Pyroptosis activates conventional type I dendritic cells to mediate the priming of highly functional anticancer T cells.

BACKGROUND: Initiation of antitumor immunity is reliant on the stimulation of dendritic cells (DCs) to present tumor antigens to naïve T cells and generate effector T cells that can kill cancer cells. Induction of immunogenic cell death after certain types of cytotoxic anticancer therapies can stimulate T cell-mediated immunity. However, cytotoxic therapies simultaneously activate multiple types of cellular stress and programmed cell death; hence, it remains unknown what types of cancer cell death confer superior antitumor immunity. METHODS: Murine cancer cells were engineered to activate apoptotic or pyroptotic cell death after Dox-induced expression of procell death proteins. Cell-free supernatants were collected to measure secreted danger signals, cytokines, and chemokines. Tumors were formed by transplanting engineered tumor cells to specifically activate apoptosis or pyroptosis in established tumors and the magnitude of immune response measured by flow cytometry. Tumor growth was measured using calipers to estimate end point tumor volumes for Kaplan-Meier survival analysis. RESULTS: We demonstrated that, unlike apoptosis, pyroptosis induces an immunostimulatory secretome signature. In established tumors pyroptosis preferentially activated CD103+ and XCR1+ type I conventional DCs (cDC1) along with a higher magnitude and functionality of tumor-specific CD8+ T cells and reduced number of regulatory T cells within the tumor. Depletion of cDC1 or CD4+ and CD8+ T cells ablated the antitumor response leaving mice susceptible to a tumor rechallenge. CONCLUSION: Our study highlights that distinct types of cell death yield varying immunotherapeutic effect and selective activation of pyroptosis can be used to potentiate multiple aspects of the anticancer immunity cycle.

Determinants for Antitumor and Protumor Effects of Programmed Cell Death.

Cytotoxic anticancer therapies activate programmed cell death in the context of underlying stress and inflammatory signaling to elicit the emission of danger signals, cytokines, and chemokines. In a concerted manner, these immunomodulatory secretomes stimulate antigen presentation and T cell-mediated anticancer immune responses. In some instances, cell death-associated secretomes attract immunosuppressive cells to promote tumor progression. As it stands, cancer cell death-induced changes in the tumor microenvironment that contribute to antitumor or protumor effects remain largely unknown. This is complicated to examine because cell death is often subverted by tumors to circumvent natural, and therapy-induced, immunosurveillance. Here, we provide insights into important but understudied aspects of assessing the contribution of cell death to tumor elimination or cancer progression, including the role of tumor-associated genetics, epigenetics, and oncogenic factors in subverting immunogenic cell death. This perspective will also provide insights on how future studies may address the complex antitumor and protumor immunologic effects of cell death, while accounting for variations in tumor genetics and underlying microenvironment.

HDACi-dependent Microenvironmental Normalization Overcomes Tumor Burden-induced T-cell Exhaustion.

PURPOSE: T-cell exhaustion limits immunotherapy for the treatment of solid tumors. Although immune checkpoint blockade and adoptive T-cell therapy (ACT) can mediate tumor regression, their potency is often determined by tumor burden. Here, we identified tumor burden-related pathway changes that are conducive to T-cell exhaustion. We then determined whether microenvironmental reprogramming via epigenetic modulation could reverse T-cell exhaustion and improve immunotherapeutic responsiveness. EXPERIMENTAL DESIGN: We developed a murine syngeneic tumor model wherein an increased burden ablated therapeutic responsiveness to ACT, which corresponded with systemic induction of T-cell exhaustion. Transcriptome analysis of these large tumors allowed us to characterize changes to immunosuppressive pathway expression during class I histone deacetylase inhibitor MS-275 treatment. We then measured the therapeutic impact of MS-275 during ACT and assessed T-cell exhaustion by transcriptome/phenotypic analysis. RESULTS: ACT durably regressed small tumors but failed to control large tumors, which were associated with systemic T-cell exhaustion and ablation of T-cell responses. Large tumors were defined by an immunosuppressive pathway signature. MS-275 reversed this pathway signature and promoted durable regression of large tumors during ACT. Prototypical exhaustion marker Tim-3 was selectively upregulated in transferred T cells despite displaying a reduced exhaustion signature. Instead, we observed enhanced activation-dependent signaling correlating with enrichment of the IL2-STAT5 signaling axis. Activated CD8+ T-cell responses were predominantly skewed toward terminal effector cell-like CD44+ Tim-3hi TCF1- CD127- KLRG1+ differentiation. CONCLUSIONS: Tumor burden-induced pathway changes can be reversed through epigenetic reprogramming, enabling the conversion from T-cell exhaustion to effector lineage differentiation.

HDACi promotes inflammatory remodeling of the tumor microenvironment to enhance epitope spreading and antitumor immunity.

Adoptive cell therapy (ACT) with tumor-specific memory T cells has shown increasing efficacy in regressing solid tumors. However, tumor antigen heterogeneity represents a longitudinal challenge for durable clinical responses due to the therapeutic selective pressure for immune escape variants. Here, we demonstrated that delivery of the class I histone deacetylase inhibitor MS-275 promoted sustained tumor regression by synergizing with ACT in a coordinated manner to enhance cellular apoptosis. We found that MS-275 altered the tumor inflammatory landscape to support antitumor immunoactivation through the recruitment and maturation of cross-presenting CD103+ and CD8+ DCs and depletion of Tregs. Activated endogenous CD8+ T cell responses against nontarget tumor antigens were critically required for the prevention of tumor recurrence. Importantly, MS-275 altered the immunodominance hierarchy by directing epitope spreading toward the endogenous retroviral tumor-associated antigen p15E. Our data suggest that MS-275 in combination with ACT multimechanistically enhanced epitope spreading and promoted long-term clearance of solid tumors.

Using a Prime-Boost Vaccination Strategy That Proved Effective for High Resolution Epitope Mapping to Characterize the Elusive Immunogenicity of Survivin.

Survivin is a member of the inhibitor of apoptosis family of proteins and has been reported to be highly expressed in a variety of cancer types, making it a high priority target for cancer vaccination. We previously described a heterologous prime-boost strategy using a replication-deficient adenovirus, followed by an oncolytic rhabdovirus that generates unprecedented antigen-specific T cell responses. We engineered each vector to express a mutated version of full-length murine survivin. We first sought to uncover the complete epitope map for survivin-specific T cell responses in C57BL/6 and BALB/c mice by flow cytometry. However, no T cell responses were detected by intracellular cytokine staining after re-stimulation of T cells. Survivin has been found to be expressed by activated T cells, which could theoretically cause T cell-mediated killing of activated T cells, known as fratricide. We were unable to recapitulate this phenomenon in experiments. Interestingly, the inactivated survivin construct has been previously shown to directly kill tumor cells in vitro. However, there was no evidence in our models of induction of death in antigen-presenting cells due to treatment with a survivin-expressing vector. Using the same recombinant virus-vectored prime-boost strategy targeting the poorly immunogenic enhanced green fluorescent protein proved to be a highly sensitive method for mapping T cell epitopes, particularly in the context of identifying novel epitopes recognized by CD4+ T cells. Overall, these results suggested there may be unusually robust tolerance to survivin in commonly used mouse strains that cannot be broken, even when using a particularly potent vaccination platform. However, the vaccination method shows great promise as a strategy for identifying novel and subdominant T cell epitopes.

The Role of Type I Interferon Signaling in Regulating Cytokine Production and Cell Survival in Bone Marrow-Derived Macrophages.

During viral infections, cells produce type I interferons (IFNs), which are detected by the IFNα/ß receptor (IFNAR). To survive in hosts, viruses have strategies to downregulate IFN-mediated signaling. We hypothesized that macrophages, which are among the first myeloid cells to respond to viral infections, would produce a different cytokine profile if responding to ligation of pattern recognition receptors (PRRs) while IFNAR-mediated signaling was compromised. Specifically, IFNAR-mediated regulation of interleukin (IL)-1α, IL-6, IL-12, and tumor necrosis factor-α was studied in cultured murine bone marrow-derived macrophages. Since viruses like vesicular stomatitis virus can ligate PRRs such as Toll-like receptor (TLR)4 and 7, macrophages were stimulated with the TLR4 and TLR7 agonists lipopolysaccharide (LPS) or imiquimod, respectively, with or without antibody-mediated IFNAR-blockade. Cytokines and viability were assessed for 3 days poststimulation. Blocking IFNARs acutely exacerbated cytokine production by macrophages and aided their survival when they were treated with LPS. In contrast, cytokine concentrations were unaffected or slightly reduced by IFNAR blockade, but macrophages died at a greater rate when imiquimod was the stimulus. This demonstrated a differential role of IFNAR signaling in regulating PRR-induced cytokines. This suggests potential mechanisms whereby macrophages responding to viruses that inhibit type I IFN responses might contribute to excessive inflammation in some cases and inappropriately low-magnitude responses in others. This also provides a well-defined cell-based model for further dissecting the role of type I IFN signaling in macrophages responding to viral and other infections.

Endogenous T cells prevent tumor immune escape following adoptive T cell therapy.

While the outcome of adoptive T cell therapy (ACT) is typically correlated with the functionality of the inoculated T cells, the role of the endogenous T cells is unknown. The success of checkpoint blockade therapy has demonstrated the potentially curative value of preexisting tumor-primed T cells in cancer treatment. Given the results from checkpoint blockade therapy, we hypothesized that endogenous T cells contribute to long-term survival following ACT. Here, we describe a therapeutic approach combining ACT with an oncolytic vaccine that allows simultaneous analysis of antitumor immunity mediated by transferred and endogenous T cells. We found that, in addition to promoting the expansion and tumor infiltration of the transferred T cells, oncolytic vaccines boosted tumor-primed host T cells. We determined that transferred T cells contributed to rapid destruction of large tumor masses while endogenous T cells concurrently prevented the emergence of antigen-loss variants. Moreover, while transferred T cells disappeared shortly after tumor regression, endogenous T cells secured long-term memory with a broad repertoire of antigen specificity. Our findings suggest that this combination strategy may exploit the full potential of ACT and tumor-primed host T cells to eliminate the primary tumor, prevent immune escape, and provide long-term protective memory.

The U3 and Env Proteins of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Both Contribute to Tissue Tropism.

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are small-ruminant betaretroviruses that share high nucleotide and amino acid identity, utilize the same cellular receptor, hyaluronoglucosaminidase 2 (Hyal2) for entry, and transform tissues with their envelope (Env) glycoprotein; yet, they target discrete regions of the respiratory tract-the lung and nose, respectively. This distinct tissue selectivity makes them ideal tools with which to study the pathogenesis of betaretroviruses. To uncover the genetic determinants of tropism, we constructed JSRV-ENTV chimeric viruses and produced lentivectors pseudotyped with the Env proteins from JSRV (Jenv) and ENTV (Eenv). Through the transduction and infection of lung and nasal turbinate tissue slices, we observed that Hyal2 expression levels strongly influence ENTV entry, but that the long terminal repeat (LTR) promoters of these viruses are likely responsible for tissue-specificity. Furthermore, we show evidence of ENTV Env expression in chondrocytes within ENTV-infected nasal turbinate tissue, where Hyal2 is highly expressed. Our work suggests that the unique tissue tropism of JSRV and ENTV stems from the combined effort of the envelope glycoprotein-receptor interactions and the LTR and provides new insight into the pathogenesis of ENTV.

Type I IFN blockade uncouples immunotherapy-induced antitumor immunity and autoimmune toxicity.

Despite its success in treating melanoma and hematological malignancies, adoptive cell therapy (ACT) has had only limited effects in solid tumors. This is due in part to a lack of specific antigen targets, poor trafficking and infiltration, and immunosuppression in the tumor microenvironment. In this study, we combined ACT with oncolytic virus vaccines (OVVs) to drive expansion and tumor infiltration of transferred antigen-specific T cells and demonstrated that the combination is highly potent for the eradication of established solid tumors. Consistent with other successful immunotherapies, this approach elicited severe autoimmune consequences when the antigen targeted was a self-protein. However, modulation of IFN-α/-ß signaling, either by functional blockade or rational selection of an OVV backbone, ameliorated autoimmune side effects without compromising antitumor efficacy. Our study uncovers a pathogenic role for IFN-α/-ß in facilitating autoimmune toxicity during cancer immunotherapy and presents a safe and powerful combinatorial regimen with immediate translational applications.

HDACi Delivery Reprograms Tumor-Infiltrating Myeloid Cells to Eliminate Antigen-Loss Variants.

Immune recognition of tumor-expressed antigens by cytotoxic CD8+ T cells is the foundation of adoptive T cell therapy (ACT) and has been shown to elicit significant tumor regression. However, therapy-induced selective pressure can sculpt the antigenicity of tumors, resulting in outgrowth of variants that lose the target antigen. We demonstrate that tumor relapse from ACT and subsequent oncolytic viral vaccination can be prevented using class I HDACi, MS-275. Drug delivery subverted the phenotype of tumor-infiltrating CD11b+ Ly6Chi Ly6G- myeloid cells, favoring NOS2/ROS secretion and pro-inflammatory genes characteristic of M1 polarization. Simultaneously, MS-275 abrogated the immunosuppressive function of tumor-infiltrating myeloid cells and reprogrammed them to eliminate antigen-negative tumor cells in a caspase-dependent manner. Elevated IFN-γ within the tumor microenvironment suggests that MS-275 modulates the local cytokine landscape to favor antitumor myeloid polarization through the IFN-γR/STAT1 signaling axis. Exploiting tumor-infiltrating myeloid cell plasticity thus complements T cell therapy in targeting tumor heterogeneity and immune escape.

Truncation of the enzootic nasal tumor virus envelope protein cytoplasmic tail increases Env-mediated fusion and infectivity.

Enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) are highly related ovine betaretroviruses that induce nasal and lung tumours in small ruminants, respectively. While the ENTV and JSRV envelope (Env) glycoproteins mediate virus entry using the same cellular receptor, the glycosylphosphatidylinositol-linked protein hyaluronoglucosaminidase, ENTV Env pseudovirions mediate entry into cells from a much more restricted range of species than do JSRV Env pseudovirions. Unlike JSRV Env, ENTV Env does not induce cell fusion at pH 5.0 or above, but rather requires a much lower pH (4.0-4.5) for fusion to occur. The cytoplasmic tail of retroviral envelope proteins is a key modulator of envelope-mediated fusion and pseudotype efficiency, especially in the context of virions composed of heterologous Gag proteins. Here we report that progressive truncation of the ENTV Env cytoplasmic tail improves transduction efficiency of pseudotyped retroviral vectors and that complete truncation of the ENTV Env cytoplasmic tail increases transduction efficiency to wild-type JSRV Env levels by increasing fusogenicity without affecting sensitivity to inhibition by lysosomotropic agents, subcellular localization or efficiency of inclusion into virions. Truncation of the cytoplasmic domain of ENTV Env resulted in a significant advantage in viral entry into all cell types tested, including foetal ovine lung and nasal cells. Taken together, we demonstrate that the cytoplasmic tail modulates the fusion activity of the ENTV Env protein and that truncation of this region enhances Eenv-mediated entry into target cells.

Seroconversion of sheep experimentally infected with enzootic nasal tumor virus.

BACKGROUND: Enzootic nasal tumor virus (ENTV-1) is an exogenous betaretrovirus of sheep that transforms epithelial cells lining the ethmoid turbinates leading to a disease called enzootic nasal adenocarcinoma (ENA). A unique feature of ENA is the apparent absence of a specific humoral immune response to the virus, despite the highly productive infection in nasal tumors. The sheep genome contains approximately 27 copies of endogenous ovine betaretroviral sequences (enJSRVs) and expression of enJSRVs in the ovine placenta and uterine endometrium throughout gestation is thought to induce immunological tolerance to exogenous ovine betaretroviruses, a factor that may influence the likelihood of exogenous ENTV infection and disease outcome. Nevertheless, we recently demonstrated the presence of neutralizing antibodies directed against the ENTV-1 envelope glycoprotein in sheep naturally exposed to ENTV-1. FINDINGS: Here, we employed an ENTV-1 envelope glycoprotein surface subunit specific ELISA and a virus neutralization assay to monitor serum antibody responses to ENTV-1 in a group of lambs experimentally infected with ENTV-1 virus containing filtered ENA tumor homogenate. Seroconversion and development of neutralizing antibodies was detected in one of six experimentally infected lambs. CONCLUSIONS: Our results demonstrate that sheep can respond immunologically and seroconvert following ENTV-1 infection suggesting that anti-viral immune responses may play a role in the development of ENA.

Construction of a molecular clone of ovine enzootic nasal tumor virus.

BACKGROUND: Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep. Transmission experiments performed using virus isolated from cell free nasal tumor homogenates suggest that ENTV-1 is the causative agent of ENA; however, this etiological relationship has not been conclusively proven due to the fact that the virus cannot be propagated in vitro nor is there an infectious molecular clone of the virus. METHODS: Here we report construction of a molecular clone of ENTV-1 and demonstrate that transfection of this molecular clone into HEK 293T cells produces mature virus particles. RESULTS: Analysis of recombinant virus particles derived from the initial molecular clone revealed a defect in the proteolytic processing of Gag; however, this defect could be corrected by co-expression of the Gag-Pro-Pol polyprotein from the highly related Jaagsiekte sheep retrovirus (JSRV) suggesting that the polyprotein cleavage sites in the ENTV-1 molecular clone were functional. Mutagenesis of the molecular clone to correct amino acid variants identified within the pro gene did not restore proteolytic processing; whereas deletion of one proline residue from a polyproline tract located in variable region 1 (VR1) of the matrix resulted in production of CA protein of the mature (cleaved) size strongly suggesting that normal virion morphogenesis and polyprotein cleavage took place. Finally, electron microscopy revealed the presence of spherical virus particles with an eccentric capsid and an average diameter of about 100 nm. CONCLUSION: In summary, we have constructed the first molecular clone of ENTV-1 from which mature virus particles can be produced. Future experiments using virus produced from this molecular clone can now be conducted to fulfill Koch's postulates and demonstrate that ENTV-1 is necessary and sufficient to induce ENA in sheep.

Capitalizing on Cancer Specific Replication: Oncolytic Viruses as a Versatile Platform for the Enhancement of Cancer Immunotherapy Strategies.

The past decade has seen considerable excitement in the use of biological therapies in treating neoplastic disease. In particular, cancer immunotherapy and oncolytic virotherapy have emerged as two frontrunners in this regard with the first FDA approvals for agents in both categories being obtained in the last 5 years. It is becoming increasingly apparent that these two approaches are not mutually exclusive and that much of the therapeutic benefit obtained from the use of oncolytic viruses (OVs) is in fact the result of their immunotherapeutic function. Indeed, OVs have been shown to recruit and activate an antitumor immune response and much of the current work in this field centers around increasing this activity through strategies such as engineering genes for immunomodulators into OV backbones. Because of their broad immunostimulatory functions, OVs can also be rationally combined with a variety of other immunotherapeutic approaches including cancer vaccination strategies, adoptive cell transfer and checkpoint blockade. Therefore, while they are important therapeutics in their own right, the true power of OVs may lie in their ability to enhance the effectiveness of a wide range of immunotherapies.

Mechanistic insights into the oncolytic activity of vesicular stomatitis virus in cancer immunotherapy.

Immunotherapy and oncolytic virotherapy have both shown anticancer efficacy in the clinic as monotherapies but the greatest promise lies in therapies that combine these approaches. Vesicular stomatitis virus is a prominent oncolytic virus with several features that promise synergy between oncolytic virotherapy and immunotherapy. This review will address the cytotoxicity of vesicular stomatitis virus in transformed cells and what this means for antitumor immunity and the virus' immunogenicity, as well as how it facilitates the breaking of tolerance within the tumor, and finally, we will outline how these features can be incorporated into the rational design of new treatment strategies in combination with immunotherapy.

Development of an ante-mortem diagnostic test for enzootic nasal tumor virus and detection of neutralizing antibodies in host serum.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the nasal mucosa of sheep and goats and is associated with enzootic nasal tumour virus (ENTV). As ENA is a common disease in North America and there are no vaccines against ENTV-1, diagnostic tests that can identify infected animals and assist with eradication and disease surveillance efforts are greatly needed. In this study, we endeavoured to develop a novel, non-invasive diagnostic tool that could be used not only to validate clinical signs of ENA but also to detect ENTV-1 infection prior to the onset of disease signs (i.e. pre-clinical diagnosis). Cytology, serology and reverse transcription (RT)-PCR-based diagnostic methods were investigated. Although the cytology-based assay was able to detect ENTV-1 infection in some animals, it had poor sensitivity and specificity and thus was not developed further as an ante-mortem diagnostic method. Three different assays, including ELISA, Western blotting and virus neutralization, were developed to detect the presence of ENTV-1-specific antibodies in sheep serum. Whilst a surprisingly large number of sheep mounted an antibody-mediated immune response against ENTV-1, and in some cases neutralizing, correlation with disease status was poor. In contrast, RT-PCR on RNA extracted from nasal swabs reliably detected exogenous ENTV-1 sequences, did not amplify endogenous ovine betaretroviral sequences, demonstrated high concordance with immunohistochemical staining for ENTV-1 envelope protein, and had perfect sensitivity and specificity. This report describes a practical and highly specific RT-PCR technique for the detection of clinical and pre-clinical ENA that may prove beneficial in future control or eradication programmes.

Burning mouth syndrome: patch test results from a large case series.

BACKGROUND: Burning mouth syndrome (BMS) is a burning or sore mouth in the absence of changes in the oral mucosa. It is often difficult to diagnose and treat. Numerous theories of the etiology have been suggested, including contact allergy. OBJECTIVE: To determine the clinical utility of patch testing in patients with BMS. METHODS: We retrospectively reviewed the charts of patients diagnosed with BMS who had patch testing performed between January 1, 2008, and July 31, 2012. RESULTS: Of 142 consecutive patients with BMS, 132 consented to patch testing; 89 (67%) had allergic patch test reactions. Of the patients with positive results, 66 (74%) had results that were deemed to have possible relevance. The most common allergens detected were nickel sulfate 2.5%, dodecyl gallate 0.3%, octyl gallate 0.3%, fragrance mix 8%, benzoyl peroxide 1%, and cinnamic alcohol 1%. CONCLUSIONS: Our findings suggest that contact allergy may be an etiologic factor in some patients with BMS. Patch testing is a useful investigation for BMS patients.

Jaagsiekte sheep retrovirus detected in human lung cancer tissue arrays.

BACKGROUND: Adenocarcinoma is the most common type of non-small cell lung cancer and is frequently observed in non-smoking patients. Adenocarcinoma in-situ (formerly referred to as bronchioloalveolar carcinoma) is a subset of lung adenocarcinoma characterized by growth along alveolar septae without evidence of stromal, vascular, or pleural invasion, that disproportionately affects never-smokers, women, and Asians. Adenocarcinoma in-situ is morphologically and histologically similar to a contagious lung neoplasm of sheep called ovine pulmonary adenocarcinoma (OPA). OPA is caused by infection with the exogenous betaretrovirus, jaagsiekte sheep retrovirus (JSRV), whose envelope protein (Env) is a potent oncogene. Several studies have reported that a proportion of human lung adenocarcinomas are immunopositive for an antigen related to the Gag protein of JSRV, however other groups have been unable to verify these observations by PCR. METHODS: Here we examine human lung cancer tissue arrays (TA) for evidence of JSRV Env protein and DNA by immunohistochemical staining and PCR, respectively. RESULTS: Our results reveal that a subset of human lung cancers express an antigen that reacts with a JSRV Env-specific monoclonal antibody in immunohistochemistry and that exogenous JSRV-like env and gag sequences can be amplified from TA tumor samples, albeit inefficiently. CONCLUSIONS: While a causative role has not been established, these data suggest that a JSRV-like virus might infect humans. With next generation sequencing approaches, a JSRV-like virus in human lung cancers may be identified which could have profound implications for prevention, diagnosis and therapy.

Experimental transmission of enzootic nasal adenocarcinoma in sheep.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the secretory epithelial cells of the nasal mucosa of sheep and goats. It is associated with the betaretrovirus, enzootic nasal tumor virus (ENTV), but a causative relationship has yet to be demonstrated. In this study, 14-day-old lambs were experimentally infected via nebulization with cell-free tumor filtrates derived from naturally occurring cases of ENA. At 12 weeks post-infection (wpi), one of the five infected lambs developed clinical signs, including continuous nasal discharge and open mouth breathing, and was euthanized. Necropsy revealed the presence of a large bilateral tumor occupying the nasal cavity. At 45 wpi, when the study was terminated, none of the remaining infected sheep showed evidence of tumors either by computed tomography or post-mortem examination. ENTV-1 proviral DNA was detected in the nose, lung, spleen, liver and kidney of the animal with experimentally induced ENA, however there was no evidence of viral protein expression in tissues other than the nose. Density gradient analysis of virus particles purified from the experimentally induced nasal tumor revealed a peak reverse transcriptase (RT) activity at a buoyant density of 1.22 g/mL which was higher than the 1.18 g/mL density of peak RT activity of virus purified from naturally induced ENA. While the 1.22 g/mL fraction contained primarily immature unprocessed virus particles, mature virus particles with a similar morphology to naturally occurring ENA could be identified by electron microscopy. Full-length sequence analysis of the ENTV-1 genome from the experimentally induced tumor revealed very few nucleotide changes relative to the original inoculum with only one conservative amino acid change. Taken together, these results demonstrate that ENTV-1 is associated with transmissible ENA in sheep and that under experimental conditions, lethal tumors are capable of developing in as little as 12 wpi demonstrating the acutely oncogenic nature of this ovine betaretrovirus.

Genetic characterization of small ruminant lentiviruses circulating in naturally infected sheep and goats in Ontario, Canada.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are related members of a group of small ruminant lentiviruses (SRLVs) that infect sheep and goats. SRLVs are endemic in many countries, including Canada. However, very little is known about the genetic characteristics of Canadian SRLVs, particularly in the province of Ontario. Given the importance of surveillance and eradication programs for the control of SRLVs, it is imperative that the diagnostic tests used to identify infected animals are sensitive to local strains of SRLVs. The aim of this work was to characterize SRLV strains circulating in Ontario and to evaluate the variability of the immunodominant regions of the Gag protein. In this study, the nearly complete gag sequence of 164 SRLVs, from 130 naturally infected sheep and 32 naturally infected goats from Ontario, was sequenced. Animals belonged to distantly located single and mixed species (sheep and goats) farms. Ovine lentiviruses from the same farm tended to cluster more closely together than did caprine lentiviruses from the same farm. Sequence analysis revealed a higher degree of heterogeneity among the caprine lentivirus sequences with an average inter-farm pairwise DNA distance of 10% and only 5% in the ovine lentivirus group. Interestingly, amplification of SRLVs from ELISA positive sheep was successful in 81% of cases, whereas amplification of SRLV proviral DNA was only possible in 55% of the ELISA positive goat samples; suggesting that a significant portion of caprine lentiviruses circulating in Ontario possess heterogeneity at the primer binding sites used in this study. Sequences of sheep and goat SRLVs from Ontario were assembled into phylogenetic trees with other known SRLVs and were found to belong to sequence groups A2 and B1, respectively, as defined by Shah et al. (2004a). A novel caprine lentivirus with a pairwise genetic difference of 15.6-25.4% relative to other group B subtypes was identified. Thus we suggest the designation of a novel subtype, B4, within the caprine lentivirus-like cluster. Lastly, we demonstrate evidence of recombination between ovine lentiviruses. These results emphasize the broad genetic diversity of SRLV strains circulating in the province of Ontario and show that the gag region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.