Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy.

Intravesical treatment using either reovirus or natural killer (NK) cells serves as an efficient strategy for the treatment of bladder cancer cells (BCCs); however, corresponding monotherapies have often shown modest cytotoxicity. The potential of a locoregional combination using high-dose reovirus and NK cell therapy in an intravesical approach has not yet been studied. In this study, we evaluated the effectiveness of reoviruses and expanded NK cells (eNK) as potential strategies for the treatment of bladder cancer. The anti-tumor effects of mono-treatment with reovirus type 3 Dearing strain (RC402 and RP116) and in combination with interleukin (IL)-18/-21-pretreated eNK cells were investigated on BCC lines (5637, HT-1376, and 253J-BV) using intravesical therapy to simulate in vitro model. RP116 and IL-18/-21-pretreated eNK cells exhibited effective cytotoxicity against grade 1 carcinoma (5637 cells) when used alone, but not against HT-1376 (grade 2 carcinoma) and 253J-BV cells (derived from a metastatic site). Notably, combining RP116 with IL-18/-21-pretreated eNK cells displayed effective cytotoxicity against both HT-1376 and 253J-BV cells. Our findings underscore the potential of a combination therapy using reoviruses and NK cells as a promising strategy for treating bladder cancer.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

Reovirus infection of tumor cells reduces the expression of NKG2D ligands, leading to impaired NK-cell cytotoxicity and functionality.

In recent years, reoviruses have been of major interest in immunotherapy because of their oncolytic properties. Preclinical and clinical trials, in which reovirus was used for the treatment of melanoma and glioblastoma, have paved the way for future clinical use of reovirus. However, little is known about how reovirus infection affects the tumor microenvironment and immune response towards infected tumor cells. Studies have shown that reovirus can directly stimulate natural killer (NK) cells, but how reovirus affects cellular ligands on tumor cells, which are ultimately key to tumor recognition and elimination by NK cells, has not been investigated. We tested how reovirus infection affects the binding of the NK Group-2 member D (NKG2D) receptor, which is a dominant mediator of NK cell anti-tumor activity. Using models of human-derived melanoma and glioblastoma tumors, we demonstrated that NKG2D ligands are downregulated in tumor cells post-reovirus-infection due to the impaired translation of these ligands in reovirus-infected cells. Moreover, we showed that downregulation of NKG2D ligands significantly impaired the binding of NKG2D to infected tumor cells. We further demonstrated that reduced recognition of NKG2D ligands significantly alters NK cell anti-tumor cytotoxicity in human primary NK cells and in the NK cell line NK-92. Thus, this study provides novel insights into reovirus-host interactions and could lead to the development of novel reovirus-based therapeutics that enhance the anti-tumor immune response.

Modulation of Reoviral Cytolysis (I): Combination Therapeutics.

Patients with stage IV gastric cancer suffer from dismal outcomes, a challenge especially in many Asian populations and for which new therapeutic options are needed. To explore this issue, we used oncolytic reovirus in combination with currently used chemotherapeutic drugs (irinotecan, paclitaxel, and docetaxel) for the treatment of gastric and other gastrointestinal cancer cells in vitro and in a mouse model. Cell viability in vitro was quantified by WST-1 assays in human cancer cell lines treated with reovirus and/or chemotherapeutic agents. The expression of reovirus protein and caspase activity was determined by flow cytometry. For in vivo studies, athymic mice received intratumoral injections of reovirus in combination with irinotecan or paclitaxel, after which tumor size was monitored. In contrast to expectations, we found that reoviral oncolysis was only poorly correlated with Ras pathway activation. Even so, the combination of reovirus with chemotherapeutic agents showed synergistic cytopathic effects in vitro, plus enhanced reovirus replication and apoptosis. In vivo experiments showed that reovirus alone can reduce tumor size and that the combination of reovirus with chemotherapeutic agents enhances this effect. Thus, we find that oncolytic reovirus therapy is effective against gastric cancer. Moreover, the combination of reovirus and chemotherapeutic agents synergistically enhanced cytotoxicity in human gastric cancer cell lines in vitro and in vivo. Our data support the use of reovirus in combination with chemotherapy in further clinical trials, and highlight the need for better biomarkers for reoviral oncolytic responsiveness.

Modulation of Reoviral Cytolysis (II): Cellular Stemness.

Oncolytic viruses (OVs) are an emerging cancer therapeutic that are intended to act by selectively targeting and lysing cancerous cells and by stimulating anti-tumour immune responses, while leaving normal cells mainly unaffected. Reovirus is a well-studied OV that is undergoing advanced clinical trials and has received FDA approval in selected circumstances. However, the mechanisms governing reoviral selectivity are not well characterised despite many years of effort, including those in our accompanying paper where we characterize pathways that do not consistently modulate reoviral cytolysis. We have earlier shown that reovirus is capable of infecting and lysing both certain types of cancer cells and also cancer stem cells, and here we demonstrate its ability to also infect and kill healthy pluripotent stem cells (PSCs). This led us to hypothesize that pathways responsible for stemness may constitute a novel route for the modulation of reoviral tropism. We find that reovirus is capable of killing both murine and human embryonic and induced pluripotent stem cells. Differentiation of PSCs alters the cells' reoviral-permissive state to a resistant one. In a breast cancer cell line that was resistant to reoviral oncolysis, induction of pluripotency programming rendered the cells permissive to cytolysis. Bioinformatic analysis indicates that expression of the Yamanaka pluripotency factors may be associated with regulating reoviral selectivity. Mechanistic insights from these studies will be useful for the advancement of reoviral oncolytic therapy.

Nonstructural protein NS17 of grass carp reovirus Honghu strain promotes virus infection by mediating cell-cell fusion and apoptosis.

Fusion-associated small transmembrane (FAST) proteins can promote cell fusion, alter membrane permeability and trigger apoptosis to promote virus proliferation in orthoreoviruses. However, it is unknown whether FAST proteins perform these functions in aquareoviruses (AqRVs). Non-structural protein 17 (NS17) carried by grass carp reovirus Honghu strain (GCRV-HH196) belongs to the FAST protein family, and we preliminarily explored its relevance to virus infection. NS17 has similar domains to FAST protein NS16 of GCRV-873, comprising a transmembrane domain, a polybasic cluster, a hydrophobic patch and a polyproline motif. It was observed in the cytoplasm and the cell membrane. Overexpression of NS17 enhanced the efficiency of cell-cell fusion induced by GCRV-HH196 and promoted virus replication. Overexpression of NS17 also led to DNA fragmentation and reactive oxygen species (ROS) accumulation, and it triggered apoptosis. The findings illuminate the functions of NS17 in GCRV infection, and provide a reference for the development of novel antiviral strategies.

Deciphering molecular mechanisms stabilizing the reovirus-binding complex.

Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.

Metabolomics in rare minnow (Gobiocypris rarus) after infection by attenuated and virulent grass carp reovirus genotype â ¡.

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes hemorrhagic disease in a variety fish, seriously affecting the aquaculture industry in China. However, the pathogenesis of GCRV Ⅱ is unclear. Rare minnow is an ideal model organism to study the pathogenesis of GCRV Ⅱ. Herein, we applied liquid chromatography-tandem mass spectrometry metabolomics to investigate metabolic responses in the spleen and hepatopancreas of rare minnow injected with virulent GCRV Ⅱ isolate DY197 and attenuated isolate QJ205. Results indicated that marked metabolic changes were identified in both the spleen and hepatopancreas after GCRV Ⅱ infection, and the virulent DY197 strain induced more significantly different metabolites (SDMs) than the attenuated QJ205 strain. Moreover, most SDMs were downregulated in the spleen and tend to be upregulated in hepatopancreas. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that tissue-specific metabolic responses were identified after viruses infection, and the virulent DY197 strain induced more SDMs involved in amino acid metabolism in the spleen, especially the tryptophan metabolism, cysteine and methionine metabolism, which were essential for immune regulation in host; Meanwhile, nucleotide metabolism, protein synthesis and metabolism related pathways were enriched in the hepatopancreas by both virulent and attenuated strains. Our findings revealed the large scale metabolic alterations in rare minnow in response to attenuated and virulent GCRV Ⅱ infection, which will lead to a better understanding of the pathogenesis of viruses and host-pathogens interactions.

Assessing the role of Piscine orthoreovirus in disease and the associated risk for wild Pacific salmon.

This paper is a response to Polinski, M. P. et al. Innate antiviral defense demonstrates high energetic efficiency in a bony fish. BMC Biology 19, 138 (2021). https://doi.org/10.1186/s12915-021-01069-2.

An aquatic virus exploits the IL6-STAT3-HSP90 signaling axis to promote viral entry.

Viral seasonality in the aquaculture industry is an important scientific issue for decades. While the molecular mechanisms underpinning the temperature-dependent pathogenesis of aquatic viral diseases remain largely unknown. Here we report that temperature-dependent activation of IL6-STAT3 signaling was exploited by grass carp reovirus (GCRV) to promote viral entry via increasing the expression of heat shock protein 90 (HSP90). Deploying GCRV infection as a model system, we discovered that GCRV induces the IL6-STAT3-HSP90 signaling activation to achieve temperature-dependent viral entry. Further biochemical and microscopic analyses revealed that the major capsid protein VP7 of GCRV interacted with HSP90 and relevant membrane-associated proteins to boost viral entry. Accordingly, exogenous expression of either IL6, HSP90, or VP7 in cells increased GCRV entry in a dose-dependent manner. Interestingly, other viruses (e.g., koi herpesvirus, Rhabdovirus carpio, Chinese giant salamander iridovirus) infecting ectothermic vertebrates have evolved a similar mechanism to promote their infection. This work delineates a molecular mechanism by which an aquatic viral pathogen exploits the host temperature-related immune response to promote its entry and replication, instructing us on new ways to develop targeted preventives and therapeutics for aquaculture viral diseases.

Oncolytic Reoviruses: Can These Emerging Zoonotic Reoviruses Be Tamed and Utilized?

Orthoreovirus is a nonenveloped double-stranded RNA virus under the Reoviridae family. This group of viruses, especially mammalian orthoreovirus (MRV), are reported with great therapeutic values due to their oncolytic effects. In this review, the life cycle and oncolytic effect of MRV and a few emerging reoviruses were summarized. This article also highlights the challenges and strategies of utilizing MRV and the emerging reoviruses, avian orthoreovirus (ARV) and pteropine orthoreovirus (PRV), as oncolytic viruses (OVs). Besides, the emergence of potential ARV and PRV as OVs were discussed in comparison to MRV. Finally, the risk of reovirus as zoonosis or reverse zoonosis (zooanthroponosis) were debated, and concerns were raised in this article, which warrant continue surveillance of reovirus (MRV, ARV, and PRV) in animals, humans, and the environment.

Duck cGAS inhibits DNA and RNA virus replication by activating IFNs and antiviral ISGs.

Cyclic GMP-AMP Synthase (cGAS) is a pivotal adaptor of the signaling pathways involving the pattern recognition receptors and plays an important role in apoptosis and immune regulation. The cGAS function in mammals has been investigated extensively; however, the function of duck cGAS (du-cGAS) in response to viral infections is still unclear. This study aimed to clone the mallard (Anas platyrhynchos) cGAS homolog to investigate the function of duck cGAS (du-cGAS) in host antiviral innate immunity. The results showed that the open reading frame (ORF) region of the du-cGAS gene was 1296 bp, encoding 432 amino acids (aa) and exhibiting similar functional domains with its chicken counterpart. Knockdown of the endogenous du-cGAS by specific sgRNA strongly increased the replication of DNA viruses, including duck adenovirus B2 (DAdV B2) and duck short beak and dwarfism syndrome virus (SBDSV). However, the knockout did not impair the replication of novel duck reovirus (NDRV), an RNA virus. Furthermore, the mRNA expressions of type I interferon (IFNs) and vital interferon-stimulated genes (ISGs) were remarkably reduced in the du-cGAS knockout DEF cell line. Inversely, du-cGAS overexpression greatly activated the transcription of IFN-α, IFN-ß, and vital ISGs, and impaired the replication of DAdV B2, SBDSV, and NDRV in the DEF cell line. Importantly, we found that a deletion of 68 aa in the N terminus didn't impair the antiviral function of du-cGAS. Overexpressing NTase Core, C-Domain (Mab21), or Zinc-Ribbon domain independently had no antiviral effects. Generally, these results reveal that du-cGAS is a vital component of the innate immune system of ducks, with a universal antiviral activity, and provides a useful strategy for the control of waterfowl viral diseases.

The µ2 and λ1 Proteins of Mammalian Reovirus Modulate Early Events Leading to Induction of the Interferon Signaling Network.

It has been previously shown that amino acid polymorphisms in reovirus proteins µ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of ß-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of µ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells.

Grass Carp Reovirus triggers autophagy enhancing virus replication via the Akt/mTOR pathway.

Autophagy impacts the replication cycle of many viruses. Grass Carp Reovirus (GCRV) is an agent that seriously affects the development of the grass carp aquaculture industry. The role of autophagy in GCRV infection is not clearly understood. In this study, we identified that GCRV infection triggered autophagy in CIK cells, which was demonstrated by transmission electron microscopy, the conversion of LC3B I to LC3B II and the level of autophagy substrate p62. Furthermore, we found that GCRV infection activated Akt-mTOR signaling pathway, and the conversion of LC3B I to LC3B II was increased by inhibiting mTOR with rapamycin (Rap) but decreased by activating Akt with insulin. We then assessed the effects of autophagy on GCRV replication. We found that inducing autophagy with Rap promoted GCRV proliferation but inhibiting autophagy with 3 MA or CQ inhibited GCRV replication in CIK cells. Moreover, it was found that enhancing Akt-mTOR activity by insulin, GCRV VP7 protein and viral titers of GCRV were decreased. Collectively, these results indicated that GCRV infection induced autophagy involved in GCRV replication via the Akt-mTOR signal pathway. Thus, new insights into GCRV pathogenesis and antiviral treatment strategies are provided.

Pteropine Orthoreovirus, PRV7S (Sikamat Virus) Demonstrates Oncolysis in Nasopharyngeal Carcinoma Cell Lines.

BACKGROUND: Oncolytic properties had been demonstrated in Mammalian Orthoreovirus (MRV) and Avian Orthorevirus (ARV). Besides MRV and ARV, Pteropine Orthoreovirus (PRV) is also categorized under the genus Orthoreovirus. PRV7S (Sikamat virus) is an orthoreovirus isolated in Malaysia. Present study aims to investigate the oncolytic effects of PRV7S on ranges of nasopharyngeal carcinoma (NPC) cells through apoptosis in comparison to MRV3. METHODS: Non-cancerous nasopharyngeal (NCNP) and NPC cells were infected by PRV7S and MRV3. The effects of PRV7S on the proliferation inhibition and apoptotic activity of NPC cells was examined using MTT assay and flow cytometry. Additionally, western blot assay was performed to analyze the expression of RAS and apoptotic protein. Lastly, qPCR assay was performed to demonstrate that PRV7S and MRV3 replicated in infected-NPC and infected-NCNP cells. RESULTS: The proliferation of NPC cells were significantly inhibited after PRV7S infection in a time dependent manner in comparison to infected-NCPC cells. Flow cytometry analysis showed that PRV7S infection was able to induce apoptosis on NPC cells at 48 hpi. Western blot results showed that upon PRV7S infection, N/H/K RAS protein expression was reduced, whereas caspase-3 protein expression increased in NPC cells. qPCR assay showed higher viral load of PRV7S found in infected-NPC compared to infected-NCNP cells. CONCLUSIONS: PRV7S inhibits the proliferation and induces apoptosis of NPC cells similar to MRV3. Therefore, PRV7S is a potential oncolytic virus.

The quantitative proteomic analysis of rare minnow, Gobiocypris rarus, infected with virulent and attenuated isolates of grass carp reovirus genotype â ¡.

Grass carp reovirus genotype Ⅱ (GCRV II) causes severe hemorrhagic disease in grass carp and affects the aquaculture industry in China. GCRV Ⅱ isolates have been collected from different epidemic areas in China, and these isolates can lead to different degrees of hemorrhagic symptoms in grass carp. Rare minnow (Gobiocypris rarus) is widely used as a model fish to study the mechanism of hemorrhagic disease because of its high sensitivity to GCRV. In this study, the protein levels in the spleen of rare minnow after infection with GCRV virulent isolate JZ809 and attenuated isolate XT422 were investigated using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. 109 and 50 differentially expressed proteins (DEPs) in the virulent and attenuated infection groups were obtained, respectively, among which 40 DEPs were identified in both groups. Combining protein expression profiling with gene ontology (GO) annotation, the responses of rare minnow to the two genotypes GCRV Ⅱ in terms of upregulated proteins were similar, focusing on ATP synthesis, in which ATP can serve as a "danger" signal to activate an immunoreaction in eukaryotes. Meanwhile, the virulent genotype JZ809 induced more immunoproteins and increased the levels of ubiquitin-proteasome system members to adapt to virus infection. However, together with a persistent and excessive inflammatory response and declining carbon metabolism, rare minnow presented more severe hemorrhagic disease and mortality after infection with virulent JZ809 than with attenuated XT422. The results provide a valuable information that will increase our understanding of the pathogenesis of viruses with different levels of virulence and the mechanism of interaction between the virus and host. Furthermore, the 6 proteins that were only significantly upregulated in the XT422 infection group all belonged to cluster 2, and 28 of 30 proteins that were only upregulated in JZ809 infection group were clustered into cluster 1. For the downregulated proteins, all DEPs in the XT422 infection group were clustered into cluster 4, and 25 of 39 proteins that were only significantly downregulated in the JZ809 infection group belonged to cluster 3. The results indicated that the DEPs in the attenuated XT422 infection group might be sensitive and their abundance changed more quickly when fish experienced virus infection.

Dynamics of Polarized Macrophages and Activated CD8+ Cells in Heart Tissue of Atlantic Salmon Infected With Piscine Orthoreovirus-1.

Piscine orthoreovirus (PRV-1) infection causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is also associated with focal melanized changes in white skeletal muscle where PRV-1 infection of macrophages appears to be important. In this study, we studied the macrophage polarization into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes during experimentally induced HSMI. The immune response in heart with HSMI lesions was characterized by CD8+ and MHC-I expressing cells and not by polarized macrophages. Fluorescent in situ hybridization (FISH) assays revealed localization of PRV-1 in a few M1 macrophages in both heart and skeletal muscle. M2 type macrophages were widely scattered in the heart and were more abundant in heart compared to the skeletal muscle. However, the M2 macrophages did not co-stain for PRV-1. There was a strong cellular immune response to the infection in the heart compared to that of the skeletal muscle, seen as increased MHC-I expression, partly in cells also containing PRV-1 RNA, and a high number of cytotoxic CD8+ granzyme producing cells that targeted PRV-1. In skeletal muscle, MHC-I expressing cells and CD8+ cells were dispersed between myocytes, but these cells did not stain for PRV-1. Gene expression analysis by RT-qPCR complied with the FISH results and confirmed a drop in level of PRV-1 following the cell mediated immune response. Overall, the results indicated that M1 macrophages do not contribute to the initial development of HSMI. However, large numbers of M2 macrophages reside in the heart and may contribute to the subsequent fast recovery following clearance of PRV-1 infection.

PRV-1 Infected Macrophages in Melanized Focal Changes in White Muscle of Atlantic Salmon (Salmo salar) Correlates With a Pro-Inflammatory Environment.

Melanized focal changes in white skeletal muscle of farmed Atlantic salmon, "black spots", is a quality problem affecting on average 20% of slaughtered fish. The spots appear initially as "red spots" characterized by hemorrhages and acute inflammation and progress into black spots characterized by chronic inflammation and abundant pigmented cells. Piscine orthoreovirus 1 (PRV-1) was previously found to be associated with macrophages and melano-macrophages in red and black spots. Here we have addressed the inflammatory microenvironment of red and black spots by studying the polarization status of the macrophages and cell mediated immune responses in spots, in both PRV-1 infected and non-infected fish. Samples that had been collected at regular intervals through the seawater production phase in a commercial farm were analyzed by multiplex fluorescent in situ hybridization (FISH) and RT-qPCR methods. Detection of abundant inducible nitric oxide synthase (iNOS2) expressing M1-polarized macrophages in red spots demonstrated a pro-inflammatory microenvironment. There was an almost perfect co-localization with the iNOS2 expression and PRV-1 infection. Black spots, on the other side, had few iNOS2 expressing cells, but a relatively high number of arginase-2 expressing anti-inflammatory M2-polarized macrophages containing melanin. The numerous M2-polarized melano-macrophages in black spots indicate an ongoing healing phase. Co-localization of PRV-1 and cells expressing CD8+ and MHC-I suggests a targeted immune response taking place in the spots. Altogether, this study indicates that PRV-1 induces a pro-inflammatory environment that is important for the pathogenesis of the spots. We do not have indication that infection of PRV-1 is the initial causative agent of this condition.

Role of Myeloid Cells in Oncolytic Reovirus-Based Cancer Therapy.

Oncolytic reovirus preferentially targets and kills cancer cells via the process of oncolysis, and additionally drives clinically favorable antitumor T cell responses that form protective immunological memory against cancer relapse. This two-prong attack by reovirus on cancers constitutes the foundation of its use as an anticancer oncolytic agent. Unfortunately, the efficacy of these reovirus-driven antitumor effects is influenced by the highly suppressive tumor microenvironment (TME). In particular, the myeloid cell populations (e.g., myeloid-derived suppressive cells and tumor-associated macrophages) of highly immunosuppressive capacities within the TME not only affect oncolysis but also actively impair the functioning of reovirus-driven antitumor T cell immunity. Thus, myeloid cells within the TME play a critical role during the virotherapy, which, if properly understood, can identify novel therapeutic combination strategies potentiating the therapeutic efficacy of reovirus-based cancer therapy.

Asymmetric profiles of infection and innate immunological responses in human iPS cell-derived small intestinal epithelial-like cell monolayers following infection with mammalian reovirus.

The intestinal mucosa plays an important role as an immune barrier due to its continual exposure to invading pathogens, including viruses. It is thus highly important to evaluate virus infection profiles in the intestinal mucosa for prevention of virus infection and development of antivirus medicines; however, only a few enterocyte lines are available as in vitro intestinal models for the evaluation of virus infection. In this study, we evaluated profiles of infection and innate immune responses following infection with a mammalian orthoreovirus (hereafter reovirus), which has often been used as a tractable model for studies of viral pathogenesis, in human iPS cell-derived small intestinal epithelial-like cell (hiPS-SIEC) monolayers and cells of a human colon adenocarcinoma cell line, Caco-2. The levels of reovirus infection were similar between hiPS-SIEC and Caco-2 cell monolayers, which are often used as an intestinal model, after apical and basolateral infection. In hiPS-SIEC monolayers, more efficient replication of the virus genome was observed following basolateral infection than apical infection, while apical infection resulted in higher levels of virus protein expression and progeny virus production than basolateral infection. Reovirus significantly induced innate immune responses, including expression of type I and III interferons (IFNs), in hiPS-SIEC monolayers more efficiently than Caco-2 cells. Higher levels of type I and III interferon (IFN) expression were found in hiPS-SIEC monolayers following apical infection than basolateral infection. These results suggested that hiPS-SIECs are a promising in vitro model for the evaluation of virus infection.