Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

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.

Oral immunization with recombinant L. lactis expressing GCRV-II VP4 produces protection against grass carp reovirus infection.

The hemorrhagic disease causing by grass carp reovirus (GCRV) infection, is associated with major economic losses and significant impact on aquaculture worldwide. VP4 of GCRV is one of the major outer capsid proteins which can induce an immune response in the host. In this study, pNZ8148-VP4/L. lactis was constructed to express recombinant VP4 protein of GCRV, which was confirmed by the Western-Blot and enzyme-linked immunosorbent assay. Then we performed the oral immunization for rare minnow model and the challenge with GCRV-II. After oral administration, pNZ8148-VP4/L. lactis can continuously reside in the intestinal tract to achieve antigen presentation. The intestinal and spleen samples were collected at different time intervals after immunization, and the expression of immune-related genes was detected by real-time fluorescence quantitative PCR. The results showed that VP4 recombinant L. lactis could induce complete cellular and humoral immune responses in the intestinal mucosal system, and effectively regulate the immunological effect of the spleen. The immunogenicity and the protective efficacy of the oral vaccine was evaluated by determining IgM levels and viral challenge to vaccinated fish, a significant level (P < 0.01) of antigen-specific IgM with GCRV-II neutralizing activity was able to be detected, which provided a effective protection in the challenge experiment. These results indicated that an oral probiotic vaccine with VP4 expression can provide effective protection for grass carp against GCRV-II challenge, suggesting a promising vaccine strategy for fish.

Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines.

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation.

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.

Grass carp peroxiredoxin 5 and 6-mediated autophagy inhibit grass carp reovirus replication and mitigate oxidative stress.

Peroxiredoxins (Prxs) are a family of antioxidant enzymes crucial for shielding cells against oxidative damage from reactive oxygen species (ROS). In this study, we cloned and analyzed two grass carp peroxiredoxin genes, CiPrx5 and CiPrx6. These genes exhibited ubiquitous expression across all sampled tissues, with their expression levels significantly modulated upon exposure to grass carp reovirus (GCRV). CiPrx5 was localized in the mitochondria, while CiPrx6 was uniformly distributed in the whole cells. Transfection or transformation of CiPrx5 and CiPrx6 into fish cells or E. coli significantly enhanced host resistance to H2O2 and heavy metals, leading to increased cell viability and reduced cell apoptosis rates. Furthermore, purified recombinant CiPrx5 and CiPrx6 proteins effectively protected DNA against oxidative damage. Notably, overexpression of both peroxiredoxins in fish cells effectively inhibited GCRV replication, reduced intracellular ROS levels induced by GCRV infection and H2O2 treatment, and induced autophagy. Significantly, these functions of CiPrx5 and CiPrx6 in GCRV replication and ROS mitigation were abolished upon treatment with an autophagy inhibitor. In summation, our findings suggest that grass carp Prx5 and Prx6 promote autophagy to inhibit GCRV replication, decrease intracellular ROS, and provide protection against oxidative stress.

Human cytokeratin 1 plays a role in the interaction of Pteropine orthoreovirus with Hek293 cells but not HeLa cells.

Pteropine orthoreovirus (PRV) causes respiratory tract infections in humans. Despite its emergence as a zoonotic and respiratory virus, little is known about its cell tropism, which hampers progress in fully understanding its pathogenesis in humans. Hek293 cells are most susceptible to PRV infection, while HeLa cells are the least. Human cytokeratin 1 (CK1) was identified as the protein that interacts with PRV. The immunofluorescence assay and qPCR results revealed prior treatment with anti-CK1 may provide Hek293 cells protection against PRV. The KRT1-knockout Hek293 cells were less susceptible to PRV infection. Further study into the pathogenesis of PRV in humans is needed.

Mammalian orthoreovirus can exit cells in extracellular vesicles.

Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.

Occurrence of salmonid alphavirus and piscine orthoreovirus-1 infections in migrating salmon (Salmo salar L.) post-smolt in western Norway.

Viral diseases are a serious problem in Atlantic salmon (Salmo salar L.) farming in Norway, often leading to reduced fish welfare and increased mortality. Disease outbreaks in salmon farms may lead to spread of viruses to the surrounding environment. There is a public concern that viral diseases may negatively affect the wild salmon populations. Pancreas disease (PD) caused by salmonid alphavirus (SAV) and heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus-1 (PRV-1) are common viral diseases in salmon farms in western Norway. In the current study, we investigated the occurrence of SAV and PRV-1 infections in 651 migrating salmon post-smolt collected from three fjord systems (Sognefjorden, Osterfjorden and Hardangerfjorden) located in western Norway in 2013 and 2014 by real-time RT-PCR. Of the collected post-smolts, 303 were of wild origin and 348 were hatchery-released. SAV was not detected in any of the tested post-smolt, but PRV-1 was detected in 4.6% of them. The Ct values of PRV-1 positive fish were usually high (mean 32.0; range: 20.1-36.8). PRV-1 prevalence in post-smolts from the three fjords was 6.1% in Sognefjorden followed by 4.8% in Osterfjorden and 2.3% in Hardangerfjorden. The prevalence PRV-1 was significantly higher in wild (6.9%) compared to hatchery-released post-smolt (2.6%). The occurrence of PRV-1 infection in the fish was lowest in the Hardangerfjorden which has the highest fish farming intensity. Our results suggest that SAV infection are uncommon in migrating smolt while PRV-1 infection can be detected at low level. These findings suggest that migrating smolts were at low risk from SAV or PRV-1 released from salmon farms located in their migration routes in 2013 and 2014.

Antiviral effects of micafungin against pteropine orthoreovirus, an emerging zoonotic virus carried by bats.

Bat-borne emerging zoonotic viruses cause major outbreaks, such as the Ebola virus, Nipah virus, and/or beta coronavirus. Pteropine orthoreovirus (PRV), whose spillover event occurred from fruits bats to humans, causes respiratory syndrome in humans widely in South East Asia. Repurposing approved drugs against PRV is an effective tool to confront future PRV pandemics. We screened 2,943 compounds in an FDA-approved drug library and identified eight hit compounds that reduce viral cytopathic effects on cultured Vero cells. Real-time quantitative PCR analysis revealed that six of eight hit compounds significantly inhibited PRV replication. Among them, micafungin used clinically as an antifungal drug, displayed a prominent antiviral effect on PRV. Secondly, the antiviral effects of micafungin on PRV infected human cell lines (HEK293T and A549), and their transcriptome changes by PRV infection were investigated, compared to four different bat-derived cell lines (FBKT1 (Ryukyu flying fox), DEMKT1 (Leschenault's rousette), BKT1 (Greater horseshoe bat), YUBFKT1 (Eastern bent-wing bats)). In two human cell lines, unlike bat cells that induce an IFN-γ response pathway, an endoplasmic reticulum stress response pathway was commonly activated. Additionally, micafungin inhibits viral release rather than suppressing PRV genome replication in human cells, although it was disturbed in Vero cells. The target of micafungin's action may vary depending on the animal species, but it must be useful for human purposes as a first choice of medical care.

Comparative Transcriptomic Analysis Revealed Potential Differential Mechanisms of Grass Carp Reovirus Pathogenicity.

Grass carp reovirus (GCRV), one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella), can lead to grass carp hemorrhagic disease (GCHD). Currently, GCRV can be divided into three genotypes, but the comparison of their pathogenic mechanisms and the host responses remain unclear. In this study, we utilized the Ctenopharyngodon idella kidney (CIK) model infected with GCRV to conduct comparative studies on the three genotypes. We observed a cytopathic effect (CPE) in the GCRV-I and GCRV-III groups, whereas the GCRV-II group did not show any CPE. Moreover, a consistent trend in the mRNA expression levels of antiviral-related genes across all experimental groups of CIK cells was detected via qPCR and further explored through RNA-seq analysis. Importantly, GO/KEGG enrichment analysis showed that GCRV-I, -II, and -III could all activate the immune response in CIK cells, but GCRV-II induced more intense immune responses. Intriguingly, transcriptomic analysis revealed a widespread down-regulation of metabolism processes such as steroid biosynthesis, butanoate metabolism, and N-Glycan biosynthesis in infected CIK cells. Overall, our results reveal the CIK cells showed unique responses in immunity and metabolism in the three genotypes of GCRV infection. These results provide a theoretical basis for understanding the pathogenesis and prevention and control methods of GCRV.

Recombinant surface display vaccine enhances the immersion immune effect against grass carp reovirus in grass carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is subject to a hemorrhagic disease caused by grass carp reovirus (GCRV), which can lead to mass mortality in grass carp culture, causing significant economic loss. Vaccination is the most promising strategy for the prevention of infectious diseases. Immersion vaccination is considered the most effective disease prevention method for juvenile fish because it can be implemented on many fish at once and administered without causing stress. However, immune responses by immersion vaccination are markedly less robust due to the skin barrier and insufficient antigen uptake. The display of heterologous proteins on the cell surface has been explored as a delivery system for viral antigens in veterinary and human vaccine studies. To improve the efficacy of the immersion vaccine, the major capsid protein (VP7) of GCRV was co-displayed with Aeromonas hydrophila outer membrane protein a (OmpA) and major adhesion protein (Mah) on the outer membrane surface of nonpathogenic Escherichia coli BL21 using the anchoring motif of ice-nucleation protein (Inp). The immune responses and protection efficiency against GCRV infection via both the injection and immersion routes were evaluated. The results indicated that the activities of anti-oxidant enzymes (ACP, AKP, SOD and T-AOC), as well as the expression of immune-related genes (TNF-α, IL-1ß, MHCI and IgM) and specific VP7 antibody levels, were strongly increased in the grass carp from 7 to 21 days post-injection inoculation in a dose dependent manner. The cumulative mortality rates of injection-vaccinated groups were much lower than those of the control group after the GCRV challenge, and the relative percent survival (RPS) was greater than 80 %. Vitally, the surface co-display of vp7-Mah protein conferred marked protection to grass carp against GCRV infection after immersion administration (RPS >50 %); this was consistent with the production of high level of specific serum antibodies, non-specific immune responses, and the expression of immune-related genes. Moreover, the invasion analysis further showed that surface co-display of the vp7-Mah protein indeed significantly improved the invasion of E. coli BL21 (DE3) in vitro. Altogether, this study demonstrated that surface display GCRV core antigen vaccine system accompanied by invasion component from aquatic pathogenic microorganism is an effective prophylactic against GCRV viral diseases via the immersion administration approach.

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.

[Emerging reoviruses: the next pandemic?] / Les réovirus émergents : vers une prochaine pandémie?

As the world is experiencing the pandemic of SARS-CoV-2 responsible for COVID-19, one can wonder if members of other family of viruses could possibly emerge. Can such viruses establish a worldwide distribution with consequences similar to SARS-CoV-2? One such threat is the possible emergence of pathogenic reoviruses, especially by zoonotic transmission. Reoviruses are ubiquitous viruses exhibiting a worldwide distribution and various strains or isolates are found in many mammalian species and other vertebrates. When initially discovered, these viruses were named respiratory enteric orphan viruses (hence the acronym "reo") in order to reflect the fact that they could not be clearly associated with any given disease. However, this is not necessarily the case for all of these viruses, as clearly shown for some of these in animals. Significantly, there have been numerous reports of zoonotic transmission, especially from bats to humans. In this manuscript, pertinent properties of reoviruses will be first briefly presented followed by a review of available evidence for zoonotic transmission of pathogenic reoviruses to humans. Future work that appears to be needed for preparedness to the possible emergence of these viruses will then be briefly discussed.

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.

Type II Grass Carp Reovirus Rapidly Invades Grass Carp (Ctenopharyngodon idella) via Nostril-Olfactory System-Brain Axis, Gill, and Skin on Head.

Type II grass carp reovirus (GCRV-II) with high pathogenicity and infectivity causes severe hemorrhagic disease, which leads to extensive death in the grass carp and black carp aquaculture. However, the early invasion portal remains unclear. In this study, we explored the invasion portal, time, and pathway of GCRV-II by immersion infection in grass carp. Through the detection of the infected grass carp external body surface tissues, most of them could be detected to carry GCRV-II within 45 min except for the skin covered by scales. Further shortening the duration of infection, we proved that GCRV-II rapidly invades through the nostril (especially), gill, and skin on head at only 5 min post-immersion, rather than merely by adhesion. Subsequently, visual localization investigations of GCRV-II were conducted on the nostril, olfactory system (olfactory bulb and olfactory tract), and brain via immunofluorescence microscopy and transmission electron microscopy. We found that few viruses were located in the nostril at 5 min post-immersion infection, while a significantly increased quantity of viruses were distributed in all of the examined tissues at 45 min. Furthermore, the semi-qRT-PCR and Western blotting results of different infection times confirmed that GCRV-II invades grass carp via the nostril-olfactory system-brain axis and then viral replication unfolds. These results revealed the infection mechanism of GCRV-II in terms of the invasion portal, time, and pathway in grass carp. This study aims to understand the invasion mode of GCRV-II in grass carp, thus providing theoretical support for the prevention and control strategies of hemorrhagic disease.

Emerging reoviruses: the next pandemic?

As the world is experiencing the pandemic of SARS-CoV-2 responsible for COVID-19, one can wonder if members of other family of viruses could possibly emerge. Can such viruses establish a worldwide distribution with consequences similar to SARS-CoV-2? One such threat is the possible emergence of pathogenic reoviruses, especially by zoonotic transmission. Reoviruses are ubiquitous viruses exhibiting a worldwide distribution and various strains or isolates are found in many mammalian species and other vertebrates. When initially discovered, these viruses were named respiratory enteric orphan viruses (hence the acronym "reo") in order to reflect the fact that they could not be clearly associated with any given disease. However, this is not necessarily the case for all of these viruses, as clearly shown for some of these in animals. Significantly, there have been numerous reports of zoonotic transmission, especially from bats to humans. In this manuscript, pertinent properties of reoviruses will be first briefly presented followed by a review of available evidence for zoonotic transmission of pathogenic reoviruses to humans. Future work that appears to be needed for preparedness to the possible emergence of these viruses will then be briefly discussed.

NgR1 binding to reovirus reveals an unusual bivalent interaction and a new viral attachment protein.

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.