data demonstrate that ORFV002 inhibits the NF-κB activation through its interaction with S100A4 along with its nucleus translocation.

Genome analysis of orf virus isolates from goats in the Fujian Province of southern China.

Orf virus (ORFV), a species of the genus Parapoxvirus of the family Poxviridae, causes non-systemic, highly contagious, and eruptive disease in sheep, goat, and other wild and domestic ruminants. Our previous work shows orf to be ubiquitous in the Fujian Province of China, a region where there is considerable heterogeneity among ORFVs. In this study, we sequenced full genomes of four Fujian goat ORFV strains (OV-GO, OV-YX, OV-NP, and OV-SJ1). The four strains were 132-139 kb in length, with each containing 124-132 genes and about 64% G+C content. The most notable differences between the four strains were found near the genome termini. OV-NP lacked seven and OV-SJ1 lacked three genes near the right terminus when compared against other ORFVs. We also investigated the skin-virulence of the four Fujian ORFVs in goats. The ORFVs with gene deletions showed low virulence while the ORFVs without gene deletions showed high virulence in goats suggesting gene deletion possibly leads to attenuation of ORFVs. Gene 134 was disrupted in OV-NP genome due to the lack of initial code. The phylogenetic tree based on complete Parapoxviruse genomes showed that sheep originated and goat originated ORFVs formed distinctly separate branches with 100% bootstrap. Based on the single gene phylogenetic tree of 132 genes of ORFVs, 47 genes can be easily distinguished as having originated from sheep or goats. In order to further reveal genetic variation presented in goat ORFVs and sheep ORFVs, we analyzed the deduced amino acid sequences of gene 008, multiple alignment of amino acid sequences of gene 008 from the genome of five goat ORFVs and four sheep ORFVs revealed 33 unique amino acids differentiating it as having sheep or goats as host. The availability of genomic sequences of four Fujian goat ORFVs aids in our understanding of the diversity of orf virus isolates in this region and can assist in distinguishing between orf strains that originate in sheep and goats.

Coinfection with multiple strains of bovine papular stomatitis virus.

Bovine papular stomatitis virus (BPSV) infects cattle and, occupationally, humans. Prevalent subclinical infections, frequent reinfections, and virus persistence in healthy animals compound a poorly understood, but likely complex, scenario of BPSV perpetuation and transmission in nature. Here, we report the isolation of multiple BPSV strains coinfecting a single animal. Whole-genome analysis of isolated BPSV strains revealed genomic variability likely affecting virus virulence and infectivity. Further, incongruent phylogenetic relationships between viruses suggested genomic recombination. These results have significant implications for parapoxvirus infection biology and virus evolution in nature.