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Mammalian reovirus (MRV) is a non-enveloped, gene segmented double-stranded RNA (dsRNA) virus. It is an important zoonotic pathogen that infects many mammals and vertebrates that act as natural hosts and causes respiratory and digestive tract diseases. Studies have reported that RIG-I and MDA5 in the innate immune cytoplasmic RNA-sensing RIG-like receptor (RLR) signaling pathway can recognize dsRNA from MRV and promote antiviral type I interferon (IFN) responses. However, the mechanism by which many MRV-encoded proteins evade the host innate immune response remains unclear. Here, we show that exogenous μ1 protein promoted the proliferation of MRV in vitro, while knockdown of MRV μ1 protein expression by shRNA could impair MRV proliferation. Specifically, μ1 protein inhibited MRV or poly(I:C)-induced IFN-β expression, and attenuated RIG-I/MDA5-mediated signaling axis transduction during MRV infection. Importantly, we found that μ1 protein significantly decreased IFN-β mRNA expression induced by MDA5, RIG-I, MAVS, TBK1, IRF3(5D), and degraded the protein expression of exogenous MDA5, RIG-I, MAVS, TBK1 and IRF3 via the proteasomal and lysosomal pathways. Additionally, we show that μ1 protein can physically interact with MDA5, RIG-I, MAVS, TBK1, and IRF3 and attenuate the RIG-I/MDA5-mediated signaling cascades by blocking the phosphorylation and nuclear translocation of IRF3. In conclusion, our findings reveal that MRV outer capsid protein μ1 is a key factor in antagonizing RLRs signaling cascades and provide new strategies for effective prevention and treatment of MRV infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases., (© 2020 Blackwell Verlag GmbH.)

Mammalian orthoreoviruses (MRVs) infect almost all mammals, and there are some reports on MRVs in China. In this study, a novel strain was identified, which was designated as HLJYC2017. The results of genetic analysis showed that MRV HLJYC2017 is a reassortant strain. According to biological information analysis, different serotypes of MRV contain specific amino acid insertions and deletions in the σ1 protein. Neutralizing antibody epitope analysis revealed partial cross-protection among MRV1, MRV2, and MRV3 isolates from China. L3 gene recombination in MRV was identified for the first time in this study. The results of this study provide valuable information on MRV reassortment and evolution.

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease. IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease., (Copyright © 2018 American Society for Microbiology.)

In the course of previous works, it was observed that the virus laboratory stock (T3D S ) differs in sequence from the virus encoded by the ten plasmids currently in use in many laboratories (T3D K ), and derived from a different original virus stock. Seven proteins are affected by these sequence differences. In the present study, replication of T3D K was shown to be more sensitive to the antiviral effect of interferon. Infection by the T3D K virus was also shown to induce the production of higher amount of β and α-interferons compared to T3D S . Two proteins, the μ2 and λ2 proteins, were found to be responsible for increased sensitivity to interferon while both μ2 and λ1 are responsible for increased interferon secretion. Altogether this supports the idea that multiple reovirus proteins are involved in the control of induction of interferon and virus sensitivity to the interferon-induced response. While interrelated, interferon induction and sensitivity can be separated by defined gene combinations. While both μ2 and λ2 were previously suspected of a role in the control of the interferon response, other proteins are also likely involved, as first shown here for λ1. This also further stresses that due caution should be exerted when comparing different virus isolates with different genetic background., (Copyright © 2018 Elsevier B.V. All rights reserved.)

New therapeutic interventions are essential for improved management of patients with metastatic colorectal cancer (mCRC). This is especially critical for those patients whose tumors harbor a mutation in the KRAS oncogene (40-45% of all patients). This patient cohort is excluded from receiving anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Reovirus, a double stranded (ds) RNA virus is in clinical development for patients with chemotherapy refractory KRAS mutated tumors. Toll Like Receptor (TLR) 3, a member of the toll like receptor family of the host innate immune system is the pattern recognition motif for dsRNA pathogens. Using TLR3 expressing commercial HEK-BlueTM-hTLR3 cells we confirm that TLR3 is the host pattern recognition motif responsible for the detection of reovirus. Further, our investigation with KRAS mutated HCT116 cell line showed that effective expression of host TLR3 dampens the infection potential of reovirus by mounting a robust innate immune response. Down regulation of TLR3 expression with siRNA improves the anticancer activity of reovirus. In vivo experiments using human CRC cells derived xenografts in athymic mice further demonstrate the beneficial effects of TLR3 knock down by improving tumor response rates to reovirus. Strategies to mitigate the TLR3 response pathway can be utilized as a tool towards improved reovirus efficacy to specifically target the dissemination of KRAS mutated CRC.

Mucosal surfaces are protected from infection by both structural and sentinel cells, such as mast cells. The mast cell's role in antiviral responses is poorly understood; however, they selectively recruit natural killer (NK) cells following infection. Here, the ability of virus-infected mast cells to enhance NK cell functions was examined. Cord blood-derived human mast cells infected with reovirus (Reo-CBMC) and subsequent mast cell products were used for the stimulation of human NK cells. NK cells upregulated the CD69 molecule and cytotoxicity-related genes, and demonstrated increased cytotoxic activity in response to Reo-CBMC soluble products. NK cell interferon (IFN)-γ production was also promoted in the presence of interleukin (IL)-18. In vivo, SCID mice injected with Reo-CBMC in a subcutaneous Matrigel model, could recruit and activate murine NK cells, a property not shared by normal human fibroblasts. Soluble products of Reo-CBMC included IL-10, TNF, type I and type III IFNs. Blockade of the type I IFN receptor abrogated NK cell activation. Furthermore, reovirus-infected mast cells expressed multiple IFN-α subtypes not observed in reovirus-infected fibroblasts or epithelial cells. Our data define an important mast cell IFN response, not shared by structural cells, and a subsequent novel mast cell-NK cell immune axis in human antiviral host defense., (© 2016 S. Karger AG, Basel.)

Intestinal epithelial cells (IECs) constitute the primary barrier that separates us from the outside environment. These cells, lining the surface of the intestinal tract, represent a major challenge that enteric pathogens have to face. How IECs respond to viral infection and whether enteric viruses have developed strategies to subvert IECs innate immune response remains poorly characterized. Using mammalian reovirus (MRV) as a model enteric virus, we found that the intermediate subviral particles (ISVPs), which are formed in the gut during the natural course of infection by proteolytic digestion of the reovirus virion, trigger reduced innate antiviral immune response in IECs. On the contrary, infection of IECs by virions induces a strong antiviral immune response that leads to cellular death. Additionally, we determined that virions can be sensed by both TLR and RLR pathways while ISVPs are sensed by RLR pathways only. Interestingly, we found that ISVP infected cells secrete TGF-β acting as a pro-survival factor that protects IECs against virion induced cellular death. We propose that ISVPs represent a reovirus strategy to initiate primary infection of the gut by subverting IECs innate immune system and by counteracting cellular-death pathways., (© 2016 John Wiley & Sons Ltd.)

In the last few years, the development of a plasmid-based reverse genetics system for mammalian reovirus has allowed the production and characterization of mutant viruses. This could be especially significant in the optimization of reovirus strains for virotherapeutic applications, either as gene vectors or oncolytic viruses. The genome of a mutant virus exhibiting increased sensitivity to interferon was completely sequenced and compared with its parental virus. Viruses corresponding to either the parental or mutant viruses were then rescued by reverse genetics and shown to exhibit the expected phenotypes. Systematic rescue of different viruses harboring either of the four parental genes in a mutant virus backbone, or reciprocally, indicated that a single amino acid substitution in one of λ2 methyltransferase domains is the major determinant of the difference in interferon sensitivity between these two viruses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Viral infections activate many host signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which has recently attracted considerable interest due to its central role in modulating virus replication. This study demonstrated that the sero-type 3 reovirus strain Masked Palm Civet/China/2004 (MPC/04) could transiently activate the PI3K/Akt pathway in A549 cells at earlier time points of infection. The blockage of PI3K/Akt activation increased viral RNA synthesis and yield. The role of the downstream effectors MDM2/p53 of PI3K/Akt in regulating reovirus replication was further analyzed. We found that during reovirus infection, the level of phosphorylated MDM2 (p-MDM2) was increased and the expression of p53 was reduced. In addition, the blockage of PI3K/Akt by Ly294002 or knockdown of Akt by siRNA reduced the level of p-MDM2 and increased the level of p53. Both indicated that the downstream effectors MDM2/p53 of PI3K/Akt were activated. Pre-treatment with Nutlin, which can destroy MDM2 and p53 cross-talk and increase the expression of p53 RNA and protein, dose-dependently enhanced reovirus replication. Additionally, the overexpression of p53 alone also supported reovirus replication, and knockdown of p53 significantly inhibited viral replication. This study demonstrates that PI3K/Akt/p53 activated by mammalian reovirus can serve as a pathway for inhibiting virus replication/infection, yet the precise mechanism of this process remains under further investigation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Reovirus has gained much attention as an anticancer agent; however, the mechanism of the tumor cell-specific replication of reovirus is not fully understood. Although Ras activation is known to be crucial for tumor cell-specific replication of reovirus, it remains controversial which cellular factors are required for the reovirus-mediated tumor cell killing. In this study, we systematically investigated which cellular factors determined the efficiencies of reovirus-mediated tumor cell killing in various human cultured cell lines. The efficiency of reovirus-mediated cell killing varied widely among the cell lines. Junction adhesion molecule-A, a reovirus receptor, was highly expressed in almost all cell lines examined. Ras activation levels were largely different between the cell lines; however, there were no apparent correlations among the reovirus-mediated cell killing efficiencies and Ras activation status. On the other hand, activity levels of the cysteine proteases cathepsins B and L, which are crucial for proteolytic disassembly of the outer capsid proteins of reovirus, showed a tendency to be correlated with the efficiency of reovirus-mediated cell killing. These results indicate that the activity of cathepsins B and L is the most suitable as a biomarker for the efficacy of reovirus-mediated oncolysis among the factors examined in this study.

Background: Because mammalian reoviruses are isolated from the respiratory tract we modeled the natural history of respiratory infection of adult and suckling mice with T1 Lang (T1L) and T3 Dearing (T3D) reoviruses., Methods: Adult and suckling Balb/c mice were infected by the intranasal route and were assessed for dose response of disease as well as viral replication in the lung and other organs. Viral antigen was assessed by immunofluorescence and HRP staining of tissue sections and histopathology was assessed on formalin fixed, H + E stained tissue sections., Results: Intranasal infection of adult mice resulted in fatal respiratory distress for high doses (10(7) pfu) of T1L but not T3D. In contrast both T1L and T3D killed suckling mice at moderate viral dosages (10(5) pfu) but differed in clinical symptoms where T1L induced respiratory failure and T3D caused encephalitis. Infections caused transient viremia that resulted in spread to peripheral tissues where disease correlated with virus replication, and pathology. Immunofluorescent staining of viral antigens in the lung showed reovirus infection was primarily associated with alveoli with lesser involvement of bronchiolar epithelium. Immunofluorescent and HRP staining of viral antigens in brain showed infection of neurons by T3D and glial cells by T1L., Conclusions: These mouse models of reovirus respiratory infection demonstrated age and strain dependent disease that are expected to be relevant to understanding and modulating natural and therapeutic reovirus infections in humans.

In this study, we investigated humoral and cellular immune responses in mice to DNA vaccines containing individual S or M genes of a new type of reovirus (nRV) isolate from a severe acute respiratory syndrome (SARS) patient in Beijing, China. Mice were immunized intramuscularly (i.m.) with 100 μg of S1, S2, S3, S4, M1, M2, and M3 DNA vaccine each 4 times in 2-week intervals and assayed for humoral IgG, IgG1, IgG2, and IgG2b antibodies by ELISA and for cellular immune response, particularly IFN-γ induction by ELISpot assay. Moreover, CD4+ and CD8+ T cell levels in peripheral blood mononuclear cells were assayed by flow cytometry. We found that all DNA vaccines induced IgG antibodies, predominantly of the IgG2a class and S3 DNA vaccine was the strongest inducer. M2 and S3 DNA vaccines elicited Th1- and Th2-based immune responses, respectively, while S1 and M3 DNA vaccines induced a mixed Th1/Th2 response. M1, S2, and S4 DNA vaccines were poorly immunogenic. To our knowledge, this is the first report characterizing mammalian reovirus DNA vaccines applied to a mouse model.

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 aim of this study is to clarify the effects of administrated interferon (IFN)-α on Reovirus type-2 (Reo-2)-triggered autoimmune insulitis. Newborn DBA/1J mice infected with Reo-2 (on day 0) showed the highest titre in the pancreas on day 5 and thereafter the titre declined. Similar viral growth curve with lower titre was found in the spleen and thymus. Insulitis with impaired glucose tolerance developed in infected mice on day 10, but not on day 5. IFN-α was produced in the blood on days 3 and 5, but not on days 7 and 10. During the virus growth phase, IFN-α positive((+)) cells were detected in some pancreatic islet cells and infiltrated dendritic cells (DCs) in interstitium. Virus antigen positive cells were detected in islet cells, but not in DCs. Administration with IFN-α (10(2) , 10(3) or 10(4) international unit) on day 7, which is the time of the disappearance of virus from the pancreas and IFN-α from the blood, exacerbated insulitis with increased glucose values compared to only infected mice. IFN-α administration at the same time of infection did not develop insulitis. In addition, IFN-α administration in uninfected mice on day 7 did not cause any damage to islet cells. The present study suggests that IFN-α may have a possibility to exacerbate Reo-2-tiggered mild insulitis., (© 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.)

The aim of the present study was to determine whether or not the development of a helper T (Th) 1 response induced by Reovirus type-2 (Reo-2) infection would protect against the development of Th2-mediated late allergic asthma. This hypothesis was examined by infecting one day old neonatal DB A/1J mice with Reo-2 in an ovalbumin (OVA)-induced late asthma model. Compared with the controls (either infected or uninfected mice with or without OVA sensitization and/or OVA challenge), Reo-2 infection lessened the magnitude of the subsequent allergic Th2-mediated late asthma. In infected mice with allergic late asthma, there was decreased infiltration of interleukin (IL)-4(+), IL-5(+), IL-13(+) and very late antigen (VLA)-4(+) lymphocytes, and eotaxin-2(+) and VLA-4(+) eosinophils, in both bronchial and bronchiolar lesions. Also the expression of vascular cell adhesion molecule (VCAM)-1 and eotaxin-2 on vascular endothelial cells was reduced. Moreover, the systemic production of IL-4, IL-5, tumour necrosis factor-α and OVA-specific IgE was reduced, whereas systemic IFN-γ production was increased. In addition, there was no increase in IFN-α production. Thus the present study suggests that systemic Reo-2 infection at birth may reduce the development of subsequent late allergic asthma by the induction of a Th1 response. Therefore the potential suppressive mechanism(s) that might be induced by Reo-2 infection in newborn mice and their effects on the development of late allergic asthma are discussed., (© 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.)

For many encephalitis cases, the cause remains unidentified. After 2 children (from the same family) received a diagnosis of acute necrotizing encephalopathy at Centre Hospitalier Universitaire (Tours, France), we attempted to identify the etiologic agent. Because clinical samples from the 2 patients were negative for all pathogens tested, urine and throat swab specimens were added to epithelial cells, and virus isolates detected were characterized by molecular analysis and electron microscopy. We identified a novel reovirus strain (serotype 2), MRV2Tou05, which seems to be closely related to porcine and human strains. A specific antibody response directed against this new reovirus strain was observed in convalescent-phase serum specimens from the patients, whereas no response was observed in 38 serum specimens from 38 healthy adults. This novel reovirus is a new etiologic agent of encephalitis.

Bronchiolitis obliterans organizing pneumonia (BOOP) and acute respiratory distress syndrome (ARDS) are two clinically and histologically distinct syndromes sharing the presence of an inflammatory and fibrotic component. Apoptosis via the Fas/Fas ligand (FasL) pathway plays an important role in the development of acute lung injury and fibrosis characteristic of these and other pulmonary inflammatory and fibrotic syndromes. We evaluated the role of apoptosis via the Fas/FasL pathway in the development of pulmonary inflammation and fibrosis in reovirus 1/L-induced BOOP and ARDS. CBA/J mice were intranasally inoculated with saline, 1 x 10(6) (BOOP), or 1 x 10(7) (ARDS) PFU reovirus 1/L, and evaluated at various days postinoculation for in situ apoptosis by TUNEL analysis and Fas/FasL expression. Our results demonstrate the presence of apoptotic cells and up-regulation of Fas/FasL expression in alveolar epithelium and in infiltrating cells during the inflammatory and fibrotic stages of both reovirus 1/L-induced ARDS and BOOP. Treatment of mice with the caspase 8 inhibitor, zIETD-fmk, inhibited apoptosis, inflammation, and fibrotic lesion development in reovirus 1/L-induced BOOP and ARDS. However, CBA/KlJms-Fas(lpr-cg)/J mice, which carry a point mutation in the Fas cytoplasmic region that abolishes the ability of Fas to transduce an apoptotic signal, do not develop pulmonary inflammation and fibrotic lesions associated with reovirus 1/L-induced BOOP, but still develop inflammation and fibrotic lesions associated with reovirus 1/L-induced ARDS. These results suggest a differential role for the Fas/FasL apoptotic pathway in the development of inflammation and fibrotic lesions associated with BOOP and ARDS.

Mammalian orthoreoviruses (reoviruses) are ubiquitous viral agents that infect cells in respiratory and enteric tracts. The frequency and nature of human cellular immunoregulatory responses against reovirus are unknown. Here we establish systems to detect and quantify reovirus-induced cytokine and chemokine recall responses using primary cultures of virus-infected peripheral blood mononuclear cells (PBMC) and two widely used reovirus serotypes, type 1 Lang (T1L) and type 3 Dearing (T3D) reexposure in vitro. In cultures from 44 healthy adults, reovirus induced exceptionally strong CD4 and CD8 T-cell-dependent gamma interferon (IFN-gamma) recall responses concomitant with intense interleukin 10 (IL-10) production. These responses were elicited independently of viral replication. Surprisingly, paired analyses of subject responses to these two common serotypes revealed that while both elicit intense Th1-dominated immunity, median T3D-driven responses were 2.2-fold weaker (P = 0.0004) than those elicited by T1L. Recall responses evoked by these viral serotypes differed markedly in their mechanism of regulation. T3D IL-10 and IFN-gamma responses were CD4 and CD8 dependent and blocked by interfering with CD86 costimulation but were CD80 independent. T1L responses were consistently CD28 and CD80/86 independent. Thus, despite extensive genetic and morphological similarities between reovirus serotypes, the nature and intensity of the human recall responses as well as the control mechanisms regulating them are clearly distinct.

We hypothesized that consumption of the (n-3) PUFA, docosahexaenoic acid (DHA), modulates the mucosal immune response to enteric infection with respiratory enteric orphan virus (reovirus), a model intestinal pathogen. Mice were fed either AIN-93G control diet, containing 10 g/kg corn oil and 60 g/kg high oleic acid safflower oil, or AIN-93G, containing 10 g/kg corn oil and 60 g/kg DHA-enriched fish oil, for 4 wk and then orally gavaged with reovirus strain Type 1 Lang, (T1/L). Reovirus-specific IgA antibody was first detectable in the feces of mice fed a control diet at 6 d postinfection (PI) and was further elevated at 8 and 10 d PI. IgA responses in DHA-fed mice were similar at 6 and 8 d PI but greater at 10 d PI (P < 0.05). Both reovirus-specific serum IgA and IgG(2a) were comparably induced in mice fed control or DHA diets. Reovirus-specific IgA and IgG(2a) secretion by ex vivo Peyer's patch, lamina propria, and spleen cultures derived from control and DHA groups were comparable. Although both groups carried similar numbers of reovirus plaque forming units per intestine, DHA-fed mice shed nearly 10 times more viral RNA in feces than control mice at 2, 4, and 6 d PI (P < 0.05). However, viral RNA was not detectable in either group at 8 and 10 d. Taken together, these data suggest that DHA consumption did not markedly alter mucosal or systemic Ig responses to reovirus but delayed clearance of the virus from the intestinal tract.

We defined the function of type I interferons (IFNs) in defense against reovirus strain type 1 Lang (T1L), which is a double-stranded RNA virus that infects Peyer's patches (PPs) after peroral inoculation of mice. T1L induced expression of mRNA for IFN-alpha, IFN-beta, and Mx-1 in PPs and caused localized intestinal infection that was cleared in 10 d. In contrast, T1L produced fatal systemic infection in IFNalphaR1 knockout (KO) mice with extensive cell loss in lymphoid tissues and necrosis of the intestinal mucosa. Studies of bone-marrow chimeric mice indicated an essential role for hematopoietic cells in IFN-dependent viral clearance. Dendritic cells (DCs), including conventional DCs (cDCs), were the major source of type I IFNs in PPs of reovirus-infected mice, whereas all cell types expressed the antiviral protein Mx-1. Neither NK cells nor signaling via Toll-like receptor 3 or MyD88 were essential for viral clearance. These data demonstrate a requirement for type I IFNs in the control of an intestinal viral infection and indicate that cDCs are a significant source of type I IFN production in vivo. Therefore, innate immunity in PPs is an essential component of host defense that limits systemic spread of pathogens that infect the intestinal mucosa.

The interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases and accumulates in the cytoplasm. MxA is a key component of the innate antiviral response and has previously been shown to inhibit several viruses with single-stranded RNA genomes of both polarities and a DNA virus. In addition, MxA also targets two double-stranded RNA viruses, Infectious bursal disease virus and a mammalian reovirus as shown in this study. Thus, the antiviral spectrum of human MxA is broader than hitherto suspected. Interestingly, virus growth was not affected in cells expressing MxA(E645R), a mutant form of MxA that showed antiviral activity against orthomyxoviruses.

Exposure to immunosuppressive environmental contaminants is a possible contributing factor to increased occurrence of viral respiratory diseases. The objective of this study was to test the hypothesis that the trichothecene mycotoxin T-2 toxin (T-2), a frequent food contaminant, alters host resistance to lung infection by reovirus, a model respiratory virus. Balb/c mice (4 week old) were treated intraperitoneally with T-2 toxin (1.75 mg/kg bw) or saline vehicle and then intranasally instilled 2 h later with 10(7) plaque forming unit (PFU) of reovirus, strain Lang (T1/L) or saline vehicle. At 10 days post-instillation (PI), both virus plaque-forming responses and reovirus L2 gene expression were 10-fold higher in lungs of T-2-treated mice compared to controls. No-effect and lowest-effect levels for T-2-induced suppression of reovirus clearance were 20 and 200 microg/kg bw, respectively. Respiratory reovirus infection resulted in a mild bronchiolitis with minimal alveolitis, which was markedly exacerbated by T-2 pretreatment. Reovirus exposure induced marked increases in total cells, neutrophils and lymphocytes at 3 and 7 days PI in bronchial alveolar lavage fluid (BALF) whereas macrophages were increased only at 7 days PI. Although prior T-2 exposure attenuated total cell and macrophage counts in BALF of control and infected mice at 3 days PI, the toxin potentiated total cell, macrophage, neutrophil and lymphocyte counts in infected mice at 7 days PI. At 3 days PI, T-2 suppressed reovirus-induced IFN-gamma elevation in BALF, but enhanced production of IL-6 and MCP-1. T-2 pretreatment also suppressed reovirus-specific mucosal IgA responses in lung and enteric tract, but potentiated serum IgA and IgG responses. Taken together, T-2 increased lung viral burden, bronchopneumonia and pulmonary cellular infiltration in reovirus-infected mice. These effects might be attributable to reduced alveolar macrophage levels as well as modulated cytokine and mucosal Ig responses.

Mammalian Orthoreoviruses are important models for studies of viral pathogenesis. In the rat lung, Reovirus strain type 3 Dearing (T3D) induces substantially more inflammation than does strain type 1 Lang (T1L). To better understand mechanisms underlying differences in the host inflammatory response elicited by T1L and T3D, we characterized cytokine expression patterns induced by those strains after infection of THP-1 monocyte cells. THP-1 cells were adsorbed with either viable or ultraviolet- inactivated T1L and T3D and assayed for mRNA and protein production of growth-regulated oncogene-alpha (GRO-alpha), interleukin-8 (IL-8), or tumor necrosis factor-alpha (TNF-alpha). T3D stimulated mRNA and protein production of all three cytokines, whereas T1L stimulated mRNA and protein production of IL-8 and TNF-alpha but not GRO-alpha. In each case, T3D induced greater cytokine mRNA and protein expression than did T1L. Nonviable virus did not stimulate detectable cytokine secretion, suggesting a requirement for viral RNA synthesis in cytokine induction by THP-1 cells. A greater percentage of THP-1 cells was infected with T1L than T3D as assessed by infectious center assay, and T1L achieved higher yields of infectious progeny than did T3D in infected THP-1 cells as determined by plaque assay. These strain-dependent differences in cytokine responses and corresponding replication patterns in monocyte cells parallel findings made in studies of rat models of pneumonia and provide clues about how Reovirus interfaces with the host innate immune response to produce pulmonary disease.

We have reported previously that subclinical prolonged mild T helper (Th) 1-dependent autoimmune insulitis with impaired glucose tolerance in wealing DBA/1J mice, which is induced by the combined effects of reovirus type 2 (Reo-2) and synthetic 20-base oligodeoxynucleotides with CpG motifs (CpG ODN) (control mice). Compared with the control mice, newborn mice treated with monoclonal antibody (MoAb) against mouse CD25(+) CD4(+) T cells together with Reo-2 and CpG ODN greatly reduced the absolute number of splenic CD25(+) T cells and resulted in the development of severe insulitis, leading to an overt early diabetes. Moreover, the treatment of the MoAb increased production of interferon-gamma (IFN-gamma) and decreased that of interleukin-4 (IL-4) and transforming growth factor-beta1 (TGF-beta1) and developed high titre of autoantibodies against pancreatic islet cells. These evidences suggest that CD4(+) CD25(+) T cell may, at least in part, maintain tolerance to Reo-2-triggered and CpG ODN-induced prolonged mild Th1-dependent autoimmune insulitis, leading to the overt disease. This system may give a novel model to elucidate the mechanisms of the development of overt diabetes from borderline subclinical diabetes in virus-triggered autoimmune type I diabetes in human.

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.

Although reovirus infections are thought to be common in adults, there have been few assessments of the seroprevalence of reovirus in young children. We developed an indirect enzyme-linked immunosorbent assay to measure levels of total antireovirus immunoglobulin A, G, and M in serum specimens collected from otherwise healthy infants and children (1 month to 5 years of age) in Nashville, Tennessee. Of the 272 serum specimens evaluated, 64 (23.5%) tested positive for reovirus-specific antibodies. We observed an age-dependent increase in reovirus-specific antibodies in children 1 year of age and older, peaking at 50.0% in children 5-6 years of age. These findings suggest that reovirus infections are common during early childhood.

Intraduodenal priming of mice with reovirus serotype 1/strain Lang (reovirus 1/L) stimulates gut lymphocytes and generates precursor and effector CTLs. Our earlier studies demonstrated that germinal center and T cell Ag (GCT) is a marker which identifies reovirus 1/L-specific precursor CTL and effector CTL in Peyer's patches (PP) of reovirus 1/L-inoculated mice. In this study, we characterized the expression of the activation markers, GCT and CD11c, on reovirus 1/L-stimulated gut lymphocytes and the effector mechanisms involved in reovirus 1/L-specific cytotoxicity. We found that intraduodenal reovirus 1/L inoculation of mice induced the expression of both GCT and CD11c on PP lymphocytes (PPL), intraepithelial lymphocytes (IEL), and lamina propria lymphocytes (LPL), and these activated cells expressed Fas ligand (FasL). The majority of the GCT+ CD11c+ IEL and LPL expressed a phenotype, TCRalphabeta+ Thy-1+ CD8+ similar to that expressed on reovirus 1/L-stimulated PPL. However, splenic lymphocytes expressed GCT but not CD11c after stimulation with reovirus 1/L. Perforin, Fas-FasL, and TRAIL pathways were found to be involved in PPL, IEL, and LPL cytotoxic activity against reovirus 1/L-infected targets. In PPL, perforin and Fas-FasL pathways were more effective than TRAIL. In IEL, all three cytotoxic mechanisms were equally as effective. However, LPL prefer Fas-FasL and TRAIL over perforin. Further, we demonstrated the preferential migration of GCT+ PPL to the intraepithelial compartment and the lamina propria. These results suggest that GCT and CD11c can be used as activation markers for gut lymphocytes and CD11c can also be used to differentiate between activated gut and systemic lymphocytes.

Viral myocarditis is an important human disease, with a wide variety of viruses implicated. Cardiac myocytes are not replenished yet are critical for host survival and thus may have a unique response to infection. Previously, we determined that the extent of reovirus induction of beta interferon (IFN-beta) and IFN-beta-mediated protection in primary cardiac myocyte cultures was inversely correlated with the extent of reovirus-induced cardiac damage in a mouse model. Surprisingly, and in contrast, the IFN-beta response did not determine reovirus replication in skeletal muscle cells. Here we compared the IFN-beta response in cardiac myocytes to that in primary cardiac fibroblast cultures, a readily replenished cardiac cell type. We compared basal and reovirus-induced expression of IFN-beta, IRF-7 (an interferon-stimulated gene [ISG] that further induces IFN-beta), and another ISG (561) in the two cell types by using real-time reverse transcription-PCR. Basal IFN-beta, IRF-7, and 561 expression was higher in cardiac myocytes than in cardiac fibroblasts. Reovirus T3D induced greater expression of IFN-beta in cardiac myocytes than in cardiac fibroblasts but equivalent expression of IRF-7 and 561 in the two cell types (though fold induction for IRF-7 and 561 was higher in fibroblasts than in myocytes because of the differences in basal expression). Interestingly, while reovirus replicated to equivalent titers in cardiac myocytes and cardiac fibroblasts, removal of IFN-beta resulted in 10-fold-greater reovirus replication in the fibroblasts than in the myocytes. Together the data suggest that the IFN-beta response controls reovirus replication equivalently in the two cell types. In the absence of reovirus-induced IFN-beta, however, reovirus replicates to higher titers in cardiac fibroblasts than in cardiac myocytes, suggesting that the higher basal IFN-beta and ISG expression in myocytes may play an important protective role.

Nonfusogenic mammalian orthoreovirus (reovirus) is an enteric pathogen of mice and a useful model for studies of how an enteric virus crosses the mucosal barrier of its host and is subject to control by the mucosal immune system. We recently generated and characterized a new murine immunoglobulin A (IgA)-class monoclonal antibody (MAb), 1E1, that binds to the adhesin fiber, sigma1, of reovirus type 1 Lang (T1L) and thereby neutralizes the infectivity of that strain in cell culture. 1E1 is produced in hybridoma cultures as a mixture of monomers, dimers, and higher polymers and is protective against peroral challenges with T1L either when the MAb is passively administered or when it is secreted into the intestines of mice bearing subcutaneous hybridoma tumors. In the present study, selection and analysis of mutants resistant to neutralization by 1E1 identified the region of T1L sigma1 to which the MAb binds. The region bound by a previously characterized type 1 sigma1-specific neutralizing IgG MAb, 5C6, was identified in the same way. Each of the 15 mutants isolated and analyzed was found to be much less sensitive to neutralization by either 1E1 or 5C6, suggesting the two MAbs bind to largely overlapping regions of sigma1. The tested mutants retained the capacity to recognize specific glycoconjugate receptors on rabbit M cells and cultured epithelial cells, even though viral binding to epithelial cells was inhibited by both MAbs. S1 sequence determinations for 12 of the mutants identified sigma1 mutations at four positions between residues 415 and 447, which contribute to forming the receptor-binding head domain. When aligned with the sigma1 sequence of reovirus type 3 Dearing (T3D) and mapped onto the previously reported crystal structure of the T3D sigma1 trimer, the four positions cluster on the side of the sigma1 head, across the interface between two subunits. Three such interface-spanning epitopes are thus present per sigma1 trimer and require the intact quaternary structure of the head domain for MAb binding. Identification of these intersubunit epitopes on sigma1 opens the way for further studies of the mechanisms of antibody-based neutralization and protection with type 1 reoviruses.

Systemic immunity is progressively impaired in aging, predisposing to morbidity and mortality from neoplasia and infectious disease. However, the effect of aging on mucosal immunity is controversial. To assess intestinal immunity in aging, young and aged mice were orally exposed to reovirus or cholera toxin (CT) and specific antibody and reovirus-specific cytotoxic T-cell (CTL) responses were assessed. As previously reported, aged mice immunized orally with CT mounted diminished intestinal IgA responses to CT compared to young mice. In contrast, aged mice yielded two to three-fold more reovirus-specific IgA-producing cells in the Peyers's patches (PP) compared to young mice, and higher titers of reovirus-specific IgA in fragment culture supernatants. Cytotoxicity and CTL frequencies from aged mice were not different from those of young mice. Together, these results suggest a diminished potential for systemic and intestinal immunity to orally applied protein antigens in aging, but an intact ability to respond to intestinal virus infection. Infection with a replicating virus may induce inflammatory mediators and innate immune factors that potentiate the priming of mucosal immunity; overcoming aging related deficits otherwise observed following oral immunization with non-replicating antigens, and suggests the importance of antigen replication to antigen-specific immunotherapy strategies in the elderly., (Copyright 2004 Elsevier Inc.)

It is well established that the route of infection affects the nature of the adaptive immune response. However, little is known about the effects of the route of exposure on development of cytotoxic T-lymphocyte (CTL) responses. Alternative antigen-presenting cell populations, tissue-restricted expression of class I major histocompatibility complex-encoded molecules, and unique T-cell receptor (TCR)-bearing cells in mucosal tissues could influence the selection and expansion of responder T cells. This study addresses the question of whether the route of virus infection affects the selection and expansion of subpopulations of virus-specific CTLs. Mice were infected orally or in the hind footpads with reovirus, and the repertoires of TCR beta-chains expressed on virus-specific CD8(+) T cells in Peyer's patches or lymph nodes and spleens were examined. CD8(+) cells expressing the variable gene segment of the TCR beta-chain 6 (Vbeta6) expanded in the spleens of mice infected by either route and in CTL lines established from the spleens and draining lymphoid tissues. Adoptively transferred Vbeta6(+) CD8(+) T cells from orally or parenterally infected donors expanded in reovirus-infected severe combined immunodeficient recipient mice and mediated cytotoxicity ex vivo. Furthermore, recovered Vbeta6(+) cells were enriched for clones utilizing uniform complementarity-determining region 3 (CDR3) lengths. However, sequencing of CDR3beta regions from Vbeta6(+) CD8(+) cells indicated that Jbeta gene segment usage is significantly more restricted in CTLs from orally infected mice, suggesting that the route of infection affects selection and/or subsequent expansion of virus-specific CTLs.

Reovirus type 1 Lang (T1L) adheres to M cells in the follicle-associated epithelium of mouse intestine and exploits the transport activity of M cells to enter and infect the Peyer's patch mucosa. Adult mice that have previously cleared a reovirus T1L infection have virus-specific immunoglobulin G (IgG) in serum and IgA in secretions and are protected against reinfection. Our aim in this study was to determine whether secretory IgA is sufficient for protection of Peyer's patches against oral reovirus challenge and, if so, against which reovirus antigen(s) the IgA may be directed. Monoclonal antibodies (MAbs) of the IgA isotype, directed against the sigma1 protein of reovirus T1L, the viral adhesin, were produced and tested along with other, existing IgA and IgG MAbs against reovirus T1L outer capsid proteins. Anti-sigma1 IgA and IgG MAbs neutralized reovirus T1L in L cell plaque reduction assays and inhibited T1L adherence to L cells and Caco-2(BBe) intestinal epithelial cells in vitro, but MAbs against other proteins did not. Passive oral administration of anti-sigma1 IgA and IgG MAbs prevented Peyer's patch infection in adult mice, but other MAbs did not. When anti-sigma1 IgA and IgG MAbs were produced in mice from hybridoma backpack tumors, however, the IgA prevented Peyer's patch infection, but the IgG did not. The results provide evidence that neutralizing IgA antibodies specific for the sigma1 protein are protective in vitro and in vivo and that the presence of these antibodies in intestinal secretions is sufficient for protection against entry of reovirus T1L into Peyer's patches.

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by diffuse alveolar damage (DAD) secondary to an intense host inflammatory response of the lung to a pulmonary or extrapulmonary infectious or noninfectious insult. We have previously described a unique animal model in which CBA/J mice infected with reovirus 1/L develop ARDS. This model recapitulates the histopathological changes observed in human ARDS, which consist of the overlapping phases of exudation, including the formation of hyaline membranes, regeneration, and healing via repair with fibrosis. In this report, we show that the development of DAD in the acute phase of the disease and intraalveolar fibrosis in the late phase of the disease was not modulated by treatment with methylprednisolone (MPS). In the presence or absence of MPS, the majority of cells infiltrating the lungs after reovirus 1/L infection were polymorphonuclear leukocytes and macrophages. A number of key proinflammatory and anti-inflammatory cytokines/chemokines that are observed in the BAL fluid of ARDS patients were also found in the lungs of mice after reovirus 1/L infection and were not modulated by MPS. These include interferon-gamma, interleukin-10, and monocyte chemoattractant protein. The histopathology, cytokine/chemokine expression, and response to corticosteroids in reovirus 1/L-induced ARDS are similar to what is observed in human patients, making this a clinically relevant model.

Reovirus type 1 Lang (T1L) infects the mouse intestinal mucosa by adhering specifically to epithelial M cells and exploiting M-cell transport to enter the Peyer's patches. Oral inoculation of adult mice has been shown to elicit cellular and humoral immune responses that clear the infection within 10 days. This study was designed to determine whether adult mice that have cleared a primary infection are protected against viral entry upon oral rechallenge and, if so, whether antireovirus secretory immunoglobulin A (S-IgA) is a necessary component of protection. Adult BALB/c mice that were orally inoculated on day 0 with reovirus T1L produced antiviral S-IgA in feces and IgG in serum directed primarily against the reovirus sigma1 attachment protein. Eight hours after oral reovirus challenge on day 21, the Peyer's patches of previously exposed mice contained no detectable virus whereas Peyer's patches of naive controls contained up to 2,300 PFU of reovirus/mg of tissue. Orally inoculated IgA knockout (IgA(-/-)) mice cleared the initial infection as effectively as wild-type mice and produced higher levels of reovirus-specific serum IgG and secretory IgM than C57BL/6 wild-type mice. When IgA(-/-) mice were rechallenged on day 21, however, their Peyer's patches became infected. These results indicate that intestinal S-IgA is an essential component of immune protection against reovirus entry into Peyer's patch mucosa.