Interference following mixed infection of reovirus isolates is linked to the M2 gene.

Following infection by pairs of reovirus isolates consisting of combinations of reovirus T1 Lang, T2 Jones, or T3 Dearing, we found that one of the isolates interfered with the yield of progeny RNA derived from the other parents. The most significant interference was produced by T2 Jones or T3 Dearing, when mixed with T1 Lang. Genetic analysis revealed that the presence of the M2 gene in the interfering parent (in the T1 Lang x T3 Dearing pair) was linked to interference. Studies on interference in infected cells indicated that interference occurs after adsorption and penetration.     

Reovirus infection and tissue injury in the mouse central nervous system are associated with apoptosis.

Reovirus serotype 3 strains infect neurons within specific regions of the neonatal mouse brain and produce a lethal meningoencephalitis. Viral replication and pathology colocalize and have a predilection for the cortex, hippocampus, and thalamus. We have shown previously that infection of cultured fibroblasts and epithelial cells with reovirus type 3 Dearing (T3D) and other type 3 reovirus strains results in apoptotic cell death, suggesting that apoptosis is a mechanism of cell death in vivo. We now report that T3D induces apoptosis in infected mouse brain tissue. To determine whether reovirus induces apoptosis in neural tissues, newborn mice were inoculated intracerebrally with T3D, and at various times after inoculation, brain tissue was assayed for viral antigen by immunostaining and apoptosis was identified by DNA oligonucleosomal laddering and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cells were also stained with cresyl violet to detect morphological changes characteristic of apoptosis, including chromatin condensation and cell shrinkage. DNA laddering was detected in T3D- but not in mock-infected brain tissue. Apoptotic cells were restricted to the same regions of the brain in which infected cells and tissue damage were observed. These findings suggest that virus-induced apoptosis is a mechanism of cell death, tissue injury, and mortality in reovirus-infected mice. The correlation between apoptosis and pathogenesis in vivo identifies apoptosis as a potential target for molecular and pharmacological strategies designed to curtail or prevent diseases resulting from induction of this cell death pathway.     

Growth and survival of reovirus in intestinal tissue: role of the L2 and S1 genes.

Reovirus serotype 1 Lang can be recovered in high titer from the intestines of neonatal mice up to day 8 after peroral inoculation. By contrast, reovirus serotype 3 Dearing cannot be recovered from intestinal tissue past day 4 after peroral inoculation. This difference between the two reoviruses was mapped by using reassortants generated from nonmutagenized laboratory stocks. When the L2 and S1 genes of reovirus serotype 3 Dearing were present in reassortants, the reassortants behaved like serotype 3 Dearing in exhibiting a decreased capacity to be recovered from intestinal tissue. Likewise, viruses which contained the L2 and S2 genes from serotype 1 Lang exhibited an enhanced capacity to grow and survive, which is characteristic of serotype 1 Lang. Thus, the capacity of reovirus to survive in intestinal tissue was determined by the L2 and S1 genes.     

Reovirus binding determinants in junctional adhesion molecule-A

Junctional adhesion molecule-A (JAM-A) serves as a serotype-independent receptor for mammalian orthoreoviruses (reoviruses). The membrane-distal immunoglobulin-like D1 domain of JAM-A is required for homodimerization and binding to reovirus attachment protein sigma1. We employed a structure-guided mutational analysis of the JAM-A dimer interface to identify determinants of reovirus binding. We purified mutant JAM-A ectodomains for solution-phase and surface plasmon resonance binding studies and expressed mutant forms of full-length JAM-A in Chinese hamster ovary cells to assess reovirus binding and infectivity. Mutation of residues in the JAM-A dimer interface that participate in salt-bridge or hydrogen-bond interactions with apposing JAM-A monomers abolishes the capacity of JAM-A to form dimers. JAM-A mutants incapable of dimer formation form complexes with the sigma1 head that are indistinguishable from wild-type JAM-A-sigma1 head complexes, indicating that sigma1 binds to JAM-A monomers. Residues Glu(61) and Lys(63) of beta-strand C and Leu(72) of beta-strand C' in the dimer interface are required for efficient JAM-A engagement of strain type 3 Dearing sigma1. Mutation of neighboring residues alters the kinetics of the sigma1-JAM-A binding interaction. Prototype reovirus strains type 1 Lang and type 2 Jones share similar, although not identical, binding requirements with type 3 Dearing. These results indicate that reovirus engages JAM-A monomers via residues found mainly on beta-strands C and C' of the dimer interface and raise the possibility that the distinct disease phenotypes produced in mice following infection with different strains of reovirus are in part attributable to differences in contacts with JAM-A.     

Genetic variation during lytic reovirus infection: high-passage stocks of wild-type reovirus contain temperature-sensitive mutants.

Wild-type clones of reovirus serotypes 1 (Lang), 2 (Jones), and 3 (Dearing) were serially passaged in L cells at a high multiplicity of infection, and the virus population was examined at passage levels 2, 5, and 11 for the presence of temperature-sensitive (ts) mutants. By passage 11 all three serotypes contained ts mutants that were not present in the original wild-type stock. ts mutants representing three mutant groups were identified. The majority of these mutants were in group G. Our results show that high-passage stocks of reovirus consist of a genetically heterogeneous population.     

Invasion of the peripheral nervous systems of adult mice by the CVS strain of rabies virus and its avirulent derivative AvO1.

The penetration of the CVS strain of rabies virus and its avirulent derivative AvO1 into peripheral neurons was investigated after intramuscular inoculation into the forelimbs of adult mice. It was found that CVS directly penetrates both the sensitive and motor routes with equal efficiency, without prior multiplication in muscle cells. Infected neurons became detectable 18 h after infection. The second cycle of infection occurred within 2 days, and at day 3 there was a massive invasion of the spinal cord and sensory ganglia. In sensory ganglia, where it was possible to identify cell outlines, it was evident that the infection did not proceed directly from cell body to cell body. The avirulent strain AvO1 penetrated motor and sensory neurons with the same efficiency as CVS. Restriction of viral propagation was observed from the second and third cycles onwards. No further development of the infection could be seen after day 3, and by that time the lysis of primarily infected neurons seemed to occur.     

The transneuronal spread phenotype of herpes simplex virus type 1 infection of the mouse hind footpad.

The mouse hind footpad inoculation model has served as a standard laboratory system for the study of the neuropathogenesis of herpes simplex virus type 1 (HSV-1) infection. The temporal and spatial distribution of viral antigen, known as the transneuronal spread phenotype, has not previously been described; nor is it understood why mice develop paralysis in an infection that involves sensory nerves. The HSV-as-transneuronal-tracer experimental paradigm was used to define the transneuronal spread of HSV-1 in this model. A new decalcification technique and standard immunocytochemical staining of HSV-1 antigens enabled a detailed analysis of the time-space distribution of HSV-1 in the intact spinal column. Mice were examined on days 3, 4, 5, and 6 postinoculation (p.i.) of a lethal dose of wild-type HSV-1 strain 17 syn+. Viral antigen was traced retrograde into first-order neurons in dorsal root ganglia on day 3 p.i., to the dorsal spinal roots on days 4 and 5 p.i., and to second- and third-order neurons within sensory regions of the spinal cord on days 5 and 6 p.i. HSV-1 antigen distribution was localized to the somatotopic representation of the footpad dermatome within the dorsal root ganglia and spinal cord. Antigen was found in the spinal cord gray and white matter sensory neuronal circuits of nociception (the spinothalamic tract) and proprioception (the dorsal spinocerebellar tract and gracile fasciculus). Within the brain stems and brains of three paralyzed animals examined late in infection (days 5 and 6 p.i.), HSV antigen was restricted to the nucleus subcoeruleus region bilaterally. Since motor neurons were not directly involved, we postulate that hindlimb paralysis may have resulted from intense involvement of the posterior column (gracile fasciculus) in the thoracolumbar spinal cord, a region known to contain the corticospinal tract in rodents.     

Role of immune cells in protection against and control of reovirus infection in neonatal mice.

We studied the role of T cells in resistance to reovirus intestinal and central nervous system infection. Transfer of reovirus-immune adult spleen cells protected neonatal mice from (i) lethal infection with reovirus serotype 3 Dearing (T3D, footpad inoculation) and serotype 3 clone 9 (T3C9, oral inoculation) and (ii) hydrocephalus caused by serotype 1 Lang (T1L, intracranial [i.c.] inoculation). Cell-mediated protection was not serotype specific. While immune cells protected against T1L i.c., they failed to protect against 1/5,000 of the dose of T3D i.c. Two types of experiments showed that both CD4 and CD8 T cells are involved in reovirus resistance. First, immune cell-mediated protection against T3D was abrogated by in vivo treatment with anti-CD4 monoclonal antibody (MAb) and significantly inhibited by in vivo treatment with anti-CD8 MAb. Second, T3C9-infected neonatal mice treated with anti-CD4 and/or anti-CD8 developed a novel disease phenotype, an oily hair syndrome, associated with severe hepatobiliary pathology and increased viral titer in heart and liver. Immune cells and an MAb to the cell attachment protein sigma 1 (MAb G5) protected by different mechanisms. Immune cells were more effective than sigma 1 MAb G5 at controlling primary replication, while sigma 1 MAb G5 was more effective than immune cells at inhibiting neural spread of virus. We conclude that both CD4 and CD8 T cells are important for reovirus resistance, that cells and antibody act preferentially at different stages in pathogenesis in vivo, and that adoptively transferred immune cells can protect both the central nervous system and intestine.     

Differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein sigma 1.

Reoviruses are important models for studies of viral pathogenesis; however, the mechanisms by which these viruses produce cytopathic effects in infected cells have not been defined. In this report, we show that murine L929 (L) cells infected with prototype reovirus strains type 1 Lang (TIL) and type 3 Dearing (T3D) undergo apoptosis and that T3D induces apoptosis to a substantially greater extent than T1L. Using T1L x T3D reassortant viruses, we found that differences in the capacity of T1L and T3D to induce apoptosis are determined by the viral S1 gene segment, which encodes the viral attachment protein sigma 1 and the non-virion-associated protein sigma 1s. Apoptosis was induced by UV-inactivated, replication-incompetent reovirus virions, which do not contain sigma 1s and do not mediate its synthesis in infected cells. Additionally, T3D-induced apoptosis was inhibited by anti-reovirus monoclonal antibodies that inhibit T3D cell attachment and disassembly. These results indicate that sigma 1, rather than sigma 1s, is required for induction of apoptosis by the reovirus and suggest that interaction of virions with cell surface receptors is an essential step in this mechanism of cell killing.     

Reovirus core protein mu2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules

Cells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or "factories," where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L x T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the mu2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the mu2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of alpha-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of mu2 with the changes Pro-208 to Ser in a background of T1L mu2 and Ser-208 to Pro in a background of T3D mu2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the mu2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.     

Reovirus infection in rat lungs as a model to study the pathogenesis of viral pneumonia.

We undertook the present study to elucidate the pathogenesis of the pathologic response to reovirus infection in the lungs and further understand the interactions of reoviruses with pulmonary cells. We found that reoviruses were capable of causing acute pneumonia in 25- to 28-day-old Sprague-Dawley rats following intratracheal inoculation with the reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The onset of the pneumonia was rapid, marked by type I alveolar epithelial cell degeneration, type II alveolar epithelial cell hyperplasia, and the infiltration of leukocytes into the alveolar spaces. More neutrophils were recruited into the lungs during T3D infection than during T1L infection, and the serotype difference in the neutrophil response was mapped to the S1 gene of reovirus. Viral replication in the lungs was required for the development of pneumonia due to T1L and T3D infections, and replication occurred in type I alveolar epithelial cells. T1L grew to higher titers in the lungs than did either T3D or type 3 clone 9, and the S1 gene was found to play a role in determining the level of viral replication. We propose that experimental reovirus infection in the lungs can serve as a model for the pathogenesis of viral pneumonia in which pulmonary inflammation results following direct infection of lung epithelial cells.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.     

Junctional adhesion molecule a serves as a receptor for prototype and field-isolate strains of mammalian reovirus

Reovirus infections are initiated by the binding of viral attachment protein sigma1 to receptors on the surface of host cells. The sigma1 protein is an elongated fiber comprised of an N-terminal tail that inserts into the virion and a C-terminal head that extends from the virion surface. The prototype reovirus strains type 1 Lang/53 (T1L/53) and type 3 Dearing/55 (T3D/55) use junctional adhesion molecule A (JAM-A) as a receptor. The C-terminal half of the T3D/55 sigma1 protein interacts directly with JAM-A, but the determinants of receptor-binding specificity have not been identified. In this study, we investigated whether JAM-A also mediates the attachment of the prototype reovirus strain type 2 Jones/55 (T2J/55) and a panel of field-isolate strains representing each of the three serotypes. Antibodies specific for JAM-A were capable of inhibiting infections of HeLa cells by T1L/53, T2J/55, and T3D/55, demonstrating that strains of all three serotypes use JAM-A as a receptor. To corroborate these findings, we introduced JAM-A or the structurally related JAM family members JAM-B and JAM-C into Chinese hamster ovary cells, which are poorly permissive for reovirus infection. Both prototype and field-isolate reovirus strains were capable of infecting cells transfected with JAM-A but not those transfected with JAM-B or JAM-C. A sequence analysis of the sigma1-encoding S1 gene segment of the strains chosen for study revealed little conservation in the deduced sigma1 amino acid sequences among the three serotypes. This contrasts markedly with the observed sequence variability within each serotype, which is confined to a small number of amino acids. Mapping of these residues onto the crystal structure of sigma1 identified regions of conservation and variability, suggesting a likely mode of JAM-A binding via a conserved surface at the base of the sigma1 head domain.     

The Lang strain of reovirus serotype 1 and the Dearing strain of reovirus serotype 3 differ in their sensitivities to beta interferon.

Replication of the Dearing strain of reovirus serotype 3 in mouse L cells was decreased 17- to 100-fold when a saturating dose of beta interferon (1,000 IU/ml) was used. Replication of the Lang strain of reovirus serotype 1 was inhibited only two- to threefold under similar conditions. It therefore appears that closely related strains of reovirus differ in their sensitivities to beta interferon treatment of mouse L cells.     

Derivation and characterization of an efficiently myocarditic reovirus variant.

A reovirus variant, 8B, was isolated from a neonatal mouse which had been inoculated with a mixture of two reovirus strains: type 1 Lang (T1L) and type 3 Dearing (T3D) (E. A. Wenske, S.J. Chanock, L. Krata, and B. N. Fields, J. Virol. 56:613-616, 1985). 8B is a reassortant containing eight gene segments derived from the T1L parent and two gene segments derived from the T3D parent. Upon infection of neonatal mice, 8B produced a generalized infection characteristic of many reoviruses, but it also efficiently induced numerous macroscopic external cardiac lesions, unlike either of its parents. Microscopic examination of hearts from infected mice revealed myocarditis with necrotic myocytes and both polymorphonuclear and mononuclear cellular infiltration. Electron microscopy revealed viral arrays in necrotic myocytes and dystrophic calcification accompanying late lesions. Determination of viral titers in hearts from T1L-, T3D-, or 8B-infected mice indicated that growth was not the primary determinant of myocardial necrosis. Results from inoculations of athymic mice demonstrated that T cells were not a requirement for the 8B-induced myocarditis. Finally, 8B was more cytopathic than either of the parent viruses in cultured mouse L cells. Together, the data suggest that 8B-induced myocardial necrosis is due to a direct effect of reovirus on myocytes. Reovirus thus provides a useful model for the study of viral myocarditis.     

Genetic Determinants of Reovirus Pathogenesis in a Murine Model of Respiratory Infection

Many viruses invade mucosal surfaces to establish infection in the host. Some viruses are restricted to mucosal surfaces, whereas others disseminate to sites of secondary replication. Studies of strain-specific differences in reovirus mucosal infection and systemic dissemination have enhanced an understanding of viral determinants and molecular mechanisms that regulate viral pathogenesis. After peroral inoculation, reovirus strain type 1 Lang replicates to high titers in the intestine and spreads systemically, whereas strain type 3 Dearing (T3D) does not. These differences segregate with the viral S1 gene segment, which encodes attachment protein σ1 and nonstructural protein σ1s. In this study, we define genetic determinants that regulate reovirus-induced pathology following intranasal inoculation and respiratory infection. We report that two laboratory isolates of T3D, T3D^C and T3D^F, differ in the capacity to replicate in the respiratory tract and spread systemically; the T3D^C isolate replicates to higher titers in the lungs and disseminates, while T3D^F does not. Two nucleotide polymorphisms in the S1 gene influence these differences, and both S1 gene products are involved. T3D^C amino acid polymorphisms in the tail and head domains of σ1 protein influence the sensitivity of virions to protease-mediated loss of infectivity. The T3D^C polymorphism at nucleotide 77, which leads to coding changes in both S1 gene products, promotes systemic dissemination from the respiratory tract. A σ1s-null virus produces lower titers in the lung after intranasal inoculation and disseminates less efficiently to sites of secondary replication. These findings provide new insights into mechanisms underlying reovirus replication in the respiratory tract and systemic spread from the lung.     

Susceptibility and lack of evidence for a viremic state of rabies in the night owl monkey, Aotus nancymaae

ABSTRACT: BACKGROUND: Rabies causes an acute fatal encephalomyelitis in most mammals following infection with rhabdovirus of the genus Lyssavirus. Little is known about rabies virus infection in species of New World Primates. To investigate the suitability of the Aotus nancymaae owl monkey as a viable animal model for rhabdovirus candidate vaccine testing, we used clinical presentation, serology, viral isolation, and PCR to evaluate the incubation period, immunity, and pathogenesis of infected animals. We tested the hypothesis that no viremic state exists for rabies virus. METHOD: S Eight monkeys divided into two equal groups were inoculated intramuscularly either in the neck or footpad with 105 pfu of rabies virus (Pasteur/ V-13R) and observed for >130 days. Oral and blood samples were collected and analyzed. RESULTS: Two monkeys inoculated in the neck displayed classic paralytic rabies. The mean incubation period was 11.5 days. The average maximum IgG response (antibody titer >0.200 O.D.) was achieved at day 10.0 and 62.3 in the clinical rabies and non-clinical rabies cases, respectively (p=0.0429). No difference in IgM or IgG time to seroconversion or average maximum IgM level was observed between neck versus footpad inoculation groups. No viremia or viral shedding was detected by PCR or viral isolation during the observation period, including within the two symptomatic animals three days after disease onset. Tissue sections examined were unremarkable for inflammation or other histologic signs of rabies. None of the brain sections exhibited immunoreactivity for rabies virus antibody. DISCUSSION This study demonstrates there is no difference in time to immune response between inoculation sites and distance to the brain; however, immune response tends to be more rapid in cases of clinically apparent disease and prolonged in cases infected at sites further from the brain. CONCLUSIONS: Our findings support the hypothesis that a viremic state for rabies does not exist in the New World Monkey, Aotus nancymaae, and it appears that this species may be refractory to infection. The species does provide a suitable model to assess post infection immune responses. Additional studies that address the limitations of sample size, length of observation, and lack of measurable infection should be conducted.