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Serotype 3 (T3) strains of mammalian orthoreovirus (reovirus) spread to the central nervous system to infect the brain and cause lethal encephalitis in newborn mice. Although reovirus targets several regions in the brain, susceptibility to infection is not uniformly distributed. The neuronal subtypes and anatomic sites targeted throughout the brain are not precisely known. Reovirus binds several attachment factors and entry receptors, including sialic acid (SA)-containing glycans and paired immunoglobulin-like receptor B (PirB). While these receptors are not required for infection of some types of neurons, reovirus engagement of these receptors can influence neuronal infection in certain contexts. To identify patterns of T3 neurotropism, we used microbial identification after passive tissue clearance and hybridization chain reaction to stain reovirus-infected cells throughout intact, optically transparent brains of newborn mice. Three-dimensional reconstructions revealed in detail the sites targeted by reovirus throughout the brain volume, including dense infection of the midbrain and hindbrain. Using reovirus mutants incapable of binding SA and mice lacking PirB expression, we found that neither SA nor PirB is required for the infection of various brain regions. However, SA may confer minor differences in infection that vary by region. Collectively, these studies indicate that many regions in the brain of newborn mice are susceptible to reovirus and that patterns of reovirus infection are not dependent on reovirus receptors SA and PirB.IMPORTANCENeurotropic viruses invade the central nervous system (CNS) and target various cell types to cause disease manifestations, such as meningitis, myelitis, or encephalitis. Infections of the CNS are often difficult to treat and can lead to lasting sequelae or death. Mammalian orthoreovirus (reovirus) causes age-dependent lethal encephalitis in many young mammals. Reovirus infects neurons in several different regions of the brain. However, the complete pattern of CNS infection is not understood. We found that reovirus targets almost all regions of the brain and that patterns of tropism are not dependent on receptors sialic acid and paired immunoglobulin-like receptor B. These studies confirm that two known reovirus receptors do not completely explain the cell types infected in brain tissue and establish strategies that can be used to understand complete patterns of viral tropism in an intact brain., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied., Objectives: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection., Methods: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5 , LYZI , Mucin-2 , ChgA , and Villin . The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity., Results: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner., Conclusions: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Korean Society of Veterinary Science.)

Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein μ1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was nonlethal in wild-type mice, whereas more than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, as well as extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 than T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a nonlethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo , which potentiates the capacity of reovirus to cause myocarditis. IMPORTANCE Reovirus is a nonenveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the nonlethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive ( ts ) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447 , which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453 , which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453 , encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn 16 -Lys was solely responsible for the tsG453 ts phenotype. Additionally, the mutant ts C447 Ala 188 -Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

Mouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice. No rapid and accurate detection methods are currently available for this infection. In this study, we describe a rapid, simple, closed-tube, one step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for Reo-3 and compare our assay with indirect enzyme-linked immunosorbent assay (ELISA). Three sets of RT-LAMP primers were designed by sequence analysis of a specific conserved sequence of the Reo-3 S1 gene. Using RS2 primer set, the RT-LAMP assay required 60 min at 65 °C to amplify the S1 gene in one step by using Reo-3 RNA template and had no cross-reactivity with the other related pathogens, such as Sendai virus (SV), pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), Ectromelia virus (Ect), minute virus of mice (MVM), P. pneumotropica, B. bronchiseptica, K. pneumonia and P. aeruginosa. in our LAMP reaction system. The limit of detection (LOD) of our RT-LAMP assay is 4 fg/μL. The established RT-LAMP assay enabled visual detection when fluorescence detection reagents were added, and was demonstrated to be effective and efficient. We tested 30 clinical blood samples and five artificial positive samples from SPF mice, the concordance between the two methods for blood samples was 100% compared with indirect ELISA and RT-PCR. Considering its performance, specificity, sensitivity, and repeatability, the developed RT-LAMP could be a valuable tool to supply a more effective Reo-3 detection method in laboratory animal quality monitoring.

Gut microbiota could facilitate host to defense diseases, but fish-microbiota interactions during viral infection and the underlying mechanism are poorly understood. We examined interactions and responses of gut microbiota to grass carp reovirus (GCRV) infection in Ctenopharyngodon idellus, which is the most important aquaculture fish worldwide. We found that GCRV infection group with serious haemorrhagic symptoms (G7s) showed considerably different gut microbiota, especially with an abnormally high abundance of gram-negative anaerobic Cetobacterium somerae. It also showed the lowest (p < 0.05) alpha-diversity but with much higher ecological process of homogenizing dispersal (28.8%), confirming a dysbiosis of the gut microbiota after viral infection. Interestingly, signaling pathways of NOD-like receptors (NLRs), toll-like receptors (TLRs), and lipopolysaccharide (LPS) stimulation genes were significantly (q-value < 0.01) enriched in G7s, which also significantly (p < 0.01) correlated with the core gut microbial genera of Cetobacterium and Acinetobacter. The results suggested that an expansion of C. somerae initiated by GCRV could aggravate host inflammatory reactions through the LPS-related NLRs and TLRs pathways. This study advances our understanding of the interplay between fish immunity and gut microbiota challenged by viruses; it also sheds new insights for ecological defense of fish diseases with the help of gut microbiota., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine-glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy. IMPORTANCE Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus Orthoreovirus , family Reoviridae , is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1., (Copyright © 2020 American Society for Microbiology.)

Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3 PL ) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3D TD ). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3D PL produced larger plaques than T3D TD and than the T3D strain from the ATCC (T3D ATCC ) and from the Kevin Coombs laboratory (T3D KC ). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3D PL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3D PL and T3D TD Moreover, polymorphisms were discovered in T3D TD that promoted virus replication and spread in tumors, and a new T3D PL /T3D TD hybrid was generated with enhanced plaque size compared to that of T3D PL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies. IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro , which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy., (Copyright © 2020 American Society for Microbiology.)

Reovirus serotype 3 Dearing (T3D) replicates preferentially in transformed cells and is in clinical trials as a cancer therapy. Laboratory strains of T3D, however, exhibit differences in plaque size on cancer cells and differences in oncolytic activity in vivo This study aimed to determine why the most oncolytic T3D reovirus lab strain, the Patrick Lee laboratory strain (T3D PL ), replicates more efficiently in cancer cells than other commonly used laboratory strains, the Kevin Coombs laboratory strain (T3D KC ) and Terence Dermody laboratory (T3D TD ) strain. In single-step growth curves, T3D PL titers increased at higher rates and produced ∼9-fold higher burst size. Furthermore, the number of reovirus antigen-positive cells increased more rapidly for T3D PL than for T3D TD In conclusion, the most oncolytic T3D PL possesses replication advantages in a single round of infection. Two specific mechanisms for enhanced infection by T3D PL were identified. First, T3D PL exhibited higher cell attachment, which was attributed to a higher proportion of virus particles with insufficient (≤3) σ1 cell attachment proteins. Second, T3D PL transcribed RNA at rates superior to those of the less oncolytic T3D strains, which is attributed to polymorphisms in M1-encoding μ2 protein, as confirmed in an in vitro transcription assay, and which thus demonstrates that T3D PL has an inherent transcription advantage that is cell type independent. Accordingly, T3D PL established rapid onset of viral RNA and protein synthesis, leading to more rapid kinetics of progeny virus production, larger virus burst size, and higher levels of cell death. Together, these results emphasize the importance of paying close attention to genomic divergence between virus laboratory strains and, mechanistically, reveal the importance of the rapid onset of infection for reovirus oncolysis. IMPORTANCE Reovirus serotype 3 Dearing (T3D) is in clinical trials for cancer therapy. Recently, it was discovered that highly related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro , which correlates with oncolytic activity in a murine model of melanoma. The current study reveals two mechanisms for the enhanced efficiency of T3D PL in cancer cells. Due to polymorphisms in two viral genes, within the first round of reovirus infection, T3D PL binds to cells more efficiency and more rapidly produces viral RNAs; this increased rate of infection relative to that of the less oncolytic strains gives T3D PL a strong inherent advantage that culminates in higher virus production, more cell death, and higher virus spread., (Copyright © 2020 American Society for Microbiology.)

The environment represents a significant barrier to infection. Physical stressors (heat) or chemical agents (ethanol) can render virions noninfectious. As such, discrete proteins are necessary to stabilize the dual-layered structure of mammalian orthoreovirus (reovirus). The outer capsid participates in cell entry: (i) σ3 is degraded to generate the infectious subviral particle, and (ii) μ1 facilitates membrane penetration and subsequent core delivery. μ1-σ3 interactions also prevent inactivation; however, this activity is not fully characterized. Using forward and reverse genetic approaches, we identified two mutations (μ1 M258I and σ3 S344P) within heat-resistant strains. σ3 S344P was sufficient to enhance capsid integrity and to reduce protease sensitivity. Moreover, these changes impaired replicative fitness in a reassortant background. This work reveals new details regarding the determinants of reovirus stability. IMPORTANCE Nonenveloped viruses rely on protein-protein interactions to shield their genomes from the environment. The capsid, or protective shell, must also disassemble during cell entry. In this work, we identified a determinant within mammalian orthoreovirus that regulates heat resistance, disassembly kinetics, and replicative fitness. Together, these findings show capsid function is balanced for optimal replication and for spread to a new host., (Copyright © 2019 American Society for Microbiology.)

Mammalian orthoreoviruses (MRV) type 3 have been recently identified in human and several animal hosts, highlighting the apparent lack of species barriers. Here we report the identification and genetic characterization of MRVs strains in alpine chamois, one of the most abundant wild ungulate in the Alps. Serological survey was also performed by MRV neutralization test in chamois population during five consecutive years (2008-2012). Three novel MRVs were isolated on cell culture from chamois lung tissues. No respiratory or other clinical symptoms neither lung macroscopic lesions were observed in the chamois population. MRV strains were classified as MRV-3 within the lineage III, based on S1 phylogeny, and were closely related to Italian strains identified in dog, bat and diarrheic pig. The full genome sequence was obtained by next-generation sequencing and phylogenetic analyses showed that other segments were more similar to MRVs of different geographic locations, serotypes and hosts, including human, highlighting genome reassortment and lack of host specific barriers. By using serum neutralization test, a high prevalence of MRV-3 antibodies was observed in chamois population throughout the monitored period, showing an endemic level of infection and suggesting a self-maintenance of MRV and/or a continuous spill-over of infection from other animal species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Following attachment to host receptors via σ1, reovirus particles are endocytosed and disassembled to generate infectious subvirion particles (ISVPs). ISVPs undergo conformational changes to form ISVP*, releasing σ1 and membrane-targeting peptides from the viral μ1 protein. ISVP* formation is required for delivery of the viral core into the cytoplasm for replication. We characterized the properties of T3D F /T3D C S1, an S1 gene monoreassortant between two laboratory isolates of prototype reovirus strain T3D: T3D F and T3D C T3D F /T3D C S1 is poorly infectious. This deficiency is a consequence of inefficient encapsidation of S1-encoded σ1 on T3D F /T3D C S1 virions. Additionally, compared to T3D F , T3D F /T3D C S1 undergoes ISVP-to-ISVP* conversion more readily, revealing an unexpected role for σ1 in regulating ISVP* formation. The σ1 protein is held within turrets formed by the λ2 protein. To test if the altered properties of T3D F /T3D C S1 are due to a mismatch between σ1 and λ2 proteins from T3D F and T3D C , properties of T3D F /T3D C L2 and T3D F /T3D C S1L2, which express a T3D C -derived λ2, were compared. The presence of T3D C λ2 allowed more efficient σ1 incorporation, producing particles that exhibit T3D F -like infectivity. Compared to T3D F , T3D F /T3D C L2 prematurely converts to ISVP*, uncovering a role for λ2 in regulating ISVP* formation. Importantly, a virus with matching σ1 and λ2 displayed a more regulated conversion to ISVP* than either T3D F /T3D C S1 or T3D F /T3D C L2. In addition to identifying new regulators of ISVP* formation, our results highlight that protein mismatches produced by reassortment can alter virus assembly and thereby influence subsequent functions of the virus capsid. IMPORTANCE Cells coinfected with viruses that possess a multipartite or segmented genome reassort to produce progeny viruses that contain a combination of gene segments from each parent. Reassortment places new pairs of genes together, generating viruses in which mismatched proteins must function together. To test if such forced pairing of proteins that form the virus shell or capsid alters the function of the particle, we investigated properties of reovirus variants in which the σ1 attachment protein and the λ2 protein that anchors σ1 on the particle are mismatched. Our studies demonstrate that a σ1-λ2 mismatch produces particles with lower levels of encapsidated σ1, consequently decreasing virus attachment and infectivity. The mismatch between σ1 and λ2 also altered the capacity of the viral capsid to undergo conformational changes required for cell entry. These studies reveal new functions of reovirus capsid proteins and illuminate both predictable and novel implications of reassortment., (Copyright © 2018 American Society for Microbiology.)

Background: Oncolytic reovirus pelareorep might preferentially infect and destroy rat sarcoma (RAS)-activated cells, and has preclinical and early clinical activity against colorectal cancer (CRC)., Patients and Methods: After a 6-patient safety run-in, 103 patients with metastatic CRC were randomly assigned to standard first-line leucovorin/5-FU/oxaliplatin (FOLFOX6)/bevacizumab (FOLFOX/BEV) every 2 weeks with (n = 51) or without (n = 52) pelareorep 3 × 10 10 tissue culture infective dose 50 on days 1 to 5 (cycles 1, 2, 4, and alternate cycles thereafter). The primary end point was progression-free survival (PFS). Secondary end points included overall survival (OS), objective response rate (ORR), quality of life, and correlative analyses., Results: At 13 months' median follow-up, PFS was inferior in the pelareorep arm (median 7 vs. 9 months; hazard ratio [HR], 1.59 [80% confidence interval (CI), 1.18-2.15]; P = .046). There was no statistical difference in OS (median, 19.2 vs. 20.1 months; HR, 1.22; P = .38). An increased ORR was observed with pelareorep (adjusted odds ratio, 2.52 [80% CI, 1.44-4.41]; P = .03), but with a shorter median duration of response (5 vs. 9 months; P = .028). Pelareorep patients experienced more hypertension and proteinuria, and were more likely to omit bevacizumab before progression. A trend to lower dose intensity and shorter oxaliplatin and bevacizumab treatment duration was observed with pelareorep., Conclusion: Combination pelareorep with FOLFOX/BEV was tolerable with an increased ORR, but PFS was inferior. Subgroup analysis of baseline variables including Kirsten rat sarcoma oncogene did not identify subgroups with PFS benefit. Decreased treatment intensity with standard agents likely contributed to the lack of benefit with pelareorep. Future studies might consider alternate pelareorep/chemotherapy strategies or combination therapy with novel agents., (Copyright © 2018 Elsevier Inc. All rights reserved.)

While the mammalian orthoreovirus type 3 dearing (reovirus T3D) infects many different tumour cells, various cell lines resist the induction of reovirus-mediated cell death. In an effort to increase the oncolytic potency, we introduced transgenes into the S1 segment of reovirus T3D. The adenovirus E4orf4 gene was selected as transgene since the encoded E4orf4 protein induces cell death in transformed cells. The induction of cell death by E4orf4 depends in part on its binding to phosphatase 2A (PP2A). In addition to the S1-E4orf4 reovirus, two other reoviruses were employed in our studies. The reovirus rS1-RFA encodes an E4orf4 double-mutant protein that cannot interact with PP2A and the rS1-iLOV virus encoding the fluorescent marker iLOV as a reporter. The replacement of the codons for the junction adhesion molecule-A (JAM-A) binding head domain of the truncated spike protein blocks the entry of these recombinant viruses via the reovirus receptor JAM-A. Instead these viruses rely on internalization via binding to sialic acids on the cell surface. This expands their tropism and allows infection of JAM-A-deficient tumour cells. Here we not only demonstrate the feasibility of this approach but also established that the cytolytic activity of these recombinant viruses is largely transgene independent.

Background: Pelareorep, a serotype 3 reovirus, has demonstrated preclinical and early clinical activity in breast cancer and synergistic cytotoxic activity with microtubule targeting agents. This multicentre, randomized, phase II trial was undertaken to evaluate the efficacy and safety of adding pelareorep to paclitaxel for patients with metastatic breast cancer (mBC)., Methods: Following a safety run-in of 7 patients, 74 women with previously treated mBC were randomized either to paclitaxel 80 mg/m 2 intravenously on days 1, 8, and 15 every 4 weeks plus pelareorep 3 × 10 10 TCID 50 intravenously on days 1, 2, 8, 9, 15, and 16 every 4 weeks (Arm A) or to paclitaxel alone (Arm B). Primary endpoint was progression-free survival (PFS). Secondary endpoints were objective response rate, overall survival (OS), circulating tumour cell counts, safety, and exploratory correlative analyses. All comparisons used a two-sided test at an alpha level of 20%. Survival analyses were adjusted for prior paclitaxel., Results: Final analysis was performed after a median follow-up of 29.5 months. Pelareorep was well tolerated. Patients in Arm A had more favourable baseline prognostic variables. Median adjusted PFS (Arm A vs B) was 3.78 mo vs 3.38 mo (HR 1.04, 80% CI 0.76-1.43, P = 0.87). There was no difference in response rate between arms (P = 0.87). Median OS (Arm A vs B) was 17.4 mo vs 10.4 mo (HR 0.65, 80% CI 0.46-0.91, P = 0.1)., Conclusions: This first, phase II, randomized study of pelareorep and paclitaxel in previously treated mBC did not show a difference in PFS (the primary endpoint) or RR. However, there was a significantly longer OS for the combination. Further exploration of this regimen in mBC may be of interest.

Proteins that form the reovirus outer capsid play an active role in the entry of reovirus into host cells. Among these, the σ1 protein mediates attachment of reovirus particles to host cells via interaction with cell surface glycans or the proteinaceous receptor junctional adhesion molecule A (JAM-A). The μ1 protein functions to penetrate the host cell membrane to allow delivery of the genome-containing viral core particle into the cytoplasm to initiate viral replication. We demonstrate that a reassortant virus that expresses the M2 gene-encoded μ1 protein derived from prototype strain T3D in an otherwise prototype T1L background (T1L/T3DM2) infects cells more efficiently than parental T1L. Unexpectedly, the enhancement in infectivity of T1L/T3DM2 is due to its capacity to attach to cells more efficiently. We present genetic data implicating the central region of μ1 in altering the cell attachment property of reovirus. Our data indicate that the T3D μ1-mediated enhancement in infectivity of T1L is dependent on the function of σ1 and requires the expression of JAM-A. We also demonstrate that T1L/T3DM2 utilizes JAM-A more efficiently than T1L. These studies revealed a previously unknown relationship between two nonadjacent reovirus outer capsid proteins, σ1 and μ1., Importance: How reovirus attaches to host cells has been extensively characterized. Attachment of reovirus to host cells is mediated by the σ1 protein, and properties of σ1 influence the capacity of reovirus to target specific host tissues and produce disease. Here, we present new evidence indicating that the cell attachment properties of σ1 are influenced by the nature of μ1, a capsid protein that does not physically interact with σ1. These studies could explain the previously described role for μ1 in influencing reovirus pathogenesis. These studies are also of broader significance because they highlight an example of how genetic reassortment between virus strains could produce phenotypes that are distinct from those of either parent., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Mammalian Orthoreoviruses 3 (MRV3) have been described in diarrheic pigs from USA and Asia. We firstly detected MRV3 in Europe (Italy) in piglets showing severe diarrhea associated with Porcine Epidemic Diarrhea. The virus was phylogenetically related to European reoviruses of human and bat origin and to US and Chinese pig MRV3.

Pelareorep causes oncolysis in tumor cells with activated Ras. We hypothesized that pelareorep would have efficacy and immunomodulatory activity in metastatic pancreatic adenocarcinoma (MPA) when combined with carboplatin and paclitaxel. A randomized phase 2 study (NCT01280058) was conducted in treatment-naive patients with MPA randomized to two treatment arms: paclitaxel/carboplatin + pelareorep (Arm A, n = 36 evaluable patients) versus paclitaxel/carboplatin (Arm B, n = 37 evaluable patients). There was no difference in progression-free survival (PFS) between the arms (Arm A PFS = 4.9 months, Arm B PFS = 5.2 months, P = 0.6), and Kirsten rat sarcoma viral oncogene (KRAS) status did not impact outcome. Quality-adjusted Time without Symptoms or Toxicity analysis revealed that the majority of PFS time was without toxicity or progression (4.3 months). Patient immunophenotype appeared important, as soluble immune biomarkers were associated with treatment outcome (fractalkine, interleukin (IL)-6, IL-8, regulated on activation, normal T cell expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF)). Increased circulating T and natural killer (NK)-cell subsets were also significantly associated with treatment outcome. Addition of pelareorep was associated with higher levels of 14 proinflammatory plasma cytokines/chemokines and cells with an immunosuppressive phenotype (Tregs, cytotoxic T lymphocyte associated protein 4 (CTLA4)(+) T cells). Overall, pelareorep was safe but does not improve PFS when administered with carboplatin/paclitaxel, regardless of KRAS mutational status. Immunologic studies suggest that chemotherapy backbone improves immune reconstitution and that targeting remaining immunosuppressive mediators may improve oncolytic virotherapy.

Mammalian orthoreoviruses (MRVs) are widespread and infect virtually all mammals. We report here the first case of a natural mutant and reassortant serotype 3 reovirus from mink in China, known as MRV3 SD-14. Whole-genome sequence analysis showed that the MRV3 SD-14 may have resulted from a reassortment involving MRVs that infected swine, humans and mink. Interestingly, the S1 segment, which encodes the viral attachment protein σ1, which influences viral virulence and cell tropism in the host, had a stop codon mutation at amino acid 246. Surveillance of the virulence and evolution of MRVs in humans and other animals deserves more attention.

Unlabelled: Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90°C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence., Importance: Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the United States. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea., (Copyright © 2015 Thimmasandra Narayanappa et al.)

Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b(+), Gr-1(+), Ly6C(high) myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b(+), Gr-1(+), Ly6C(high) myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b(+), Gr-1(+), Ly6C(high) myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus-based anticancer immunotherapies., (Copyright © 2015 by The American Association of Immunologists, Inc.)

As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC) analyses of reovirus, strain type 3 Dearing (T3D)-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation) to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D). Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26, and 44 were significantly down-regulated by T1L, T3D, and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3, and Mx1. This study expands our understanding of reovirus-induced host responses.

Unlabelled: Wild-type mammalian orthoreovirus serotype 3 Dearing (T3wt) is nonpathogenic in humans but preferentially infects and kills cancer cells in culture and demonstrates promising antitumor activity in vivo. Using forward genetics, we previously isolated two variants of reovirus, T3v1 and T3v2, with increased infectivity toward a panel of cancer cell lines and improved in vivo oncolysis in a murine melanoma model relative to that of T3wt. Our current study explored how mutations in T3v1 and T3v2 promote infectivity. Reovirions contain trimers of σ1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantitative Western blot analysis showed that purified T3v1 and T3v2 virions had ∼ 2- and 4-fold-lower levels of σ1 fiber than did T3wt virions. Importantly, using RNA interference to reduce σ1 levels during T3wt production, we were able to generate wild-type reovirus with reduced levels of σ1 per virion. As σ1 levels were reduced, virion infectivity increased by 2- to 5-fold per cell-bound particle, demonstrating a causal relationship between virion σ1 levels and the infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1, and T3v2 uncoated the outer capsid proteins σ3 and μ1C at similar rates. However, having started with fewer σ1 molecules, a complete loss of σ1 was achieved sooner for T3v1 and T3v2. Distinct from intracellular uncoating, chymotrypsin digestion, as a mimic of natural enteric infection, resulted in more rapid σ3 and μ1C removal, unique disassembly intermediates, and a rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity toward natural versus therapeutic niches may therefore require distinct reovirus structures and σ1 levels., Importance: Wild-type reovirus is currently in clinical trials as a potential cancer therapy. Our molecular studies on variants of reovirus with enhanced oncolytic activity in vitro and in vivo now show that distinct reovirus structures promote adaptation toward cancer cells and away from conditions that mimic natural routes of infection. Specifically, we found that reovirus particles with fewer molecules of the cell attachment protein σ1 became more infectious toward transformed cells. Reduced σ1 levels conferred a benefit to incoming particles only, resulting in an earlier depletion of σ1 and a higher probability of establishing productive infection. Conversely, reovirus variants with fewer σ1 molecules showed reduced stability and infectivity and distinct disassembly when exposed to conditions that mimic natural intestinal proteolysis. These findings support a model where the mode of infection dictates the precise optimum of reovirus structure and provide a molecular rationale for considering alternative reovirus structures during oncolytic therapy., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Optimum clinical protocols require systemic delivery of oncolytic viruses in the presence of an intact immune system. We show that preconditioning with immune modulators, or loading virus onto carrier cells ex vivo, enhances virus-mediated antitumor activity. Our early trials of systemic reovirus delivery showed that after infusion reovirus could be recovered from blood cells--but not from plasma--suggesting that rapid association with blood cells may protect virus from neutralizing antibody. We therefore postulated that stimulation of potential carrier cells directly in vivo before intravenous viral delivery would enhance delivery of cell-associated virus to tumor. We show that mobilization of the CD11b(+) cell compartment by granulocyte macrophage-colony stimulating factor immediately before intravenous reovirus, eliminated detectable tumor in mice with small B16 melanomas, and achieved highly significant therapy in mice bearing well-established tumors. Unexpectedly, cytokine conditioning therapy was most effective in the presence of preexisting neutralizing antibody. Consistent with this, reovirus bound by neutralizing antibody effectively accessed monocytes/macrophages and was handed off to tumor cells. Thus, preconditioning with cytokine stimulated recipient cells in vivo for enhanced viral delivery to tumors. Moreover, preexisting neutralizing antibody to an oncolytic virus may, therefore, even be exploited for systemic delivery to tumors in the clinic.

At early times in Mammalian Orthoreovirus (MRV) infection, cytoplasmic inclusions termed stress granules (SGs) are formed as a component of the innate immune response, however, at later times they are no longer present despite continued immune signaling. To investigate the roles of MRV proteins in SG modulation we examined non-structural protein µNS localization relative to SGs in infected and transfected cells. Using a series of mutant plasmids, we mapped the necessary μNS residues for SG localization to amino acids 78 and 79. We examined the capacity of a μNS(78-79) mutant to associate with known viral protein binding partners of μNS and found that it loses association with viral core protein λ2. Finally, we show that while this mutant cannot support de novo viral replication, it is able to rescue replication following siRNA knockdown of μNS. These data suggest that μNS association with SGs, λ2, or both play roles in MRV replication., (© 2013 Published by Elsevier Inc.)

Apoptosis is a type of controlled cell death that is essential for development and tissue homeostasis. It also serves as a robust host response against infection by many viruses. The capacity of neurotropic viruses to induce apoptosis strongly correlates with virulence. However, the precise function of apoptosis in viral infection is not well understood. Reovirus is a neurotropic virus that induces apoptosis in a variety of cell types, including central nervous system neurons, leading to fatal encephalitis in newborn mice. To determine the effect of apoptosis on reovirus replication in the host, we generated two otherwise isogenic viruses that differ in a single amino acid in viral capsid protein μ1 that segregates with apoptotic capacity. Apoptosis-proficient and apoptosis-deficient viruses were compared for replication, dissemination, tropism, and tissue injury in newborn mice and for the capacity to spread to uninfected littermates. Our results indicate that apoptotic capacity enhances reovirus replication in the brain and consequent neurovirulence but reduces transmission efficiency. The replication advantage of the apoptosis-proficient strain is limited to the brain and correlates with enhanced infectivity of neurons. These studies reveal a new cell type-specific determinant of reovirus virulence.

The reovirus outer capsid protein μ1 forms a lattice surrounding the viral core. In the native state, μ1 determines the environmental stability of the viral capsid. Additionally, during cell entry, μ1 undergoes structural rearrangements that facilitate delivery of the viral cores across the membrane. To determine how the capsid-stabilizing functions of μ1 impinge on the capacity of μ1 to undergo conformational changes required for cell entry, we characterized viruses with mutations engineered at charged residues within the μ1 loop formed by residues 72 to 96 (72-96 loop). This loop is proposed to stabilize the capsid by mediating interactions between neighboring μ1 trimers and between trimers and the core. We found that mutations at Glu89 (E89) within this loop produced viruses with compromised efficiency for completing their replication cycle. ISVPs of E89 mutants converted to ISVP*s more readily than those of wild-type viruses. The E89 mutants yielded revertants with second-site substitutions within regions that mediate interaction between μ1 trimers at a site distinct from the 72-96 loop. These viruses also contained changes in regions that control interactions within μ1 trimers. Viruses containing these second-site changes displayed restored plaque phenotypes and were capable of undergoing ISVP-to-ISVP* conversion in a regulated manner. These findings highlight regions of μ1 that stabilize the reovirus capsid and demonstrate that an enhanced propensity to form ISVP*s in an unregulated manner compromises viral fitness.

Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.

Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The rotavirus RNA-dependent RNA polymerase (RdRp), VP1, contains canonical RdRp motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential import to nucleotide and divalent cation stabilization, we aligned VP1 of divergent rotaviruses and the structural homolog reovirus λ3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRp motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRp activity to particle assembly., (Published by Elsevier Inc.)

Reovirus preferentially replicates in transformed cells and is being explored as a cancer therapy. Immunological and physical barriers to virotherapy inspired a quest for reovirus variants with enhanced oncolytic potency. Using a classical genetics approach, we isolated two reovirus variants (T3v1 and T3v2) with superior replication relative to wild-type reovirus serotype 3 Dearing (T3wt) on various human and mouse tumorigenic cell lines. Unique mutations in reovirus λ2 vertex protein and σ1 cell attachment protein were associated with the large plaque-forming phenotype of T3v1 and T3v2, respectively. Both T3v1 and T3v2 exhibited higher infectivity (i.e., a higher PFU-to-particle ratio) than T3wt. A detailed analysis of virus replication revealed that virus cell binding and uncoating were equivalent for variant and wild-type reoviruses. However, T3v1 and T3v2 were significantly more efficient than T3wt in initiating productive infection. Thus, when cells were infected with equivalent input virus particles, T3v1 and T3v2 produced significantly higher levels of early viral RNAs relative to T3wt. Subsequent steps of virus replication (viral RNA and protein synthesis, virus assembly, and cell death) were equivalent for all three viruses. In a syngeneic mouse model of melanoma, both T3v1 and T3v2 prolonged mouse survival compared to wild-type reovirus. Our studies reveal that oncolytic potency of reovirus can be improved through distinct mutations that increase the infectivity of reovirus particles.

Myocarditis is indicated as the second leading cause of sudden death in young adults. Reovirus induces myocarditis in neonatal mice, providing a tractable model system for investigation of this important disease. Alpha/beta-interferon (IFN-α/β) treatment improves cardiac function and inhibits viral replication in patients with chronic myocarditis, and the host IFN-α/β response is a determinant of reovirus strain-specific differences in induction of myocarditis. Virus-induced IFN-β stimulates a signaling cascade that establishes an antiviral state and further induces IFN-α/β through an amplification loop. Reovirus strain-specific differences in induction of and sensitivity to IFN-α/β are associated with the viral M1, L2, and S2 genes. The reovirus M1 gene-encoded μ2 protein is a strain-specific repressor of IFN-β signaling, providing one possible mechanism for the variation in resistance to IFN and induction of myocarditis between different reovirus strains. We report here that μ2 amino acid 208 determines repression of IFN-β signaling and modulates reovirus induction of IFN-β in cardiac myocytes. Moreover, μ2 amino acid 208 determines reovirus replication, both in initially infected cardiac myocytes and after viral spread, by regulating the IFN-β response. Amino acid 208 of μ2 also influences the cytopathic effect in cardiac myocytes after spread. Finally, μ2 amino acid 208 modulates myocarditis in neonatal mice. Thus, repression of IFN-β signaling mediated by reovirus μ2 amino acid 208 is a determinant of the IFN-β response, viral replication and damage in cardiac myocytes, and myocarditis. These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-β response and, consequently, damage to the heart.

Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

The prognosis of gastric cancer patients with peritoneal dissemination is extremely poor, and the development of an effective treatment is necessary. The aim of this study was to investigate the efficacy of oncolytic reovirus against peritoneal metastasis in human gastric cancer using an experimental animal model. Four human gastric cancer cell lines, including MKN45p, NUGC4, MKN7 and KatoIII, a normal NIH3T3 cell line as a control, and reovirus serotype 3, were used in this study. We evaluated the cytopathic effect of reovirus and the Ras activity in each gastric cancer cell line in vitro. To evaluate oncolytic efficacy in vivo, reovirus (1x10(8) PFU) was administered into the peritoneal cavity of nude mice on days 7, 8 and 9 after inoculation with MKN45p cells. Mean volume of ascites and the total number and weight of the peritoneal tumors were measured after sacrifice. After reovirus infection, cytopathic effect was observed in all four gastric cancer cell lines, but not in the control cells. Ras activation assay showed that Ras activity in all four gastric cancer cell lines increased to a higher level than that in the control cells. In the animal model experiments, mean volume of ascites and the total number and weight of the peritoneal tumors in the reovirus treatment group were significantly lower than those in the control group. In conclusions, intraperitoneal administration of reovirus could be useful as a new modality against peritoneal metastasis in gastric cancer.

An oncolytic virus is considered a targeted cancer therapy due to its ability to specifically target, replicate in and lyse cancer cells while leaving normal cells unharmed. Over the last few years several tumor selective oncolytic viruses have been developed. These include certain DNA viruses such as adenovirus that have been genetically manipulated to target specific cancer cells by exerting restrictions on the virus at the level of cell entry, viral gene transcription and viral protein translation. There are a variety of RNA viruses being studied as possible cancer therapies including reovirus. Reovirus is intrinsically oncolytic without the need for any genetic manipulation. The inherent oncolytic properties of this virus are derived from the fact that it specifically targets cells with an activated Ras pathway found in many cancer cells. REOLYSIN® is a proprietary formulation of human reovirus type 3 Dearing developed by Oncolytics Biotech Inc. and is the only therapeutic reovirus in clinical development. This review provides an overview of the development of REOLYSIN as a potential cancer therapeutic and the growing role of in situ based molecular pathology methods in providing clinical proof of concept and in guiding clinical development., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Serotype-specific patterns of reovirus disease in the CNS of newborn mice segregate with the viral S1 gene segment, which encodes attachment protein sigma1 and nonstructural protein sigma1s. The importance of receptor recognition in target cell selection by reovirus implicates the sigma1 protein as the primary effector of disease outcome. However, the contribution of sigma1s to reovirus disease is unknown. To define the function of sigma1s in reovirus pathogenesis, we generated a sigma1s-deficient virus by altering a single nucleotide to disrupt the sigma1s translational start site. Viruses were recovered that contain nine gene segments from strain type 3 Dearing and either the wild-type or sigma1s-null S1 gene segment from strain type 1 Lang. Following peroral inoculation of newborn mice, both viruses replicated in the intestine, although the wild-type virus achieved higher yields than the sigma1s-null virus. However, unlike the wild-type virus, the sigma1s-deficient virus failed to disseminate to sites of secondary viral replication, including the brain, heart, and liver. Within the small intestine, both viruses were detected in Peyer's patches, but only the wild-type virus reached the mesenteric lymph node. Concordantly, wild-type virus, but not sigma1s-deficient virus, was detected in the blood of infected animals. Wild-type and sigma1s-null viruses produced equivalent titers following intracranial inoculation, indicating that sigma1s is dispensable for viral growth in the murine CNS. These results suggest a key role for sigma1s in virus spread from intestinal lymphatics to the bloodstream, thereby allowing the establishment of viremia and dissemination to sites of secondary replication within the infected host.

The secreted cytokine alpha/beta interferon (IFN-alpha/beta) binds its receptor to activate the Jak-STAT signal transduction pathway, leading to formation of the heterotrimeric IFN-stimulated gene factor 3 (ISGF3) transcription complex for induction of IFN-stimulated genes (ISGs) and establishment of an antiviral state. Many viruses have evolved countermeasures to inhibit the IFN pathway, thereby subverting the innate antiviral response. Here, we demonstrate that the mildly myocarditic reovirus type 1 Lang (T1L), but not the nonmyocarditic reovirus type 3 Dearing, represses IFN induction of a subset of ISGs and that this repressor function segregates with the T1L M1 gene. Concordantly, the T1L M1 gene product, mu2, dramatically inhibits IFN-beta-induced reporter gene expression. Surprisingly, T1L infection does not degrade components of the ISGF3 complex or interfere with STAT1 or STAT2 nuclear translocation as has been observed for other viruses. Instead, infection with T1L or reassortant or recombinant viruses containing the T1L M1 gene results in accumulation of interferon regulatory factor 9 (IRF9) in the nucleus. This effect has not been previously described for any virus and suggests that mu2 modulates IRF9 interactions with STATs for both ISGF3 function and nuclear export. The M1 gene is a determinant of virus strain-specific differences in the IFN response, which are linked to virus strain-specific differences in induction of murine myocarditis. We find that virus-induced myocarditis is associated with repression of IFN function, providing new insights into the pathophysiology of this disease. Together, these data provide the first report of an increase in IRF9 nuclear accumulation associated with viral subversion of the IFN response and couple virus strain-specific differences in IFN antagonism to the pathogenesis of viral myocarditis.

Human Orthoreovirus Type 3 Dearing is not pathogenic to humans and has been evaluated clinically as an oncolytic agent. Its transduction efficiency and the tumor cell selectivity may be enhanced by incorporating ligands for alternative receptors. However, the genetic modification of reoviruses has been difficult, and genetic targeting of reoviruses has not been reported so far. Here we describe a technique for generating genetically targeted reoviruses. The propagation of wild-type reoviruses on cells expressing a modified sigma 1-encoding segment embedded in a conventional RNA polymerase II transcript leads to substitution of the wild-type genome segment by the modified version. This technique was used for generating reoviruses that are genetically targeted to an artificial receptor expressed on U118MG cells. These cells lack the junction adhesion molecule-1 and therefore resist infection by wild-type reoviruses. The targeted reoviruses were engineered to carry the ligand for this receptor at the C terminus of the sigma 1 spike protein. This demonstrates that the C terminus of the sigma 1 protein is a suitable locale for the insertion of oligopeptide ligands and that targeting of reoviruses is feasible. The genetically targeted viruses can be propagated using the modified U118MG cells as helper cells. This technique may be applicable for the improvement of human reoviruses as oncolytic agents.

A low-copy component of mammalian reovirus particles is mu2, an 83-kDa protein encoded by the M1 viral genome segment and packaged within the viral core. Previous studies have identified mu2 as a nucleoside triphosphate phosphohydrolase (NTPase) as well as an RNA 5'-triphosphate phosphohydrolase (RTPase), putatively involved in reovirus RNA synthesis and/or 5'-capping. Other studies have identified mu2 as a microtubule-binding protein, which also associates with the viral factory matrix protein muNS and thereby anchors the factories to cellular microtubules during infections by most reovirus strains. To extend studies of mu2 functions during infection, we tested a small interfering RNA (siRNA) directed against the M1 plus-strand RNAs of reovirus strains Type 1 Lang (T1L) and Type 3 Dearing (T3D). The siRNA strongly suppressed mu2 expression by either strain and reduced infectious yields in a strain-dependent manner. This first strain difference was genetically mapped to the M1 genome segment and tentatively assigned to a single mu2 sequence polymorphism, Pro/Ser208, which also determines a T1L-T3D strain difference in microtubule association. The siRNA-based defect in mu2 expression was rescued by plasmids, containing silent mutations in the siRNA-targeted sequence, which encoded either T1L or T3D mu2, but the growth defect was rescued only by T1L mu2. This second strain difference was also mapped to Pro/Ser208, in that swapping this one residue between T1L and T3D mu2 reversed the rescue phenotypes. Thus, the T1L-T3D strain difference in mu2-microtubule association was correlated not only with the extent of reduction in infectious yields by the siRNA but also with the extent of rescue by plasmid-derived mu2. In addition, the rescue capacity of T1L mu2 was abrogated by nocodazole treatment, providing independent evidence for the importance of mu2-microtubule association in plasmid-based rescue. In two separate cases, the results revealed functional differences between virus- and plasmid-derived mu2. Ala substitutions within the NTP-binding motif of T1L mu2 also abrogated its rescue capacity, suggesting that the NTPase or RTPase activity of mu2 is additionally required for effective viral growth.

Usage of DNA vaccination has been limited by inefficient cellular expression of plasmid constructs used in DNA vaccines. We describe a novel system for enhancing delivery of DNA vaccine plasmids into cells and their nuclei. This delivery system uses recombinant reovirus type 3 sigma1 attachment protein genetically modified with a nuclear localization sequence (sigma1-NLS) as a targeting ligand. Purified sigma1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with sigma1 or sigma1-NLS. In L929 fibroblasts loaded with sigma1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified sigma1. Transfection of L929 cells with sigma1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using sigma1-conjugated PEI. These results suggest that sigma1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy.

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein sigma1 and membrane penetration protein mu1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a sigma1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L x T3D reassortant viruses revealed that the mu1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of sigma1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the mu1 protein plays an essential role in stimulating proapoptotic signaling.

The reovirus virion is a moderately complex structure that contains eight structural proteins organized in multiple concentric capsid layers. Mammalian orthoreovirus virions undergo partial uncoating to produce infectious subvirion particles (ISVPs) and cores. Virions and ISVPs are infectious, whereas cores are transcriptionally active, but non-infectious, presumably because cores lack entry signals present in outer capsid proteins. We generated and purified reovirus cores and exposed them to cells with and without transfection reagents to directly test whether punfied cores can establish productive infections. Cores added directly to cells were essentially non-infectious. Specific infectivity of core/lipofectamine mixtures was increased more than 10,000-fold. Similar results were found with two reovirus serotypes (T1L and T3D) and in different cell types. These experiments indicated transfection reagents enhance core particle entry into cells and that cores contain all necessary components to replicate. This method may pave the way to improved genetic manipulations and to improved usage of reovirus as an anti-cancer agent.

The mechanisms by which viruses kill susceptible cells in target organs and ultimately produce disease in the infected host remain poorly understood. Dependent upon the site of inoculation and strain of virus, experimental infection of neonatal mice with reoviruses can induce fatal encephalitis or myocarditis. Reovirus-induced apoptosis is a major mechanism of tissue injury, leading to disease development in both the brain and heart. In cultured cells, differences in the capacity of reovirus strains to induce apoptosis are determined by the S1 gene segment, which also plays a major role as a determinant of viral pathogenesis in both the heart and the central nervous system (CNS) in vivo. The S1 gene is bicistronic, encoding both the viral attachment protein sigma-1 and the nonstructural protein sigma-1-small (sigma1s). Although sigma1s is dispensable for viral replication in vitro, we wished to investigate the expression of sigma1s in the infected heart and brain and its potential role in reovirus pathogenesis in vivo. Two-day-old mice were inoculated intramuscularly or intracerebrally with either sigma1s(-) or sigma1s(+) reovirus strains. While viral replication in target organs did not differ between sigma1s(-) and sigma1s(+) viral strains, virus-induced caspase-3 activation and resultant histological tissue injury in both the heart and brain were significantly reduced in sigma1s(-) reovirus-infected animals. These results demonstrate that sigma1s is a determinant of the magnitude and extent of reovirus-induced apoptosis in both the heart and CNS and thereby contributes to reovirus pathogenesis and virulence.

Reovirus infections are typically subclinical in weaned mice, and are best detected using serologic tests. After exposure to the soiled bedding of some mice obtained from various sources, numerous sentinel mice tested reovirus seropositive by use of indirect immunofluorescence assays (IIFA) in our institution. A major commercial rodent pathogen testing laboratory verified our IIFA results, but since the same samples were reovirus seronegative using their "more specific" enzyme-linked immunosorbent assay (ELISA), the IIFA results were reported as "false positives." As past in-house observations suggested transmission of the virus to sentinel and other animals, we sought to determine whether the IIFA results were always "false positives." An opportunity to test this notion arose after receipt of reovirus IIFA-positive transgenic mice from an academic source. Using reverse transcriptase-polymerase chain reaction (RT-PCR) assays, the presence of reovirus RNA was detected in fecal specimens taken from some sentinel animals that subsequently seroconverted from IIFA-negative to IIFA-positive for reovirus. The virus could not be isolated by use of tissue culturing methods. Nucleotide sequence analysis established the presence of unique reovirus sequences. These results indicate that contemporary reovirus infections may not be detected by use of some serologic tests, and that RT-PCR analysis may be useful for confirmation of active reovirus infection in certain situations.

Mammalian reoviruses are non-enveloped viruses that contain a segmented, double-stranded RNA genome. Reoviruses infect most mammalian species, although infection with these viruses in humans is usually asymptomatic. We report the isolation of a novel reovirus strain from a 6.5-week-old child with meningitis. Hemagglutination and neutralization assays indicated that the isolate is a serotype 3 strain, leading to the designation T3/Human/Colorado/1996 (T3C/96). Sequence analysis of the T3C/96 S1 gene segment, which encodes the viral attachment protein, sigma 1, confirmed the serotype assignment for this strain and indicated that T3C/96 is a novel reovirus isolate. T3C/96 is capable of systemic spread in newborn mice after peroral inoculation and produces lethal encephalitis. These results suggest that serotype 3 reoviruses can cause meningitis in humans.

Reovirus type 3 (Reo-3) can infect numerous rodent species and induces the clinical syndrome 'oily skin disease' in neonatal mice, and is a common contaminant of biological materials. The reverse transcriptase polymerase chain reaction (RT-PCR) assay has proven useful for the detection of Reo-3 in rodents and contaminated biological materials. Fluorogenic nuclease reverse transcriptase polymerase chain reaction assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, an fnRT-PCR assay specific for Reo-3 was developed by targeting primer and probe sequences to a unique region of the Reo-3 M3 gene. The fnRT-PCR detected both strains of Reo-3 (Dearing and Abney), but did not detect Reovirus types 1 or 2, other viruses in the family Reoviridae, or other RNA viruses that naturally infect rodents. The fnRT-PCR detected less than 1 fg of target template and detected viral RNA in tissues obtained from mice experimentally infected with Reo-3. The assay also displayed comparable sensitivity when compared to the mouse antibody production test commonly used to detect viral contamination of biological materials. In conclusion, this fnRT-PCR assay offers a potentially high-throughput diagnostic assay for detecting Reo-3 RNA in infected mice and contaminated biological materials.

Infection of neonatal mice with some reovirus strains produces a disease similar to infantile biliary atresia, but previous attempts to correlate reovirus infection with this disease have yielded conflicting results. We used isogenic reovirus strains T3SA- and T3SA+, which differ solely in the capacity to bind sialic acid as a coreceptor, to define the role of sialic acid in reovirus encephalitis and biliary tract infection in mice. Growth in the intestine was equivalent for both strains following peroral inoculation. However, T3SA+ spread more rapidly from the intestine to distant sites and replicated to higher titers in spleen, liver, and brain. Strikingly, mice infected with T3SA+ but not T3SA- developed steatorrhea and bilirubinemia. Liver tissue from mice infected with T3SA+ demonstrated intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in humans, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ bound 100-fold more efficiently than T3SA- to human cholangiocarcinoma cells. These observations suggest that the carbohydrate-binding specificity of a virus can dramatically alter disease in the host and highlight the need for epidemiologic studies focusing on infection by sialic acid-binding reovirus strains as a possible contributor to the pathogenesis of neonatal biliary atresia.

Oral inoculation of reovirus type 3 Abney (T3A) into neonatal mice induces hepatitis and the biliary atresia-associated oily fur syndrome (OFS), a phenotype previously linked to the S1 gene. We found that following oral inoculation, none of three T3A mutants, JH2, JH3, and JH4, containing different single amino acid substitutions in the M2 gene, induced the OFS or extensive liver necrosis. Similarly, reassortant viruses containing both a JH4-S1 and a JH4-M2 gene segment did not induce the OFS, whereas another reassortant containing a JH4-S1 gene and a M2 gene from reovirus type 3 Dearing fully recovered this capacity. Together, these results constitute the first evidence for the involvement of the M2 gene in the S1 gene-associated capacity of T3A to induce hepatobiliary disease in neonatal mice.

To better understand the mechanisms by which neurotropic viruses invade peripheral nerve pathways and produce CNS disease, we defined the type 3 (T3) reovirus genes that are determinants of the capacity of reovirus T3 strain Dearing (T3D) and T3 clone 9 (C9) to infect the spinal cord and kill mice after hindlimb injection. T3D and C9 viruses are both highly virulent (LD(50) < 10(1) PFU) after intracranial injection of neonatal mice. However, C9 is significantly more lethal than T3D after either intramuscular injection (LD(50) < 10(1) vs LD(50) 10(4) PFU) or peroral injection (LD(50) 10(3.4) vs LD(50) > 10(8.3) PFU). Using reassortant viruses containing different combinations of genes derived from T3D and C9, we found that the S1 gene, encoding the cell attachment protein sigma 1 and the nonstructural protein sigma 1s, and the L3 gene, encoding the core shell protein lambda 1 were the primary determinants of lethality after intramuscular injection. The L3 gene and the L2 gene encoding spike protein, lambda 2, determined differences in spinal cord titer after intramuscular injection. A C9 x T3D mono-reassortant containing all T3D genes except for the C9-derived L3 was lethal after peroral injection. These studies indicate that the S1, L2, and L3 genes all play a potential role in neuroinvasiveness and provide the first identification of a role in pathogenesis for the L3 gene.