The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis.

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.

Intertypic reassortment of mammalian orthoreovirus identified in wastewater in Japan.

Mammalian orthoreovirus (MRV), a non-enveloped virus with a ten-segmented double-stranded RNA genome, infects virtually all mammals, including humans. Human infection with MRV seems to be common in early childhood, but is rarely symptomatic. Despite the ubiquitous presence of MRV in mammals as well as in environmental waters, the molecular characterisation of the MRV genome remains to be fully elucidated. In this study, two novel strains, MRV-2 THK0325 and MRV-1 THK0617, were unintentionally isolated from wastewater in Japan via an environmental surveillance of enteric viruses. Homology and phylogenetic analysis demonstrated that all the segments of THK0325 were closely related to the MRV-2 Osaka strains, which were recently proposed to have existed for at least two decades in Japan. Most of the segments in THK0617 also showed a close relationship with the MRV-2 Osaka strains, but the M2, S1, and S3 segments belong to another MRV cluster. According to the S1 sequence, the determinant of serotype THK0617 was classified as MRV-1, and both the M2 and S3 segments were closely related to MRV-1 and -3 from the tree shrew in China. These results suggest that the MRV-2 Osaka-like strain spread widely throughout Japan, accompanied by intertypic reassortment occurring in East Asia.

Bat mammalian orthoreoviruses cause severe pneumonia in mice.

Mammalian orthoreovirus (MRV) infections are ubiquitous in mammals. Increasing evidence suggests that some MRVs can cause severe respiratory disease and encephalitis in humans and other animals. Previously, we isolated six bat MRV strains. However, the pathogenicity of these bat viruses remains unclear. In this study, we investigated the host range and pathogenicity of 3 bat MRV strains (WIV2, 3 and 7) which represent three serotypes. Our results showed that all of them can infect cell lines from different mammalian species and displayed different replication efficiency. The BALB/c mice infected by bat MRVs showed clinical symptoms with systematic infection especially in lung and intestines. Obvious tissue damage were found in all infected lungs. One of the strains, WIV7, showed higher replication efficiency in vitro and vivo and more severe pathogenesis in mice. Our results provide new evidence showing potential pathogenicity of bat MRVs in animals and probable risk in humans.

Anti-tumour activity of oncolytic reovirus against canine histiocytic sarcoma cells.

Canine histiocytic sarcoma is an aggressive, fatal neoplastic disease with a poor prognosis. Lomustine is generally accepted as the first-line systemic therapy, although this compound does not provide complete regression. Therefore, research into a novel approach against canine histiocytic sarcoma is needed. However, anti-tumour effects of oncolytic therapy using reovirus against histiocytic sarcoma are unknown. Here, we showed that reovirus has oncolytic activity in canine histiocytic sarcoma cell lines in vitro and in vivo. We found that reovirus can replicate and induce caspase-dependent apoptosis in canine histiocytic sarcoma cell lines. A single intra-tumoural injection of reovirus completely suppressed the growth of subcutaneously grafted tumours in NOD/SCID mice. Additionally, we demonstrated that susceptibility to reovirus-induced cell death was attributable to the extent of expression of type I interferons induced by reovirus infection in vitro. In conclusion, oncolytic reovirus appears to be an effective treatment option for histiocytic sarcoma, and therefore warrants further investigation in early clinical trials.

An Orchestra of Reovirus Receptors: Still Searching for the Conductor.

Viruses are constantly engaged in a molecular arms race with the host, where efficient and tactical use of cellular receptors benefits critical steps in infection. Receptor use dictates initiation, establishment, and spread of viral infection to new tissues and hosts. Mammalian orthoreoviruses (reoviruses) are pervasive pathogens that use multiple receptors to overcome protective host barriers to disseminate from sites of initial infection and cause disease in young mammals. In particular, reovirus invades the central nervous system (CNS) with serotype-dependent tropism and disease. A single viral gene, encoding the attachment protein σ1, segregates with distinct patterns of CNS injury. Despite the identification and characterization of several reovirus receptors, host factors that dictate tropism via interaction with σ1 remain undefined. Here, we summarize the state of the reovirus receptor field and discuss open questions toward understanding how the reovirus attachment protein dictates CNS tropism.

Persistent mammalian orthoreovirus, coxsackievirus and adenovirus co-infection in a child with a primary immunodeficiency detected by metagenomic sequencing: a case report.

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.

Quantitative Temporal in Vivo Proteomics Deciphers the Transition of Virus-Driven Myeloid Cells into M2 Macrophages.

Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host-virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G-, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G-, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral-host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection.

Reovirus µ1 Protein Affects Infectivity by Altering Virus-Receptor Interactions.

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.

Characterization and pathogenicity of a novel mammalian orthoreovirus from wild short-nosed fruit bats.

Mammalian orthoreoviruses (MRVs) have a wide range of geographic distribution and have been isolated from humans and various animals. This study describes the isolation, molecular characterization and analysis of pathogenicity of MRV variant B/03 from wild short-nosed fruit bats. Negative stain electron microscopy illustrated that the B/03 strain is a non-enveloped icosahedral virus with a diameter of 70nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) migration patterns showed that the B/03 viral genome contains 10 segments in a 3:3:4 arrangement. The isolate belongs to MRV serotype 1 based on S1 gene nucleotide sequence data. BALB/c mice experimentally infected with B/03 virus by intranasal inoculation developed severe respiratory distress with tissue damage and inflammation. Lastly, B/03 virus has an increased transmission risk between bats and humans or animals.

Comparison of three neurotropic viruses reveals differences in viral dissemination to the central nervous system.

Neurotropic viruses initiate infection in peripheral tissues prior to entry into the central nervous system (CNS). However, mechanisms of dissemination are not completely understood. We used genetically marked viruses to compare dissemination of poliovirus, yellow fever virus 17D (YFV-17D), and reovirus type 3 Dearing in mice from a hind limb intramuscular inoculation site to the sciatic nerve, spinal cord, and brain. While YFV-17D likely entered the CNS via blood, poliovirus and reovirus likely entered the CNS by transport through the sciatic nerve to the spinal cord. We found that dissemination was inefficient in adult immune-competent mice for all three viruses, particularly reovirus. Dissemination of all viruses was more efficient in immune-deficient mice. Although poliovirus and reovirus both accessed the CNS by transit through the sciatic nerve, stimulation of neuronal transport by muscle damage enhanced dissemination only of poliovirus. Our results suggest that these viruses access the CNS using different pathways.

Viruses, apoptosis, and neuroinflammation--a double-edged sword.

Apoptosis, or programmed cell death, is a fundamental and widespread cell biological process that is distinct from cell necrosis and can be induced by a wide variety of stimuli including viral infections. Apoptosis may occur via either the intrinsic or extrinsic pathways and confers several advantages to the virally infected host including the prevention of further viral propagation and the potential inhibition and resolution of inflammatory processes. Several viruses have been shown to have the capacity to induce apoptosis in susceptible cells including herpes simplex virus, Varicella-zoster virus, rabies virus, human immunodeficiency virus, and reovirus. Apoptosis has also been observed in human African trypanosomiasis which is an infection caused by a protozoan parasite. The mechanisms leading to apoptosis may differ depending on the type of infection. Apoptosis has been reported in several neurodegenerative diseases and also psychiatric disorders but the true clinical significance of such observations is not certain, and, though interesting, it is very difficult to ascribe causation in these conditions. The presence of inflammation in the central nervous system in any neurological condition, including those associated with a viral infection, is not necessarily an absolute marker of serious disease and the notion of 'good' versus 'bad' inflammation is considered to be valid in some circumstances. The precise relationship between viruses, apoptosis, and inflammation is viewed as a complex one requiring further investigation to unravel and understand its nature.

Reovirus type-2-triggered autoimmune cholangitis in extrahepatic bile ducts of weanling DBA/1J mice.

BACKGROUND: Reovirus is a proposed cause of infantile biliary atresia. However, mechanistic insight regarding Reo-2 as a potential cholangiotropic virus is lacking. Furthermore, it is unknown whether Reo-2 infection can induce autoimmune-mediated bile duct injury. METHODS: Lesions of bile ducts in newborn DBA/1J mice infected with Reo-2 were analyzed immunopathologically. RESULTS: Damage to biliary epithelia occurs after Reo-2 infection. In addition, nonsuppurative cholangitis with fibrosis in extrahepatic (especially septal) bile ducts developed following complete viral clearance from the liver. At the inflamed ducts, major histocompatibility complex class I expressing((+)) and FAS(+) cholangiocytes were associated with FAS ligand(+) lymphocytes and tumor necrosis factor-α(+) mononuclear cells (macrophages and lymphocytes). These cholangiocytes were apoptotic and necrotic. Moreover, affected ducts were infiltrated by CD3(+), CD4(+), CD8(+), IFN-γ(+), and FAS(+) lymphocytes. Analysis of blood from Reo-2-infected mice revealed that they developed anticholangiocyte cytoplasm antibodies and had high serum IFN-γ concentration. Notably, there was no increase in Foxp3(+) lymphocytes at inflamed ducts, lymph nodes, and thymi. CONCLUSION: Reo-2 infection induced T-helper cell type 1-dependent injury to bile ducts in weanling mice. The lesions observed in mice may be analogous to those associated with human infantile biliary atresia, which are caused by an autoimmune-mediated process.

Emerging infectious diseases associated with bat viruses.

Bats play important roles as pollen disseminators and pest predators. However, recent interest has focused on their role as natural reservoirs of pathogens associated with emerging infectious diseases. Prior to the outbreak of severe acute respiratory syndrome (SARS), about 60 bat virus species had been reported. The number of identified bat viruses has dramatically increased since the initial SARS outbreak, and most are putative novel virus species or genotypes. Serious infectious diseases caused by previously identified bat viruses continue to emerge throughout in Asia, Australia, Africa and America. Intriguingly, bats infected by these different viruses seldom display clinical symptoms of illness. The pathogenesis and potential threat of bat-borne viruses to public health remains largely unknown. This review provides a brief overview of bat viruses associated with emerging human infectious diseases.

Quantification of the host response proteome after mammalian reovirus T1L infection.

All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular "transcriptome." We used SILAC (stable isotope labeling by amino acids in cell culture), combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L). 3,076 host proteins were detected at 6 hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24 hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.

Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1.

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.

Recruitment of cellular clathrin to viral factories and disruption of clathrin-dependent trafficking.

The viral factories of mammalian reovirus (MRV) are cytoplasmic structures that serve as sites of viral genome replication and particle assembly. A 721-aa MRV non-structural protein, µNS, forms the factory matrix and recruits other viral proteins to these structures. In this report, we show that µNS contains a conserved C-proximal sequence (711-LIDFS-715) that is similar to known clathrin-box motifs and is required for recruitment of clathrin to viral factories. Clathrin recruitment by µNS occurs independently of infecting MRV particles or other MRV proteins. Ala substitution for a single Leu residue (mutation L711A) within the putative clathrin-binding motif of µNS inhibits clathrin recruitment, but does not prevent formation or expansion of viral factories. Notably, clathrin-dependent cellular functions, including both endocytosis and secretion, are disrupted in cells infected with MRV expressing wild-type, but not L711A, µNS. These results identify µNS as a novel adaptor-like protein that recruits cellular clathrin to viral factories, disrupting normal functions of clathrin in cellular membrane trafficking. To our knowledge, this is the only viral or bacterial protein yet shown to interfere with clathrin functions in this manner. The results additionally establish a new approach for studies of clathrin functions, based on µNS-mediated sequestration.

A proapoptotic peptide derived from reovirus outer capsid protein {micro}1 has membrane-destabilizing activity.

The reovirus outer capsid protein µ1 is responsible for cell membrane penetration during virus entry and contains determinants necessary for virus-induced apoptosis. Residues 582 to 611 of µ1 are necessary and sufficient for reovirus-induced apoptosis, and residues 594 and 595 independently regulate the efficiency of viral entry and reovirus-induced cell apoptosis, respectively. Two of three α-helices within this region, helix 1 (residues 582 to 611) and helix 3 (residues 644 to 675), play a role in reovirus-induced apoptosis. Here, we chemically synthesized peptides representing helix 1 (H1), H1:K594D, H1:I595K, and helix 3 (H3) and examined their biological properties. We found that H1, but not H3, was able to cause concentration- and size-dependent leakage of molecules from small unilamellar liposomes. We further found that direct application of H1, but not H1:K594D, H1:I595K, or H3, to cells resulted in cytotoxicity. Application of the H1 peptide to L929 cells caused rapid elevations in intracellular calcium concentration that were independent of phospholipase C activation. Cytotoxicity of H1 was not restricted to eukaryotic cells, as the H1 peptide also had bactericidal activity. Based on these findings, we propose that the proapoptotic function of the H1 region of µ1 is dependent on its capacity to destabilize cellular membranes and cause release of molecules from intracellular organelles that ultimately induces cell necrosis or apoptosis, depending on the dose.

Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak.

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein µ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed µ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of µ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for µ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of µ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing µ1, indicating that caspases were not required. Additionally, µ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.

Localization of mammalian orthoreovirus proteins to cytoplasmic factory-like structures via nonoverlapping regions of microNS.

Virally induced structures called viral factories form throughout the cytoplasm of cells infected with mammalian orthoreoviruses (MRV). When expressed alone in cells, MRV nonstructural protein microNS forms factory-like structures very similar in appearance to viral factories, suggesting that it is involved in forming the structural matrix of these structures. microNS also associates with MRV core particles; the core proteins mu2, lambda1, lambda2, lambda3, and sigma2; and the RNA-binding nonstructural protein sigmaNS. These multiple associations result in the recruitment or retention of these viral proteins or particles at factory-like structures. In this study, we identified the regions of microNS necessary and sufficient for these associations and additionally examined the localization of viral RNA synthesis in infected cells. We found that short regions within the amino-terminal 220 residues of microNS are necessary for associations with core particles and necessary and sufficient for associations with the proteins mu2, lambda1, lambda2, sigma2, and sigmaNS. We also found that only the lambda3 protein associates with the carboxyl-terminal one-third of microNS and that viral RNA is synthesized within viral factories. These results suggest that microNS may act as a cytoplasmic scaffolding protein involved in localizing and coordinating viral replication or assembly intermediates for the efficient production of progeny core particles during MRV infection.