Tanapox Virus Research Articles

Oncolytic Tanapoxvirus Variants Expressing mIL-2 and mCCL-2 Regress Human Pancreatic Cancer Xenografts in Nude Mice.

Pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer-related death and presents the lowest 5-year survival rate of any form of cancer in the US. Only 20% of PDAC patients are suitable for surgical resection and adjuvant chemotherapy, which remains the only curative treatment. Chemotherapeutic and gene therapy treatments are associated with adverse effects and lack specificity/efficacy. In this study, we assess the oncolytic potential of immuno-oncolytic tanapoxvirus (TPV) recombinants expressing mouse monocyte chemoattractant protein (mMCP-1 or mCCL2) and mouse interleukin (mIL)-2 in human pancreatic BxPc-3 cells using immunocompromised and CD-3+ T-cell-reconstituted mice. Intratumoral treatment with TPV/∆66R/mCCL2 and TPV/∆66R/mIL-2 resulted in a regression in BxPc-3 xenograft volume compared to control in immunocompromised mice; mCCL-2 expressing TPV OV resulted in a significant difference from control at p < 0.05. Histological analysis of immunocompromised mice treated with TPV/∆66R/mCCL2 or TPV/∆66R/mIL-2 demonstrated multiple biomarkers indicative of increased severity of chronic, active inflammation compared to controls. In conclusion, TPV recombinants expressing mCCL2 and mIL-2 demonstrated a therapeutic effect via regression in BxPc-3 tumor xenografts. Considering the enhanced oncolytic potency of TPV recombinants demonstrated against PDAC in this study, further investigation as an alternative or combination treatment option for human PDAC may be warranted.

Tanapox Virus and Yaba Monkey Tumor Virus K3 Orthologs Inhibit Primate Protein Kinase R in a Species-Specific Fashion.

Yaba monkey tumor virus (YMTV) and Tanapox virus (TPV) are members of the Yatapoxvirus genus and can infect humans and other primates. Despite the threat posed by yatapoxviruses, the factors determining their host range are poorly understood. In this study, we analyzed the ability of YMTV and TPV orthologs of vaccinia virus K3 (called 012 in YMTV and TPV), which share 75% amino acid identity with one another, to inhibit PKR from 15 different primate species. We first used a luciferase-based reporter, and found that YMTV and TPV K3 orthologs inhibited PKR in a species-specific manner and showed distinct PKR inhibition profiles. TPV 012 inhibited PKR from 11 primates, including humans, substantially better than YMTV 012. In contrast, both K3 orthologs inhibited the other four primate PKRs comparably well. Using YMTV 012 and TPV 012 hybrids, we mapped the region responsible for the differential PKR inhibition to the C- terminus of the K3 orthologs. Next, we generated chimeric vaccinia virus strains to investigate whether TPV K3 and YMTV K3 orthologs could rescue the replication of a vaccinia virus strain that lacks PKR inhibitors K3L and E3L. Virus replication in primate-derived cells generally correlated with the patterns observed in the luciferase-based assay. Together, these observations demonstrate that yatapoxvirus K3 orthologs have distinct PKR inhibition profiles and inhibit PKR in a species-specific manner, which may contribute to the differential susceptibility of primate species to yatapoxvirus infections.

Multiple Administration Routes, Including Intramuscular Injection, of Oncolytic Tanapoxvirus Variants Significantly Regress Human Melanoma Xenografts in BALB/c Nude Mice Reconstituted with Splenocytes from Normal BALB/c Donors.

Human melanoma is the most aggressive form of skin cancer and is responsible for the most deaths of all skin cancers. Localized tumors, and those which have limited spread, have 5-year survival rates of over 90%, with those numbers steadily rising over the past decade. However, metastatic melanomas have a sharp decrease in 5-year survival rates and are still an area of need for new, successful therapies. Immuno-oncolytic viruses (OVs) have emerged as a promising class of immunovirotherapy that can potentially address this disease. The Food and Drug Administration in the United States has approved one oncolytic herpes simplex virus expressing granulocyte-macrophage colony-stimulating factor (Talimogene Laherparepvec) for the treatment of metastatic melanoma, and others could soon follow for this and other cancers. In previous studies, Tanapoxvirus (TPV) recombinants expressing mouse interleukin-2 (mIL-2) and another expressing bacterial flagellin from Salmonella typhimurium (FliC) have demonstrated anti-tumor efficacy in nude mouse models. TPV replicates only in humans and monkeys, including tumor cells, which makes the use of syngeneic tumor models impossible for the study of this OV in a standard immunocompetent system. In this study, TPV/Δ66R/mIL-2 and TPV/Δ2L/Δ66R/FliC were tested for their ability to treat human melanoma xenografts (SK-MEL3) in a BALB/c nude mouse model reconstituted with splenocytes from genetically compatible, normal BALB/c donor mice. Two SK-MEL3 tumors were transplanted into each flank of BALB/c nude mice, and the larger tumor was treated intratumorally (IT) with virus or mock injection. In one set of animals, mice received adoptive transfers of splenocytes from BALB/c mice on day 4 to reconstitute their immune systems and allow for adaptive immune responses to occur in a xenograft model. Direct IT injection of TPV/Δ66R/mIL-2 led to significantly greater rates of tumor regression compared to reconstituted control (RC) mice in the primary and distant tumor sites, whereas TPV/Δ2L/Δ66R/FliC treatment led to significantly greater rates of tumor regression in distant tumor sites only. A second experiment used TPV/Δ66R/mIL-2 to test whether TPV could be administered intravenously (IV), intramuscularly (IM), or both routes simultaneously to exert similar anti-tumor effects in an indirectly treated tumor. A single SK-MEL3 tumor was transplanted into one flank of BALB/c nude mice and was treated either into the tail vein, the nearest rear leg to the tumor, or both. All mice then received adoptive transfers of splenocytes in the same way as previously described on day 4 and received an additional TPV treatment on day 14. The results demonstrated that TPV/Δ66R/mIL-2 treatment IV or IM had significantly greater rates of tumor regression than RC-treated mice but failed to exert this effect when both routes were used simultaneously. Data obtained through these experiments support the continued development of Tanapoxvirus for the treatment of human melanoma and using immune reconstitution to create intact adaptive immunity in a xenograft context, which can allow other tropism-limited OVs to be studied against human cancers.

Tanapoxvirus: From discovery towards oncolytic immunovirotherapy.

Insufficiency of standard cancer therapeutic agents and a high degree of toxicity associated with chemotherapy and radiotherapy have created a dearth of therapeutic options for metastatic cancers. Oncolytic viruses (OVs) are an emerging therapeutic option for the treatment of various human cancers. Several OVs, including poxviruses, are currently in preclinical and clinical studies and have shown to be effective in treating metastatic cancer types. Tanapoxvirus (TANV), a member of the Poxviridae family, is being developed as an OV for different human cancers due to its desirable safety and efficacy features. TANV causes a mild self-limiting febrile disease in humans, does not spread human to human, and its large genome makes it a relatively safer OV for use in humans. TANV is relatively well characterized at both molecular and clinical levels. Some of the TANV-encoded proteins that are a part of the virus' immune evasion strategy are also characterized. TANV replicates considerably slower than vaccinia virus. TANV has been shown to replicate in different human cancer cells in vitro and regresses human tumors in a nude mouse model. TANV is currently being developed as a therapeutic option for several human cancers including breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, retinoblastoma, and melanoma. This review provides a comprehensive summary from the discovery to the development of TANV as an OV candidate for a wide array of human cancers.

The assembly and function of poxvirus proteins

Proteins play essential roles in biological processes, and atomic details of their structure and function provide an insight into their biological roles and how they can be manipulated for therapeutic purposes. One class of proteins that are still poorly characterized at the structural level are those involved in pox virus replication. This work describes the structural and functional studies of several proteins involved in Tanapox virus replication replication. The Tanapox virus is a poxvirus that is related to the medially important vaccinia, variola and Monkeypox viruses. The variola virus is the causative agent of smallpox, which is estimated to have caused 300–500 million deaths in the 19th century alone and is a potential bioterrorism threat. Structural and functional insights into the proteins and biomolecular complexes that regulate the lifecycle of the virus will aid in the development of therapeutic agents against poxviruses and the development of poxviruses as therapeutic tools against cancer. The main goals of the study were to assess the structure and function of Tanapox proteins using biochemical and biophysical methods. Many Tanapox virus protein construct samples were generated and studied. Nuclear magnetic resonance (NMR) spectroscopy was performed on several samples to assess their structural and conformationally dynamic properties. Circular dichroism spectroscopy, size exclusion chromatography and electrophoretic mobility shift assays were used to determine the roles of these proteins molecular function. The results reveal that Tanapox virus proteins are generally insoluble when expressed alone. While biophysical studies of insoluble proteins are difficult, the samples generated are ideal for generating antibodies for in vivo functional studies.Support or Funding InformationThank you to Dr. Karim Essani for providing Tanapox genome.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oncolytic Tanapoxvirus Expressing Interleukin-2 is Capable of Inducing the Regression of Human Melanoma Tumors in the Absence of T Cells.

Oncolytic viruses (OVs), which preferentially infect cancer cells and induce host anti-tumor immune responses, have emerged as an effective melanoma therapy. Tanapoxvirus (TANV), which possesses a large genome and causes mild self-limiting disease in humans, is potentially an ideal OV candidate. Interleukin-2 (IL-2), a T-cell growth factor, plays a critical role in activating T cells, natural killer (NK) cells and macrophages in both the innate and adaptive immune system. We aimed to develop a recombinant TANV expressing mouse IL-2 (TANVΔ66R/mIL- 2), replacing the viral thymidine kinase (TK) gene (66R) with the mouse (m) mIL-2 transgene resulting in TANVΔ66R/mIL-2. Human melanoma tumors were induced in female athymic nude mice by injecting SKMEL- 3 cells subcutaneously. Mice were treated with an intratumoral injection of viruses when the tumor volumes reached 45 ± 4.5 mm3. In cell culture, expression of IL-2 attenuated virus replication of not only TANVΔ66R/ mIL-2, but also TANVGFP. It was demonstrated that IL-2 inhibited virus replication through intracellular components and without activating the interferon-signaling pathway. Introduction of mIL-2 into TANV remarkably increased its anti-tumor activity, resulting in a more significant regression than with wild-type (wt) TANV and TANVΔ66R. Histopathological studies showed that extensive cell degeneration with a significantly increased peri-tumor accumulation of mononuclear cells in the tumors treated with TANVΔ66R/mIL-2, compared to wtTANV or TANVΔ66R. We conclude that TANVΔ66R/mIL-2 is potentially therapeutic for human melanomas in the absence of T cells, and IL-2 expression resulted in an overall increase of therapeutic efficacy.

Neutralization of matrix metalloproteinase-9 potentially enhances oncolytic efficacy of tanapox virus for melanoma therapy.

Matrix metalloproteinases (MMPs), which are involved in degradation of extracellular matrix, are critical regulators in tumor progression, metastasis and angiogenesis. Although induction of MMPs is frequently observed during the viral infection, the effect of MMPs on viral replication varies between viruses. MMP-9, for instance, is upregulated and promotes the replication of some viruses, such as herpes simplex virus, but inhibits the replication of others. Here, we report that infection with tanapox virus (TPV) promotes the expression of MMP-9 in the melanoma cells. In addition, we show that MMP-9 exerts an anti-viral effect on TPV replication and plays a protective role in TPV-infected melanoma cells in vitro. Moreover, the neutralization of MMP-9 in melanoma cells remarkably enhances the TPV infection and leads to a significant reduction in cell survival. In summary, this study contributes to understanding of the role played by MMP-9 in TPV infectivity and provides more insights for using TPV as cancer virotherapy in future studies. Since TPV has shown substantial oncolytic efficacy in promoting melanoma tumor regression in animal models, identifying mechanisms that suppress MMP-9 expression upon TPV infection can potentially improve its use as a melanoma virotherapy.

Tanapoxvirus lacking the 15L gene inhibits melanoma cell growth in vitro by inducing interferon-λ1 release.

Oncolytic viruses (OVs) have emerged as a promising approach for melanoma treatment by causing tumor lysis and inducing immuno-modulatory activities. Tanapoxvirus (TPV), which causes a mild self-limiting disease in humans and contains a large DNA genome, appears as a promising OV candidate. TPV recombinants were generated with the thymidine kinase/66R gene deletion (TPVΔ66R), the 15L gene deletion (TPVΔ15L), or with both the 15L and 66R gene ablation (TPVΔ15LΔ66R). Our previous studies have shown that treatment of TPVΔ15L resulted in significant tumor regression in xenotransplanted human melanoma in nude mice. Here, we demonstrate that an anti-viral activity identified as interferon-λ1 (IFN-λ1) was secreted in a remarkably higher quantity from human lung fibroblast WI-38 and melanoma SK-MEL-3 cells infected with TPVΔ15L. Furthermore, we show that IFN-λ1 exhibits a more pronounced anti-proliferative effect in melanoma cells than IFN-α and IFN-β in vitro. Additional experiments strongly suggest that TPVΔ15L kills melanoma cells partially through inducing IFN-λ1. Taken together, our results demonstrate the immuno-modulatory activities associated with TPVΔ15L and suggest further exploration of TPVΔ15L as a melanoma virotherapy.

Tanapoxvirus lacking a neuregulin-like gene regresses human melanoma tumors in nude mice.

Neuregulin (NRG), an epidermal growth factor is known to promote the growth of various cell types, including human melanoma cells through ErbB family of tyrosine kinases receptors. Tanapoxvirus (TPV)-encoded protein TPV-15L, a functional mimic of NRG, also acts through ErbB receptors. Here, we show that the TPV-15L protein promotes melanoma proliferation. TPV recombinant generated by deleting the 15L gene (TPVΔ15L) showed replication ability similar to that of wild-type TPV (wtTPV) in owl monkey kidney cells, human lung fibroblast (WI-38) cells, and human melanoma (SK-MEL-3) cells. However, a TPV recombinant with both 15L and the thymidine kinase (TK) gene 66R ablated (TPVΔ15LΔ66R) replicated less efficiently compared to TPVΔ15L and the parental virus. TPVΔ15L exhibited more robust tumor regression in the melanoma-bearing nude mice compared to other TPV recombinants. Our results indicate that deletion of TPV-15L gene product which facilitates the growth of human melanoma cells can be an effective strategy to enhance the oncolytic potential of TPV for the treatment of melanoma.

Oncolytic tanapoxvirus expressing FliC causes regression of human colorectal cancer xenografts in nude mice.

Colorectal cancers are significant causes of morbidity and mortality and existing therapies often perform poorly for individuals afflicted with advanced disease. Oncolytic virotherapy is an emerging therapeutic modality with great promise for addressing this medical need. Herein we describe the in vivo testing of recombinant variants of the tanapoxvirus (TPV). Recombinant viruses were made ablated for either the 66R gene (encoding a thymidine kinase), the 2L gene (encoding a TNF-binding protein), or both. Some of the recombinants were armed to express mouse chemotactic protein 1 (mCCL2/mMCP-1), mouse granulocyte-monocyte colony stimulating factor (mGM-CSF), or bacterial flagellin (FliC). Tumors were induced in athymic nude mice by implantation of HCT 116 cells and subsequently treated by a single intratumoral injection of one of the recombinant TPVs. Histological examination showed a common neoplastic cell type and a range of immune cell infiltration, necrosis, and tumor cell organization. Significant regression was seen in tumors treated with virus TPV/Δ2L/Δ66R/fliC, and to a lesser extent the recombinants TPV/Δ2L and TPV/Δ66R. Our results suggest that oncolytic recombinants of the TPV armed with activators of the innate immune response may be effective virotherapeutic agents for colorectal cancers in humans and should be explored further to fully realize their potential.

The Tanapoxvirus 142R Protein is a Serine-Threonine Kinase that Phosphorylates the Tumor Suppressor p53

To effectively respond to viral infections, mammals rely on the innate and adaptive immune systems. Additionally, host cellular responses, such as apoptosis also play a vital role in the host defense mechanisms. To accomplish a successful replicative strategy in vivo, animal viruses have evolved a variety of molecular mechanisms that interfere with host responses. Poxviruses in particular, represents a prime example of where animal viruses encode a wide variety of proteins necessary for replication and subversion of the host’s immune and single cell responses. Several proteins that inhibit host immmunomodulatory cytokines and apoptosis of infected cells have been characterized in vaccinia virus (VV). Here, we describe the identification of a protein encoded by the tanapox virus genome (142R open reading frame) that is orthologous to the B1R protein from VV. We demonstrate that like B1R, TPV142R encodes a serine threonine kinase that can phosphorylate the tumor suppressor p53 and therefore has the potential for inhibiting apoptosis of infected cells.

The Tanapoxvirus 15L Protein Is a Virus-Encoded Neuregulin That Promotes Viral Replication in Human Endothelial Cells

Studies on large double-stranded DNA (dsDNA) viruses such as poxviruses have been helpful in identifying a number of viral and cellular growth factors that contribute to our broad understanding of virus-host interaction. Orthopoxviruses and leporipoxviruses are among the most studied viruses in this aspect. However, tanapoxvirus (TPV), a member of the genus Yatapoxvirus, still remains largely unexplored, as the only known hosts for this virus are humans and monkeys. Here, we describe the initial characterization of an epidermal growth factor (EGF)-like growth factor mimicking human neuregulin from TPV, expressed by the TPV-15L gene. Assays using a baculovirus-expressed and tagged TPV-15L protein demonstrated the ability to phosphorylate neuregulin receptors. Neuregulins represent a large family of EGF-like growth factors that play important roles in embryonic endocardium development, Schwann and oligodendrocyte survival and differentiation, localized acetylcholine receptor expression at the neuromuscular junction, and epithelial morphogenesis. Interestingly, certain neuregulin molecules are able to target specific tissues through interactions with heparin sulfate proteoglycans via an immunoglobulin (Ig)-like domain. Analyses of TPV-15L revealed no Ig-like domain, but it retains the ability to bind heparin and phosphorylate neuregulin receptors, providing compelling evidence that TPV-15L is a functional mimetic of neuregulin. TPV-15L knockout virus experiments demonstrate that the virus replicates in human umbilical vein endothelial cells less efficiently than wild-type TPV-Kenya, indicating that this is a nonessential protein for virus viability but can serve a stimulatory role for replication in some cultured cells. However, the precise role of this protein in host-virus interaction still remains to be deduced.

Tumor Necrosis Factor Inhibitors from Poxviruses with An Emphasis on Tanapoxvirus-2L Protein

Viruses have evolved strategies to counteract host defenses. Some tactics employ viral proteins to neutralize host immune effector proteins such as cytokines, chemokines and their receptors, which help coordinate the host responses against the virus. Tumor necrosis factor (TNF) is one of the crucial pro-inflammatory/anti-viral cytokines involved in inflammatory and autoimmune diseases. Poxvirus anti-immune proteins represent some of the most complex and efficient mechanisms of regulating TNF and its pathological effects. These proteins have considerable potential for treating TNF-related diseases. Here we discuss two major classes of poxvirus-TNF inhibitors focusing on the tanapoxvirus (TPV)-2L protein, previously called TPV-gp38. TPV-2L has been shown to interact and biologically neutralize human (h)TNF, and has been indirectly associated with the inhibition of other cytokines (hIFN-γ, hIL-2 and hIL-5). The TPV-2L protein alone has been expressed, purified and shown to bind with high affinity to hTNF, but lacked binding to the other cytokines. Further studies identified sequential binding of hβ2-microglobulin and hα2-macroglobulin to TPV-2L. The ability of a single viral protein to form multi-protein complexes suggests that TPV might also possess other novel strategies of evading the immune system. Reviewed here are patented poxvirus TNF-binding proteins and their genes to evaluate their potential therapeutic value.

Differential Susceptibility of Human Cancer Cell Lines to Wild-Type Tanapoxvirus Infection~!2009-09-05~!2010-02-23~!2010-04-08~!

Tanapoxvirus (TPV) is a member of the genus Yatapoxvirus in the family Poxviridae and is endemic to equatorial Africa. This disease is restricted to human and non-human primates, producing a mild febrile illness characterized by a single or more rarely additional pock-like lesions on the extremities. While there are several studies elucidating the replication cycle and host range of TPV, there is currently no standardized investigation comparing the ability of TPV to successfully replicate in a variety of tumor cell lines. This study examined the cytopathic effect and calculated the efficiency of TPV replication in vitro using 14 different human cancer cell lines. TPV replicates efficiently in some human tumor cells, and is restricted in others when measured by viral titer at 7 days post infection. Results described here clearly demonstrate that TPV replication in one glioblastoma cell line (U-373), and one colorectal cancer cell line (HCT-116) is more productive than in owl monkey kidney cells (OMK). Replication in two renal cancer cell lines (ACHN and Caki-1) is also increased when compared to OMK. TPV infection produced the greatest change in cellular morphology in U-373 cells, and to a much lesser degree in the breast cancer cell lines T-47D and MCF-7, and in the ovarian cancer line SK-OV3. Negligible change was noted in glioblastoma line U-87, breast cancer line MDA-MB-435, osteosarcoma line HOS, melanoma line SK-MEL5, colorectal cancer line COLO205, and prostate cancer line PC3. The cell lines least permissive to TPV replication were the glioblastoma (U-87) and melanoma (SK-MEL5) cell lines.

Interaction of human TNF and β2-microglobulin with Tanapox virus-encoded TNF inhibitor, TPV-2L

Tanapox virus (TPV) encodes and expresses a secreted TNF-binding protein, TPV-2L or gp38, that displays inhibitory properties against TNF from diverse mammalian species, including human, monkey, canine and rabbit. TPV-2L also has sequence similarity with the MHC-class I heavy chain and interacts differently with human TNF as compared to the known cellular TNF receptors or any of the known virus-encoded TNF receptor homologs derived from many poxviruses. In order to determine the TNF binding region in TPV-2L, various TPV-2L C-terminal truncations and internal deletions were created and the muteins were expressed using recombinant baculovirus vectors. C-terminal deletions from TPV-2L resulted in reduced binding affinity for human TNF and specific mutants of TNF that discriminate between TNF-R1 and TNF-R2. However, deletion of C-terminal 42 amino acid residues totally abolished the binding of human TNF and its mutants. Removal of any of the predicted internal domains resulted in a mutant TPV-2L protein incapable of binding to human TNF. Deletion of C-terminal residues also affected the ability of TPV-2L to block TNF-induced cellular cytotoxicity. In addition to TNF, TPV-2L can also form complexes with human β2-microglobulin to form a novel macromolecular complex. In summary, the TPV-2L protein is a bona fide MHC-1 heavy chain family member that binds and inhibits human TNF in a fashion very distinct from other known poxvirus-encoded TNF inhibitors, and also can form a novel complex with the human MHC-1 light chain, β2-microglobulin.

The Addition of Tumor Necrosis Factor plus Beta Interferon Induces a Novel Synergistic Antiviral State against Poxviruses in Primary Human Fibroblasts

Tumor necrosis factor (TNF) and members of the interferon (IFN) family have been shown to independently inhibit the replication of a variety of viruses. In addition, previous reports have shown that treatment with various combinations of these antiviral cytokines induces a synergistic antiviral state that can be significantly more potent than addition of any of these cytokines alone. The mechanism of this cytokine synergy and its effects on global gene expression, however, are not well characterized. Here, we use DNA microarray analysis to demonstrate that treatment of uninfected primary human fibroblasts with TNF plus IFN-beta induces a distinct synergistic state characterized by significant perturbations of several hundred genes which are coinduced by the individual cytokines alone, as well as the induction of more than 850 novel host cell genes. This synergy is mediated directly by the two ligands, not by intermediate secreted factors, and is necessary and sufficient to completely block the productive replication and spread of myxoma virus in human fibroblasts. In contrast, the replication of two other poxviruses, vaccinia virus and tanapox virus, are only partially inhibited in these cells by the synergistic antiviral state, whereas the spread of both of these viruses to neighboring cells was efficiently blocked. Taken together, our data indicate that the combination of TNF and IFN-beta induces a novel synergistic antiviral state that is highly distinct from that induced by either cytokine alone.

Tropism of Tanapox virus infection in primary human cells

Tanapox virus (TPV) belongs to the genus Yatapoxvirus and causes a relatively benign zoonotic disease in man, with symptoms that resemble a mild version of human monkeypox. In order to investigate the underlying mechanisms of TPV pathogenesis, the tropism and replication characteristics of TPV were examined in a variety of primary human cells. A GFP expressing TPV (TPV-GFP) was constructed and used to infect primary human dermal fibroblasts (pHDFs) and peripheral blood mononuclear cells (PBMCs), both of which are believed to be major in vivo targets of poxvirus infection. pHDFs fully supported productive replication and cell–cell spread of TPV-GFP. However, induction of cell cycle arrest in pHDFs by contact mediated inhibition or rapamycin treatment eliminated the ability of TPV to fully stimulate cell cycle progression and dramatically reduced viral replication. TPV-GFP-infected human PBMCs were screened for permissiveness by FACS analysis. CD14+ cells (monocytes) were the primary cellular target for TPV infection. A small proportion of CD3+ cells (T cells) were positive for GFP expression, yet TPV was not able to replicate and spread in cultured peripheral blood lymphocytes, regardless of their state of activation. Primary human monocytes, however, demonstrated robust TPV replication, yet these cells no longer supported replication of TPV once they differentiated into macrophages. This unique ex vivo tropism of TPV gives key insights into the basis for the self-limiting pathogenicity of TPV in man.

Comparative genetic analysis of genomic DNA sequences of two human isolates of Tanapox virus

Members of the genus Yatapoxvirus, which include Tanapox virus (TPV) and Yaba monkey tumor virus, infect primates including humans. Two strains of TPV isolated 50 years apart from patients infected from the equatorial region of Africa have been sequenced. The original isolate from a human case in the Tana River Valley, Kenya, in 1957 (TPV-Kenya) and an isolate from an infected traveler in the Republic of Congo in 2004 (TPV-RoC). Although isolated 50 years apart the genomes were highly conserved. The genomes differed at only 35 of 144,565 nucleotide positions (99.98% identical). We predict that TPV-RoC encodes 155 ORFs, however a single transversion (at nucleotide 10241) in TPV-Kenya resulted in the coding capacity for two predicted ORFs (11.1L and 11.2L) in comparison to a single ORF (11L) in TPV-RoC. The genomes of TPV are A + T rich (73%) and 96% of the sequence encodes predicted ORFs. Comparative genomic analysis identified several features shared with other chordopoxviruses. A conserved sequence within the terminal inverted repeat region that is also present in the other members of the Yatapoxviruses as well as members of the Capripoxviruses, Swinepox virus and an unclassified Deerpox virus suggests the existence of a conserved near-terminal sequence secondary structure. Two previously unidentified gene families were annotated that are represented by ORF TPV28L, which matched homologues in certain other chordopoxviruses, and TPV42.5L, which is highly conserved among currently reported chordopoxvirus sequences.

Variation in Ligand Binding Specificities of a Novel Class of Poxvirus-encoded Tumor Necrosis Factor-binding Protein

The Yatapoxviruses encode a distinct class of secreted TNF-binding protein (TNF-BP) that resembles an MHC class I heavy chain but distinct from any other known TNF inhibitor. Characterization of these viral TNF inhibitors from Tanapox virus, Yaba monkey tumor virus (YMTV) and a closely related version from Swinepox virus revealed dramatically differential TNF binding specificities for different mammalian species. The Tanapox virus 2L protein (TPV-2L) formed inhibitory complexes with human TNF, and interacted with monkey and canine TNF with high affinity but rabbit TNF with low affinity. On the other hand, YMTV-2L bound human and monkey TNF with high affinity but rabbit TNF with only low affinity. The TNF-BP from swinepox virus (SPV003/148) only interacted with porcine TNF with high affinity. The observed TNF binding analysis mirrored the biological activity of these TNF-binding protein to block TNF-induced cellular cytolysis. TPV-2L and YMTV-2L also inhibited the human TNF-mediated signaling in cells but TPV-2L exhibited higher affinity for human TNF (KD, 43 pm) compared with monkey (KD, 120 pm) whereas for YMTV-2L, the affinities were reversed (human TNF KD, 440 pm; monkey TNF KD, 230 pm). The interaction domain of human TNF with TNF-binding proteins is significantly different from that of TNFRs, as determined using human TNF mutants. We conclude that these poxvirus TNF-binding proteins represent a new class of TNF inhibitors and are distinct from the viral TNF receptor homologues characterized to date.

Real-time PCR assay for the detection of tanapox virus and yaba-like disease virus

The yatapoxvirus genus contains three members: tanapox virus (TPV), yaba-like disease virus (YLDV) and yaba monkey tumor virus (YMTV), two of which (TPV and YLDV) may infect humans. However, only a very small number of patients have been diagnosed with TPV outside Africa. Given the increased international travel and the similarity of clinical signs during the early stages of a TPV/YLDV infection as compared to diseases caused by agents of potential biological warfare, such as smallpox, monkeypox, tularemia and anthrax, the rapid and reliable recognition of a TPV/YLDV infection is crucial. A real-time PCR assay using TaqMan ® chemistry was developed in order to identify unambiguously TPV/YLDV. Primers and probe targeting a 101 bp region of the PstI L fragment of TPV, initial optimisations steps were carried out with YLDV DNA as template. Using probit regression analysis, the lower limit of detection was calculated to be ca. 8 copies per assay. A total of five TPV strains, one YDLV strain and scab-derived DNA from a patient with a TPV infection yielded specific amplification, whereas the DNA of YMTV was not amplified. Various viral and bacterial pathogens ( n = 29) associated with rash-causing illnesses were not detected using this assay.