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BACKGROUND: We report a rare case of Mammalian orthoreovirus (MRV) infection in a child with a primary immunodeficiency (PID). Infections with Mammalian orthoreovirus are very rare and probably of zoonotic origin. Only a few cases have been described so far, including one with similar pathogenesis as in our case. CASE PRESENTATION: The patient, age 11, presented with flu-like symptoms and persistent severe diarrhea. Enterovirus has been detected over several months, however, exact typing of a positive cell culture remained inconclusive. Unbiased metagenomic sequencing then detected MRV in stool samples from several time points. The sequencing approach further revealed co-infection with a recombinant Coxsackievirus and Adenovirus. MRV-specific antibodies detected by immunofluorescence proved that the patient seroconverted. CONCLUSION: This case highlights the potential of unbiased metagenomic sequencing in supplementing routine diagnostic methods, especially in situations of chronic infection with multiple viruses as seen here in an immunocompromised host. The origin, transmission routes and implications of MRV infection in humans merit further investigation.

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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.

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Mammalian orthoreoviruses (reoviruses) are members of the Reoviridae. Reoviruses contain 10 double-stranded (ds) RNA gene segments enclosed in two concentric protein shells, called outer capsid and core. These viruses serve as a versatile experimental system for studies of viral replication events at the virus-cell interface, including engagement of cell-surface receptors, internalization and disassembly, and activation of the innate immune response, including NF-κB-dependent cellular signaling pathways. Reoviruses also provide a model system for studies of virus-induced apoptosis and organ-specific disease in vivo.Reoviruses attach to host cells via the filamentous attachment protein, σ1. The σ1 protein of all reovirus serotypes engages junctional adhesion molecule-A (JAM-A), an integral component of intercellular tight junctions. The σ1 protein also binds to cell-surface carbohydrate, with the type of carbohydrate bound varying by serotype. Following attachment to JAM-A and carbohydrate, reovirus internalization is mediated by ß1 integrins, most likely via clathrin-dependent endocytosis. In the endocytic compartment, reovirus outer-capsid protein σ3 is removed by acid-dependent cysteine proteases in most cell types. Removal of σ3 results in the exposure of a hydrophobic conformer of the viral membrane-penetration protein, µ1, which pierces the endosomal membrane and delivers transcriptionally active reovirus core particles into the cytoplasm.Reoviruses induce apoptosis in both cultured cells and infected mice. Perturbation of reovirus disassembly using inhibitors of endosomal acidification or protease activity abrogates apoptosis. The µ1-encoding M2 gene is genetically linked to strain-specific differences in apoptosis-inducing capacity, suggesting a function for µ1 in induction of death signaling. Reovirus disassembly leads to activation of transcription factor NF-κB, which modulates apoptotic signaling in numerous types of cells. Inhibition of NF-κB nuclear translocation using either pharmacologic agents or expression of transdominant forms of IκB blocks reovirus-induced apoptosis, suggesting an essential role for NF-κB activation in the death response. Multiple effector pathway s downstream of NF-κB-directed gene expression execute reovirus-induced cell death. This chapter will focus on the mechanisms by which reovirus attachment and disassembly activate NF-κB and stimulate the cellular proapoptotic machinery.

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Avian reovirus (ARV) strain S1133 causes apoptosis in host cells in the middle to late stages of infection. This study investigated the early-stage biological response and intracellular signaling in ARV S1133-infected Vero and chicken cells. Treatment with conditioned medium from ARV S1133-infected cells increased the chemotactic activity of U937 cells. Neutralizing antibodies against IL-1beta and IL-6 showed that both cytokines contribute to viral-induced inflammation but neither affect cell survival. Inhibition of Akt, NF-kappaB, and Stat3 released the chemotactic activity and anti-apoptotic effect elicited by ARV S1133. ARV S1133 activated PI 3-kinase-dependent Akt/NF-kappaB and p70 S6 kinase, as well as Stat3; however, p70 S6 kinase was not involved in ARV S1133-mediated effects. DF1 cells over-expressing constitutively active PI 3-kinase and Stat3 showed association with enhancement of anti-apoptotic activity. In conclusion, in the early stages of ARV S1133 infection, activation of cell survival signals contributes to virus-induced inflammation and anti-apoptotic response.

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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.

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Poult enteritis and mortality syndrome (PEMS) is an acute, infectious intestinal disease of turkey poults, characterized by high mortality and 100% morbidity, that decimated the turkey industry in the mid-1990s. The etiology of PEMS is not completely understood. This report describes the testing of various filtrates of fecal material from control and PEMS-affected poults by oral inoculation into poults under experimental conditions, the subsequent isolation of a reovirus, ARV-CU98, from one of the PEMS fecal filtrates, and in vivo and in vitro studies conducted to determine the pathogenicity of ARV-CU98 in turkey poults. In order to identify a filtrate fraction of fecal material containing a putative etiologic agent, poults were challenged in two independent experiments with 220- and 100-nm filtrates of fecal material from PEMS-negative and PEMS-positive poults. The 100-nm filtrate was chosen for further evaluation because poults inoculated with this filtrate exhibited mortality and significantly lower (P < or = 0.05) body weight and relative bursa weight, three clinical signs associated with PEMS. These results were confirmed in a third experiment with 100-nm fecal filtrates from a separate batch of PEMS fecal material. In Experiment 3, body weight and relative bursa and thymus weights were significantly lower (P < or = 0.05) in poults inoculated with 100-nm filtrate of PEMS fecal material as compared with poults inoculated with 100-nm filtrate of control fecal material. Subsequently, a virus was isolated from the 100-nm PEMS fecal filtrate and propagated in liver cells. This virus was identified as a reovirus on the basis of cross-reaction with antisera against avian reovirus (FDO strain) as well as by electrophoretic analysis and was designated ARV-CU98. When inoculated orally into poults reared under controlled environmental conditions in isolators, ARV-CU98 was associated with a higher incidence of thymic hemorrhaging and gaseous intestines. In addition, relative bursa and liver weights were significantly lower (P < or = 0.05) in virus-inoculated poults as compared with controls. Virus was successfully reisolated from virus-challenged poults but not from control birds. Furthermore, viral antigen was detected by immunofluorescence in liver sections from virus-challenged poults at 3 and 6 days postinfection and virus was isolated from liver at 6 days postinfection, suggesting that ARV-CU98 replicates in the liver. In addition to a decrease in liver weight, there was a functional degeneration as indicated by altered plasma alanine aminotransferase and aspartate aminotransferase activities in virus poults as compared with controls. Although this reovirus does not induce fulminating PEMS, our results demonstrated that ARV-CU98 does cause some of the clinical signs in PEMS, including intestinal alterations and significantly lower relative bursa and liver weights. ARV-CU98 may contribute directly to PEMS by affecting the intestine, bursa, and liver and may contribute indirectly by increasing susceptibility to opportunistic pathogens that facilitate development of clinical PEMS.

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Groups of sucking Swiss albino mice were inoculated by the intracerebral (i.c.), intraperitoneal (i.p.) or oral route with a trypsin-sensitive avian reovirus (TR1) or a trypsin-resistant (R2) reovirus. The viruses caused a number of effects, the most severe occurring after i.c. inoculation and the least after oral inoculation. They included incoordination and tremors, oiliness of the hair, and retarded growth. Patterns of viral persistence in tissues were similar for the two viruses, with high titres in the brain on days 3 and 6 after i.c. or i.p. injection. Both viruses were still present in the brain 21 days after i.c. injection. No virus was found in any tissue when TR1 was given orally. All groups "seroconverted" except the one infected orally with TR1, but neutralization titres were low. The effects resembled those described for mammalian reoviruses in mice. The results indicate that, for short periods, wild mice may be capable of transmitting avian reoviruses between poultry flocks. Furthermore, in the production of monoclonal antibodies to avian reoviruses in mice, it is possible that pathological changes will occur.

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Respiratory virus infections are a serious health challenge. While models exist to study immune mechanisms of the respiratory tract, they have not allowed analysis of the interaction of the lower respiratory tract with other components of the mucosal immune system. This study demonstrates that reovirus 1/Lang, an effective gut mucosal immunogen, also provides a useful model of respiratory mucosal infection. Intra-nasal infection of Balb/c mice resulted in severe viral bronchopneumonia. Major components of the cellular inflammatory response in the lung interstitium and alveolar spaces were CD8 lymphocytes. Lung lymphocyte populations exhibited lysis of reovirus-infected, but not uninfected target cells after in vitro culture. The GCT antigen, a germinal center B-cell and CD8 T-cell marker, was present on 21-60% of the inflammatory lymphocytes. A novel population of GCT-expressing CD4+ lymphocytes unique to reovirus-stimulated lung alveolar and interstitial lymphocyte populations was identified.

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This study examined the relationship of avian reovirus spread and replication to induction of lesions and the relevant role of the S1 segment encoding a virus-neutralizing antigen. One-day-old broiler chickens were infected via footpad or orally with two virus strains (883 and 176) that differ greatly in virulence and a reassortant (R44) that has the S1 segment from 176 and the remaining genome segments from 883. Virus replication and histological lesions in various tissues (heart, liver, spleen, kidney, bursa, hock joint, and bone marrow) were measured at 2-day intervals until day 8 postinoculation. The virulent strain 176 spread to and replicated efficiently in all tissues examined and caused extensive and severe lesions, whereas the mild strain 883 was detected only in tissues near inoculation sites and caused only minimal lesions. The appearance of lesions correlated with the presence of viral replication in each tissue tested. Together, these results indicate that induction of lesions, or pathogenicity, is directly related to virus spread and replication. Reassortant R44 behaved like strain 176 in chicken embryo fibroblasts (CEFs), i.e., both replicated much faster and produced larger plaques than strain 883. In broiler chickens, however, R44 behaved like strain 883, replicating and inducing lesions to an extent that was fat lower than that of strain 176. These results suggest that the S1 segment alone is capable of determining viral replication and plaque formation in cultured CEFs but is not sufficient to determine the virus spread and replication and the pathological change in broiler chickens.

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Reovirus type 1, strain Lang (1/L), can infect hepatocytes in vivo only after hepatocellular damage is induced by hepatotoxins, surgical trauma, resection, or profound immunosuppression. To examine the role of cell cycle and cellular differentiation on liver cell susceptibility to reovirus infection, a murine hepatocarcinoma cell line, Hepa 1/A1, was infected with reovirus and assayed for the presence of infectious virus or reovirus antigen in cells. Despite a > 95% binding of reovirus to hepatocarcinoma cells as indicated by cytometric analysis; only 10% of hepatoma cells contained infectious virus by infectious center assay. In comparison, 100% of L cells were infected. Analysis of intracellular reovirus antigen revealed its presence in dividing but not in quiescent hepatocytes. This correlation of cellular division and cell capacity to support viral replication suggests that induction of hepatocyte proliferation may be a mechanism for liver susceptibility to reovirus infection.

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We used a recently isolated and characterized panel of monoclonal antibodies (MAbs) specific for cross-reactive determinants on reovirus outer capsid proteins to define mechanisms of antibody-mediated protection in vivo. We studied the capacities of MAbs to protect against lethal infection with reoviruses which differ in site of primary replication, route of spread, and central nervous system tropism. We found the following. (i) MAbs specific for each of the viral outer capsid proteins (sigma 1, sigma 3, and mu 1) and the core spike protein (lambda 2) were protective under certain circumstances. (ii) In vitro properties of MAbs, including isotype, neutralization of viral infectivity, inhibition of virus-induced hemagglutination, and avidity of binding, were poorly predictive of the capacities of MAbs to protect in vivo. (iii) MAbs did not act at a single stage during pathogenesis to mediate protection; instead, protective MAbs were capable of altering a variety of stages in reovirus pathogenesis. (iv) MAbs protective against one reovirus also protected against other reoviruses that utilized different pathogenetic strategies, suggesting that the viral epitope bound by an antibody rather than the pathogenetic strategy employed by the virus is a critical determinant of antibody-mediated protection in vivo. (v) A prominent mechanism of protective MAb action is inhibition of viral spread through nerves from a site of primary replication (e.g., the intestine or muscle tissue) to the central nervous system.

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Pathogenesis studies in animals can uncover details concerning viral replication, growth, and access to target organs, in vivo. This, in turn, reveals opportunities for antiviral intervention that may be otherwise missed by limiting analysis to growth of virus in tissue culture. In this report, reovirus infection of mice is used as a model. Three general aspects of reovirus behavior in mice are presented and each demonstrates a property of the virus that could easily have been missed by studies in tissue culture.

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Chuzan virus at 2 to 3 passage levels in cell cultures after isolation was inoculated intravenously into 15 seronegative pregnant cows at 89 to 150 days of gestation. All of the cows developed viremia a few days after inoculation and antibodies 2 weeks after inoculation. No clinical signs, except leukopenia, were observed throughout the experimental period. These 15 cows delivered 15 calves after normal gestation. One of the calves which was born to a dam inoculated at 120 days of gestation, showed impairment of movement, and the remaining 14 were healthy. Postmortem examination revealed that this calf had hydranencephaly- cerebellar hypoplasia (HCH) syndrome and that the remaining calves were normal. Two of the 15 calves, including the one that had HCH syndrome, had antibody to Chuzan virus in their precolostral sera. These findings provide additional evidence that Chuzan virus is the etiological agent of an epizootic of congenital abnormalities with HCH syndrome of calves in Japan, 1985 to 1986. We propose to name the HCH syndrome caused by Chuzan virus infection Chuzan disease.

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To explore how bacteria and their products may modulate viral infection, we investigated the effect of a well-characterized and highly purified product of Bordetella pertussis, a pertussis toxin, also known as lymphocytosis promoting factor (LPF), on enteric reovirus infection. LPF is known to have a variety of effects, including modulation of circulation and homing of lymphoid cells. When adult mice are inoculated with reovirus type 1 perorally, reovirus first enters the Peyer's patches (PP) through M cells, and then spreads to mesenteric lymph nodes (MLN) and spleen with minimal dissemination to other peripheral tissues. In view of the profound effect of LPF on lymphoid tissues, we evaluated whether LPF might influence the early stages of type-1 reovirus infection following peroral inoculation. Pretreatment of adult BALB/c mice with LPF significantly inhibited the spread of reovirus in a manner dependent upon the route of inoculation; LPF inhibited the extra-intestinal spread of virus from PP to MLN after intragastric inoculation; in contrast there was enhancement of the spread of blood-borne viruses to MLN after intravenous inoculation. This result, together with the fact that the efferent lymph from PP reaches MLN, suggests that a proportion of reoviruses were conveyed from PP to MLN in association with lymphoid cells along the lymphatic channels and that LPF affects reovirus, in part, by blocking cell movement.

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At 15 days of age and in the presence of measurable levels of maternal antibody against infectious bursal disease virus serotype I (1:170 virus-neutralization geometric mean titer), a recent isolate (U-28) and a prototype virulent isolate (Edgar) of the same virus caused subclinical infections in commercial broiler chickens. Isolate U-28 caused a significant reduction in the size of the bursa of Fabricius, whereas the Edgar isolate produced splenomegaly. Both isolates reduced the serological response to Newcastle disease virus. The experimental immunosuppressive potential and pathogenicity of isolate U-28 in broiler chickens confirms the role of this virus in recent infectious bursal disease outbreaks.

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The mammalian reoviruses provide a model for studying specific aspects of the immunopathogenesis of viral infection. We have used two serotype 3 reoviruses to define stages in the pathogenesis of central nervous system (CNS) infection at which a mAb specific for the reoviral cell attachment protein sigma 1 (sigma 1mAbG5) acts to protect mice against lethal disease. sigma 1mAbG5 administered either before or at the time of footpad inoculation with reovirus T3D prevented entry of T3D into the CNS. sigma 1mAbG5 also inhibited the spread of reovirus T3C9 from the gastrointestinal tract to the CNS after peroral inoculation with T3C9. These effects occurred in the absence of a significant effect of sigma 1mAbG5 on primary replication in skeletal muscle (T3D) or the gastrointestinal tract (T3C9). sigma 1mAbG5 administered after T3D had reached the spinal cord inhibited subsequent spread of infectious virus from spinal cord to brain. Even after direct intracerebral inoculation of T3D, sigma 1mAbG5 prevented both growth in the brain and spread of infectious virus from brain to eye, spinal cord, and muscle. Treatment with sigma 1mAbG5 after intracerebral inoculation with T3D prevented neuronal necrosis and resulted in a delayed and topographically restricted inflammatory response. We detected no antibody-resistant T3D variants in vivo after treatment with sigma 1mAbG5. We conclude that systemic IgG does not play a significant role at the primary site of infection with reoviruses, while it clearly acts to prevent infection of the CNS and extension of infection with the CNS. Further study will be directed to defining what components of the immune system do act at primary sites of infection, and to defining the mechanisms by which antibody acts at defined stages in pathogenesis.

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The pathogenesis of infectious bursal disease (IBD) in chickens neonatally chemically bursectomized (CB) by cyclophosphamide and subsequently inoculated with various numbers of bursal cells was examined. CB chickens inoculated with at least 62.5 X 10(6) bursal cells were as susceptible to IBD clinical manifestations (as determined by gross and microscopic evaluation of bursal tissues, virus recovery from spleen, and antibody titer) as intact chickens following inoculation with virus at 5 weeks of age. In contrast, CB chickens inoculated with 2.5 X 10(6) or fewer bursal cells were refractory to the IBD clinical manifestations compared with intact chickens or CB chickens inoculated with 62.5 X 10(6) or more bursal cells. Results from this study suggest that the availability of a large number of bursal cells is an essential factor in the development of IBD.

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In 10-day-old suckling and adult mice, reovirus type 1 adheres selectively to and penetrates membranous epithelial (M) cells. To determine when M cells first appear, when they first transport reovirus, and if reovirus adheres to and is endocytosed by other epithelial cells in the first postnatal week, we examined neonatal mouse intestine by transmission electron microscopy after reovirus type 1 exposure. At 2 days M cells accounted for 0.9% of dome epithelial cells. By 9 days M cells had increased to 7.4%. Reovirus type 1 adherence to the surface of villus and dome epithelial cells showed marked variation in 2-6-day-old animals, but by 7 days only a few absorptive cell profiles had adherent reovirus. Adherence to greater than 50% of M-cell profiles occurred in all but 2 animals, but adherence to the majority of Peyer's patch absorptive cell profiles was present only in some 4- and 5-day-old animals. Adherence to a majority of undifferentiated cell profiles occurred in some animals at all ages. Membranous epithelial cells endocytosed reovirus at all ages but only at 2 days did rare villus and dome absorptive cells endocytose reovirus into the apical cytoplasm. Thus, adherence of reovirus to the apical surface of mucosal epithelial cells is nonselective in newborn mice but becomes more selective within the first postnatal week with adherence by day 7 to most M-cell profiles, to a substantial but variable number of undifferentiated cell profiles, but to few absorptive cell profiles.

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Reovirus type 1, strain Lang, and type 3, strain Dearing, induced site-specific intestinal lesions in the adult mouse after intravenous inoculation. Reovirus type 1 caused inflammation and epithelial changes such as loss of nuclear polarity, villus blunting and crypt hyperplasia restricted to the ileum. In contrast, reovirus type 3 induced duodenitis, jejunitis, and ulcerative colitis. In the duodenum and jejunum, the epithelial cells appeared normal, but hemorrhage and inflammation in the lamina propria was present. In the colon, superficial ulceration, crypt abscesses, and intraluminal hemorrhage was observed. Segregation analysis using reassortant clones derived from reoviruses 1 and 3, suggested the viral hemagglutinin, encoded by genome segment S1, to be the major viral determinant of site specific intestinal disease following intravenous inoculation.

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