Murine Adenovirus Research Articles

Viral-bacterial co-infections screen in vitro reveals molecular processes affecting pathogen proliferation and host cell viability

The broadening of accessible methodologies has enabled mechanistic insights into single-pathogen infections, yet the molecular mechanisms underlying co-infections remain largely elusive, despite their clinical frequency and relevance, generally exacerbating symptom severity and fatality. Here, we describe an unbiased in vitro screening of pairwise co-infections in a murine macrophage model, quantifying pathogen proliferation and host cell death in parallel over time. The screen revealed that the majority of interactions are antagonistic for both metrics, highlighting general patterns depending on the pathogen virulence strategy. We subsequently decipher two distinct molecular interaction points: Firstly, murine Adenovirus 3 modifies ASC-dependent inflammasome responses in murine macrophages, altering host cell death and cytokine production, thereby impacting secondary Salmonella infection. Secondly, murine Adenovirus 2 infection triggers upregulation of Mprip, a crucial mediator of phagocytosis, which in turn causes increased Yersinia uptake, specifically in virus pre-infected bone-marrow-derived macrophages. This work therefore encompasses both a first-of-its-kind systematic assessment of host-pathogen-pathogen interactions, and mechanistic insight into molecular mediators during co-infection.

Human umbilical cord mesenchymal stem cell derived exosomes (HUCMSC-exos) recovery soluble fms-like tyrosine kinase-1 (sFlt-1)-induced endothelial dysfunction in preeclampsia

BackgroundPreeclampsia is a unique multisystem disorder that affects 5–8% of pregnancies. A high level of soluble fms-like tyrosine kinase-1 (sFlt-1) is a hallmark of preeclampsia that causes endothelial dysfunction. Exosomes derived from mesenchymal stem cells (MSCs) have been indicated to improve endothelial performances by transporting signals to target cells. We hypothesized that exosomes derived from MSCs have potential effects against preeclampsia.MethodsWe collected human umbilical cord MSC-derived exosomes (HUCMSC-exos) by ultracentrifugation. The size and morphology of the exosomes were examined using a transmission electron microscope and nanoparticle tracking analysis. Pregnant mice were injected with murine sFlt-1 adenovirus to build the preeclampsia-like mouse model and then treated with HUCMSC-exos. Human umbilical vein endothelial cells (HUVECs) were infected with lentiviruses expressing tet-on-sFlt-1 to obtain cells overexpressing sFlt-1. Cell proliferation and migration assays were used to measure the endothelial functions. The exosomes enriched proteins underlying mechanisms were explored by proteomic analysis.ResultsIn the current study, we successfully collected the cup-shaped HUCMSC-exos with diameters of 30–150 nm. In the sFlt-1-induced preeclampsia mouse model, HUCMSC-exos exhibited beneficial effects on adverse birth events by decreasing blood pressure and improving fetal birth weight. In addition, preeclamptic dams that were injected with HUCMSC-exos had rebuilt dense placental vascular networks. Furthermore, we observed that HUCMSC-exos partially rescued sFlt-1-induced HUVECs dysfunction in vitro. Proteomics analysis of HUCMSC-exos displayed functional enrichment in biological processes related to vesicle-mediated transport, cell communication, cell migration, and angiogenesis.ConclusionWe propose that exosomes derived from HUCMSCs contain abundant Versican and play beneficial roles in the birth outcomes of sFlt-1-induced preeclamptic mice by promoting angiogenesis.Graphical

Mastadenovirus Molecular Diversity in Waste and Environmental Waters from the Lisbon Metropolitan Area.

In face of the absence of epidemiological data regarding the circulation of human adenoviruses (HAdV) in Portugal, this study aimed at the evaluation of their molecular diversity in waste and environmental waters in the Lisbon Metropolitan Area (LMA). Using samples collected between 2018 and 2021, the HAdV hexon protein-coding sequence was partially amplified using three nested touch-down PCR protocols. The amplification products obtained were analyzed in parallel by two approaches: molecular cloning followed by Sanger sequencing and Next-Generation Sequencing (NGS) using Illumina® sequencing. The analysis of NGS-generated data allowed the identification of a higher diversity of HAdV-A (19%), -B (1%), -C (3%), -D (24%), and -F (25%) viral types, along with murine adenovirus (MAdV-2; 30%) in the wastewater treatment plant samples. On the other hand, HAdV-A (19%), -D (32%), and -F (36%) were identified in environmental samples, and possibly MAdV-2 (14%). These results demonstrate the presence of fecal contamination in environmental waters and the assessment of the diversity of this virus provides important information regarding the distribution of HAdV in LMA, including the detection of HAdV-F41, the most frequently reported in water worldwide.

Newly recruited intraepithelial Ly6A+CCR9+CD4+ T cells protect against enteric viral infection

The intestinal epithelium comprises the body's largest surface exposed to viruses. Additionally, the gut epithelium hosts a large population of intraepithelial T lymphocytes, or IELs, although their role in resistance against viral infections remains elusive. By fate-mapping Tcells recruited to the murine intestine, we observed an accumulation of newly recruited CD4+ Tcells after infection with murine norovirus CR6 and adenovirus type-2 (AdV), but not reovirus. CR6- and AdV-recruited intraepithelial CD4+ Tcells co-expressed Ly6A and chemokine receptor CCR9, exhibited T helper 1 and cytotoxic profiles, and conferred protection against AdV invivo and in an organoid model in an IFN-γ-dependent manner. Ablation of the Tcell receptor (TCR) or the transcription factor ThPOK in CD4+ Tcells prior to AdV infection prevented viral control, while TCR ablation during infection did not impact viral clearance. These results uncover a protective role for intraepithelial Ly6A+CCR9+CD4+ Tcells against enteric adenovirus.

Abstract B07: Oncolytic adenovirus 3 coding for CD40L as an enhancer of dendritic cell therapy

Abstract Dendritic cell (DC) therapy is considered a promising immunotherapeutic approach for treatment of advanced cancer. However, the immunosuppressive tumor microenvironment leads to DC dysfunction. Therefore, in clinical trials DC therapy has generally failed to fulfill its expectations. Oncolytic adenoviruses are well tolerated and have shown to activate antitumor immune responses. Importantly, they can convert immunosuppression locally into a proinflammatory state. To improve the proper activation of transferred DCs, we armed oncolytic adenovirus with CD40 ligand (CD40L). CD40L is known to play an important role in the regulation of immune cells through its capacity to stimulate dendritic cells that leads to the activation of cytotoxic T cells. Therefore, we generated a novel virus Ad3-hTERT-CMV-hCD40L, which is fully serotype 3 adenovirus (Ad3) for intravenous delivery. It features a human telomerase reverse transcriptase (hTERT) promoter for tumor specific replication and expresses human CD40L (hCD40L) under a cytomegalovirus (CMV) promoter for induction of antitumor efficacy. Of note, human and animal data have shown the ability of Ad3 to successfully reach tumors through the intravenous route. In syngeneic studies in an immunocompetent model, DC therapy with our murine CD40L-armed adenovirus showed significant antitumor immune response. This enhanced therapeutic effect is associated with increased tumor specific T cells and induction of T-helper type 1 immune response. This synergistic effect was further evaluated in mice humanized with human peripheral blood mononuclear cells. Treatment with hCD40L-armed adenovirus and human DCs showed 100% survival in conjunction with tumor control. To conclude, CD40L armed oncolytic adenovirus 3 improves DC therapy by favorable alteration of tumor microenvironment. These findings support clinical trials where DC therapy is enhanced with oncolytic adenovirus. Citation Format: João Manuel Santos, Akseli Hemminki, Mikko Siurala, Otto Hemminki, Riikka Havunen, Victor Cervera-Carrascon, Suvi Sorsa, Hongjie Wang, Andre Lieber, Tanja de Gruijl, Anna Kanerva, Sadia Zafar. Oncolytic adenovirus 3 coding for CD40L as an enhancer of dendritic cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B07.

Opportunistic sampling of wild native and invasive birds reveals a rich diversity of adenoviruses in Australia.

Little is known about the diversity of adenoviruses in wild birds and how they have evolved and are maintained in complex ecosystems. In this study, 409 samples were collected from woodland birds caught for banding (droppings), birds submitted to a wildlife hospital (droppings and tissues), silver gulls (droppings or tissues), and feral pigeons (Columbia livia; oral, cloacal swabs, or tissues) from the Greater Sydney area in NSW, Australia. Additional samples were from native pigeons and doves (swabs) presented to the Healesville Sanctuary, VIC, Australia. Samples were screened for adenovirus DNA using degenerate primers and polymerase chain reaction. Adenovirus sequences were detected in eighty-three samples representing thirty-five novel amino acid sequences. Fourteen novel sequences were atadenoviruses, seven were aviadenoviruses, twelve were siadenoviruses, and one was a mastadenovirus. Sequences from passerine birds were predominately found to form a single lineage within the atadenoviruses, a second lineage in the siadenoviruses, and a third smaller aviadenovirus lineage. These viruses appeared to have co-evolved with a diverse group of woodland birds that share similar habitat. Evidence for host/virus co-evolution in some viruses and a wide host range in others was observed. A high prevalence of adenovirus infection was found in rainbow lorikeets (Trichoglossus haematodus), galahs (Eolophus roseicapilla), and sulphur-crested cockatoos (Cacatua galerita). Sequences were either identical to or mapped to already established lineages in the Aviadenovirus, Siadenovirus, and Atadenovirus genera, suggesting a possible origin of the psittacine adenoviruses in ancestral Australian psittacine birds. The sequences of passerine and psittacine origin provided insight into diversity and structure of the Atadenovirus genus and demonstrated for the first-time viruses of passerine origin in the Aviadenovirus genus. Four unrelated adenovirus sequences were found in silver gull samples (Chroicocephalus novaehollandiae), including one of pigeon origin, suggesting environmental virus exposure. Three pigeon adenovirus types were detected in feral pigeons and infection prevalence was high. Evidence for host switching between invasive species and native species and native species and invasive species was documented. A variant of a murine adenovirus was detected in kidney tissue from two bird species suggesting mouse to bird transmission.

Analysis of adenovirus DNA detected in rodent species from the Democratic Republic of the Congo indicates potentially novel adenovirus types.

Different species of adenoviruses (AdVs) infect humans and animals and are known for their role as pathogens, especially in humans, with animals, primarily rodents, often serving as model systems. However, although we know over 100 types of human AdVs, we know comparatively little about the diversity of animal AdVs. Due to the fact that rodents are the most diverse family of mammals and a standard model system for human disease, we set out to sample African rodents native to the Democratic Republic of the Congo and test them for AdV DNA using a semi-nested consensus PCR. A total of 775 animals were tested, and viral DNA was detected in four of them. The AdV DNA found belongs to three different AdVs, all being closely related to murine adenovirus 2 (MAdV-2). Considering the genetic differences of the amplicon were 9%, 11% and 19% from MAdV-2 and at least 10% from each other, they seem to belong to up to three different novel types within the Murine mastadenovirus B species. This evidence of genetic diversity highlights the opportunities to isolate and study additional AdVs that infect rodents as models for AdV biology and pathology.

Murine adenoviruses: tools for studying adenovirus pathogenesis in a natural host.

Small laboratory animals are powerful models for investigating invivo viral pathogenesis of a number of viruses. For adenoviruses (AdVs), however, species-specificity poses limitations to studying human adenoviruses (HAdVs) in mice and other small laboratory animals. Thus, this review covers work on naturally occurring mouse AdVs, primarily mouse adenovirus type 1 (MAdV-1), a member of the species Murine mastadenovirus A. Molecular genetics, virus life cycle, cell and tissue tropism, interactions with the host immune response, persistence, and host genetics of susceptibility are described. A brief discussion of MAdV-2 (member of species Murine mastadenovirus B) and MAdV-3 (member of species Murine mastadenovirus C) is included. We report the use of MAdVs in the development of vectors and vaccines.

Structure and N-acetylglucosamine binding of the distal domain of mouse adenovirus 2 fibre.

Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple β-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.

Expression of human CD46 and trans-complementation by murine adenovirus 1 fails to allow productive infection by a group B oncolytic adenovirus\xa0in murine cancer cells

BackgroundOncolytic viruses are currently experiencing accelerated development in several laboratories worldwide, with some forty-seven clinical trials currently recruiting. Many oncolytic viruses combine targeted cytotoxicity to cancer cells with a proinflammatory cell lysis. Due to their additional potential to express immunomodulatory transgenes, they are also often known as oncolytic viral vaccines. However, several types of oncolytic viruses are human-specific and the lack of suitable immune-competent animal models complicates biologically relevant evaluation of their vaccine potential. This is a particular challenge for group B adenoviruses, which fail to infect even those immunocompetent animal model systems identified as semi-permissive for type 5 adenovirus. Here, we aim to develop a murine cell line capable of supporting replication of a group B oncolytic adenovirus, enadenotucirev (EnAd), for incorporation into a syngeneic immunocompetent animal model to explore the oncolytic vaccine potential of group B oncolytic viruses.MethodsTransgenic murine cell lines were infected with EnAd expressing GFP transgene under replication-independent or -dependent promoters. Virus mRNA expression, genome replication, and late protein expression were determined by qRT-PCR, qPCR, and immunoblotting, respectively. We also use Balb/c immune-competent mice to determine the tumourogenicity and infectivity of transgenic murine cell lines.ResultsOur results show that a broad range of human carcinoma cells will support EnAd replication, but not murine carcinoma cells. Murine cells can be readily modified to express surface human CD46, one of the receptors for group B adenoviruses, allowing receptor-mediated uptake of EnAd particles into the murine cells and expression of CMV promoter-driven transgenes. Although the early E1A mRNA was expressed in murine cells at levels similar to human cells, adenovirus E2B and Fibre mRNA expression levels were hampered and few virus genomes were produced. Unlike previous reports on group C adenoviruses, trans-complementation of group B adenoviruses by co-infection with mouse adenovirus 1 did not rescue replication. A panel of group B adenoviruses expressing individual mouse adenovirus 1 genes were also unable to rescue EnAd replication.ConclusionTogether, these results indicate that there may be major differences in the early stages of replication of group C and B adenoviruses in murine cells, and that the block to the life cycle of B adenoviruses in murine cells occurs in the early stage of virus replication, perhaps reflecting poor activity of Ad11p E1A in murine cells.

Metagenomics for the study of viruses in urban sewage as a tool for public health surveillance

The application of next-generation sequencing (NGS) techniques for the identification of viruses present in urban sewage has not been fully explored. This is partially due to a lack of reliable and sensitive protocols for studying viral diversity and to the highly complex analysis required for NGS data processing. One important step towards this goal is finding methods that can efficiently concentrate viruses from sewage samples. Here the application of a virus concentration method based on skimmed milk organic flocculation (SMF) using 10L of sewage collected in different seasons enabled the detection of many viruses. However, some viruses, such as human adenoviruses, could not always be detected using metagenomics, even when quantitative PCR (qPCR) assessments were positive. A targeted metagenomic assay for adenoviruses was conducted and 59.41% of the obtained reads were assigned to murine adenoviruses. However, up to 20 different human adenoviruses (HAdV) were detected by this targeted assay being the most abundant HAdV-41 (29.24%) and HAdV-51 (1.63%). To improve metagenomics' sensitivity, two different protocols for virus concentration were comparatively analysed: an ultracentrifugation protocol and a lower-volume SMF protocol. The sewage virome contained 41 viral families, including pathogenic viral species from families Caliciviridae, Adenoviridae, Astroviridae, Picornaviridae, Polyomaviridae, Papillomaviridae and Hepeviridae. The contribution of urine to sewage metavirome seems to be restricted to a few specific DNA viral families, including the polyomavirus and papillomavirus species. In experimental infections with sewage in a rhesus macaque model, infective human hepatitis E and JC polyomavirus were identified. Urban raw sewage consists of the excreta of thousands of inhabitants; therefore, it is a representative sample for epidemiological surveillance purposes. The knowledge of the metavirome is of significance to public health, highlighting the presence of viral strains that are circulating within a population while acting as a complex matrix for viral discovery.

Assessment of diversity of adenovirus DNA polymerase gene in recreational waters facilitated by ultracentrifugal concentration.

Adenoviruses (AdV) are related to respiratory and gastrointestinal diseases in animals and human beings. Their wide genetic diversity in water bodies and their resistance to environmental conditions allow the use of AdV as a reliable marker for detection of fecal contamination. In this work, the diversity of AdV along Belo Stream - in the city of Caxias do Sul, Rio Grande do Sul, Brazil - was evaluated. Samples were compared in both concentrated and unconcentrated forms. The identification of different AdV species was performed by amplifying a partial sequence of the DNA polymerase gene. AdV was detected in 24 out of 55 concentrated samples (43.6%) and the following species were identified: human adenovirus (HAdV) species C (4/55; 7.2%), D (6/55; 10.9%), E (2/55; 3.6%), and F (9/55; 16.3%). AdV related to other mammalian hosts, such as bovine adenovirus (1/55, 1.8%) and murine adenovirus (2/55, 3.6%), have also been identified; 23.6% (13/55) of the unconcentrated samples were positive, and identified as HAdV species C (6/55, 10.9%), D (1/55, 1.8%), and F (6/55, 10.9%). Results obtained evidenced the presence and the great diversity of AdV, mainly of human origin, circulating in Belo Stream. As expected, the concentration step performed helped to detect AdV in more samples.

An Oncolytic Adenovirus Encoding Decorin and Granulocyte Macrophage Colony Stimulating Factor Inhibits Tumor Growth in a Colorectal Tumor Model by Targeting Pro-Tumorigenic Signals and via Immune Activation.

In advanced and metastatic stages of colorectal cancer (CRC), reduced sensitivity to conventional strategies is still a major obstacle to successful treatments. Decorin is an important regulator in the development and progression of various cancers. To examine if CRC patients have altered decorin levels, expression of decorin and its target genes, Met and vascular endothelial growth factor A (VEGFA), were analyzed in their tumors. Compared to normal tissues, decorin expression was reduced in CRC patients' tumors, while there were increased Met and VEGFA levels. To develop a novel therapy for CRC, rAd.DCN.GM, an oncolytic adenovirus encoding decorin and granulocyte macrophage colony stimulating factor (GM-CSF), has been created. Several therapeutic strategies expressing GM-CSF have been employed in clinical trials for treating metastatic colorectal cancer. In this study, infection of CRC cells with rAd.DCN.GM expressed decorin and GM-CSF, and produced cytotoxicity. In murine CT26 xenografts, rAd.DCN.GM and control adenoviruses were administrated intratumorally on days 7 and 10, and tumor volumes were monitored over time. The study showed that rAd.DCN.GM inhibited the tumor growth and lung metastases significantly. rAd.DCN.GM induced apoptosis, inhibited proliferation, and downregulated angiogenesis and epithelial mesenchymal transition markers in the tumors. On day 12 and day 29, the immune-activation in the peripheral blood, tumors, and spleens were analyzed. rAd.DCN.GM increased CD8+ T lymphocytes in the blood, upregulated perforin and granzyme B in the tumors, inhibited transforming growth factor beta expression, and promoted dendritic-cell production in the spleen. In conclusion, rAd.DCN.GM inhibited the tumor growth and metastasis of CT26 tumors, downregulated multiple pro-tumorigenic pathways, and activated antitumor immune responses.

ZMPSTE24 Is Downstream Effector of Interferon-Induced Transmembrane Antiviral Activity.

The zinc metalloprotease ZMPSTE24 is a constitutively and ubiquitously expressed host restriction factor that is responsible for limiting infection by a broad spectrum of enveloped viruses, including influenza A, vesicular stomatitis, zika, ebola, Sindbis, cowpox, and vaccinia viruses, but not murine leukemia or adenovirus. Antiviral function is independent of ZMPSTE24 enzymatic activity. Protein interaction and genetic complementation studies indicate that ZMPSTE24 is a component of a common antiviral pathway that is associated with interferon-induced transmembrane proteins. In vivo studies with zmpste24-deficient mice demonstrate the importance of ZMPSTE24 for antiviral defense.

ZMPSTE24 defends against influenza and other pathogenic viruses.

Zinc metallopeptidase STE24 (ZMPSTE24) is a transmembrane metalloprotease whose catalytic activity is critical for processing lamin A on the inner nuclear membrane and clearing clogged translocons on the endoplasmic reticulum. We now report ZMPSTE24 is a virus-specific effector that restricts enveloped RNA and DNA viruses, including influenza A, Zika, Ebola, Sindbis, vesicular stomatitis, cowpox, and vaccinia, but not murine leukemia or adenovirus. ZMPSTE24-mediated antiviral action is independent of protease activity. Coimmunoprecipitation studies indicate ZMPSTE24 can complex with proteins of the interferon-induced transmembrane protein (IFITM) family. IFITM proteins impede viral entry, and ZMPSTE24 expression is necessary for IFITM antiviral activity. In vivo studies demonstrate ZMPSTE24-deficient mice display higher viral burdens, enhanced cytokine production, and increased mortality after influenza infection. Collectively, these findings identify ZMPSTE24 as an intrinsic broad-spectrum antiviral protein and provide insights into antiviral defense mechanisms.

Virucidal efficacy of a sonicated hydrogen peroxide system (trophon® EPR) following European and German test methods.

Aim: The virucidal efficacy of an automated ultrasound probe disinfector (trophon® EPR) was evaluated in a three step procedure according to European and German test methods. This system uses sonicated hydrogen peroxide mist (35%) at elevated temperature (50°C) in a closed chamber with control of all parameters within a 7 minute cycle. Methods: In the first step of examination, the peroxide solution was tested in a quantitative suspension assay according to the Guideline of Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV) e.V. and Robert Koch-Institute (RKI) and in parallel with the European Norm EN 14476 with all test viruses creating a virucidal claim. In the second step, the virucidal efficacy of the hydrogen peroxide solution was evaluated in a hard surface carrier test according to the Guideline of DVV with adenovirus, murine norovirus and parvovirus simulating practical conditions. Finally, the efficacy was evaluated by the automated system using stainless steel carriers inoculated with test virus and positioned at different levels inside the chamber. Results: A ≥4 log10 reduction of virus titre was demonstrated with all methods including carrier tests with murine norovirus, adenovirus, and parvovirus using the automated device. Conclusion: The automated device is able to inactivate test viruses of German and European norms and can therefore claim efficacy against human pathogenic enveloped and non-enveloped viruses. This includes human papillomaviruses which form part of the complete virucidal claim.

Effect of Cross-Fostering on a Variety of Viral Agents in Conventionally Bred Rats

We aimed to compare the serological profiles of the pups that were housed with their biological mothers and the ones which were transferred to foster-dams after birth, with regard to certain viral agents. The day of delivery for the pregnant rat is recognized as the postnatal day 0 (P0). The pups born from Group 1 [P(0), P(21), P(60)] pregnant rats were housed with their biological mothers. In Group 2 [P(1), P(21), P(60)], however, P(1) consisted of pups housed with their biological mother for 24 hours while P(21) and P(60) consisted of pups transferred from the their biological mother immediately at birth. Antibody response against Lymphocytic Choriomeningitis Virus (LCMV), Rat Parvo Virus (RPV), Sendai Virus (SV) and Toolan's H1 viruses was not detected in dams and pups of all the groups, whereas high rate (45%) of antibody positivity was observed in response to Kilham' Rat Virus (KRV). No statistically significant difference was determined between the pups left with their biological mother and with the foster mothers, except for Murine Adeno Virus type 1, type 2 (MAD 1 and 2), Reovirus type 3 (REO-3) and Theiler's Murine Encephalomyelitis Virus (TMEV). It was conspicuous in terms of REO-3 that for the pups it was essential to stay with their biological mothers and nursed during the first 24 hours. As a result, foster mother usage in breeding laboratory animals is found to be beneficial against a variety of viral agents for the pups particularly in the later period of their lives and we suggest to continue this implementation in routine laboratory practice whenever necessary.

Assessing the feasibility of fly based surveillance of wildlife infectious diseases.

Monitoring wildlife infectious agents requires acquiring samples suitable for analyses, which is often logistically demanding. A possible alternative to invasive or non-invasive sampling of wild-living vertebrates is the use of vertebrate material contained in invertebrates feeding on them, their feces, or their remains. Carrion flies have been shown to contain vertebrate DNA; here we investigate whether they might also be suitable for wildlife pathogen detection. We collected 498 flies in Taï National Park, Côte d’Ivoire, a tropical rainforest and examined them for adenoviruses (family Adenoviridae), whose DNA is frequently shed in feces of local mammals. Adenoviral DNA was detected in 6/142 mammal-positive flies. Phylogenetic analyses revealed that five of these sequences were closely related to sequences obtained from local non-human primates, while the sixth sequence was closely related to a murine adenovirus. Next-generation sequencing-based DNA-profiling of the meals of the respective flies identified putative hosts that were a good fit to those suggested by adenoviral sequence affinities. We conclude that, while characterizing the genetic diversity of wildlife infectious agents through fly-based monitoring may not be cost-efficient, this method could probably be used to detect the genetic material of wildlife infectious agents causing wildlife mass mortality in pristine areas.

Oncostatin M overexpression induces skin inflammation but is not required in the mouse model of imiquimod-induced psoriasis-like inflammation.

Oncostatin M (OSM) has been reported to be overexpressed in psoriasis skin lesions and to exert proinflammatory effects in vitro on human keratinocytes. Here, we report the proinflammatory role of OSM in vivo in a mouse model of skin inflammation induced by intradermal injection of murine OSM-encoding adenovirus (AdOSM) and compare with that induced by IL-6 injection. Here, we show that OSM potently regulates the expression of genes involved in skin inflammation and epidermal differentiation in murine primary keratinocytes. In vivo, intradermal injection of AdOSM in mouse ears provoked robust skin inflammation with epidermal thickening and keratinocyte proliferation, while minimal effect was observed after AdIL-6 injection. OSM overexpression in the skin increased the expression of the S100A8/9 antimicrobial peptides, CXCL3, CCL2, CCL5, CCL20, and Th1/Th2 cytokines, in correlation with neutrophil and macrophage infiltration. In contrast, OSM downregulated the expression of epidermal differentiation genes, such as cytokeratin-10 or filaggrin. Collectively, these results support the proinflammatory role of OSM when it is overexpressed in the skin. However, OSM expression was not required in the murine model of psoriasis induced by topical application of imiquimod, as demonstrated by the inflammatory phenotype of OSM-deficient mice or wild-type mice treated with anti-OSM antibodies.

Abstract B18: Adoptive T-cell transfer combined with murine cytokine-armed adenoviruses for the treatment of melanoma

Abstract Adoptive cell transfer (ACT) has shown promising results in clinical trials for melanoma, with response rates ranging from 40% to 72%. We aimed to enhance the efficacy of ACT by coupling it with adenoviruses coding for immunostimulatory cytokines. The non-replicating adenoviruses that were constructed (Ad5-CMV-mIFNg, Ad5-CMV-mIL2, Ad5-CMV-mIFNb and Ad5-CMV-mTNFa) were confirmed to produce biologically active murine cytokines in vitro. These viruses were then used in combination with adoptive transfer of OT-1 TCR transgenic T-cells to treat C57BL/6 mice bearing B16-OVA melanoma tumors. The animals were administered intraperitoneally with 1.5 x 106 CD8+-enriched OT-1 cells with or without intratumoral injections of cytokine-coding adenoviruses (1 x 109 viral particles/tumor). Combination treatment with Ad5-CMV-mIL2 and OT-1 resulted in statistically significant antitumor efficacy when compared with either monotherapy and untreated control (combination vs. virus, p=0.026; combination vs. OT1, p=0.003; combination vs. mock, p<0.001). Similar results were obtained with other cytokine-coding virus and OT-1 combinations, except for mIFNb. In further experiments a triple combination of Ad5-CMV-mIL2 + Ad5-CMV-mTNFa (1:1 ratio) and OT-1 T-cells were used to treat B16-OVA; improved antitumor efficacy was observed with the triple combination over dual agent therapies (triple combination vs. Ad5-CMV-mIL2 + OT-1, p=0.001; triple combination vs. Ad5-CMV-mTNFa + OT-1, p=0.049). In summary, these results support the further development of a novel immunotherapeutic approach for the treatment of melanoma. Citation Format: Mikko Siurala, Riikka Havunen, Simona Bramante, Dipongkor Saha, Siri Tähtinen, Markus Vähä-Koskela, Akseli Hemminki. Adoptive T-cell transfer combined with murine cytokine-armed adenoviruses for the treatment of melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B18.