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The gp41 membrane-proximal external region (MPER) is a target for broadly neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). However, replication-defective virus vaccines currently under evaluation in clinical trials do not efficiently elicit MPER-specific antibodies. Structural modeling suggests that the MPER forms an α-helical coiled coil that is required for function and immunogenicity. To maintain the native MPER conformation, we used reverse genetics to engineer replication-competent reovirus vectors that displayed MPER sequences in the α-helical coiled-coil tail domain of viral attachment protein σ1. Sequences in reovirus strain type 1 Lang (T1L) σ1 were exchanged with sequences encoding HIV-1 strain Ba-L MPER epitope 2F5 or the entire MPER. Individual 2F5 or MPER substitutions were introduced at virion-proximal or virion-distal sites in the σ1 tail. Recombinant reoviruses containing heterologous HIV-1 sequences were viable and produced progeny yields comparable to those with wild-type virus. HIV-1 sequences were retained following 10 serial passages in cell culture, indicating that the substitutions were genetically stable. Recombinant viruses engineered to display the 2F5 epitope or full-length MPER in σ1 were recognized by purified 2F5 antibody. Inoculation of mice with 2F5-containing vectors or rabbits with 2F5- or MPER-containing vectors elicited anti-reovirus antibodies, but HIV-1-specific antibodies were not detected. Together, these findings indicate that heterologous sequences that form α-helices can functionally replace native sequences in the α-helical tail domain of reovirus attachment protein σ1. However, although these vectors retain native antigenicity, they were not immunogenic, illustrating the difficulty of experimentally inducing immune responses to this essential region of HIV-1. IMPORTANCE Vaccines to protect against HIV-1, the causative agent of AIDS, are not approved for use. Antibodies that neutralize genetically diverse strains of HIV-1 bind to discrete regions of the envelope glycoproteins, including the gp41 MPER. We engineered recombinant reoviruses that displayed MPER epitopes in attachment protein σ1 (REO-MPER vectors). The REO-MPER vectors replicated with wild-type efficiency, were genetically stable, and retained native antigenicity. However, we did not detect HIV-1-specific immune responses following inoculation of the REO-MPER vectors into small animals. This work provides proof of principle for engineering reovirus to express antigenic epitopes and illustrates the difficulty in eliciting MPER-specific immune responses.

Positive health outcomes are related to adults' religious congregational participation. For parents of children with chronic disease, structured daily care routines and/or strict infection control precautions may limit participation. For this exploratory study, we examined the relationship between congregational support and religious coping by parents of children with cystic fibrosis (CF) compared to parents for whom child health issues were not significant stressors. CF parents reported higher levels of emotional support from congregation members and use of religious coping. Within-group differences were found for CF parents by denominational affiliation. Congregational support for parents dealing with child chronic disease is important.

Introduction: Chronic lung infection with Pseudomonas aeruginosa (PsA) is associated with more-rapid pulmonary decline and acute pulmonary exacerbations in patients with cystic fibrosis (CF). The treatment and eradication of this organism from CF airways is particularly difficult, making it the focus of many therapeutic endeavors. Inhaled antibiotics used for PsA treatment help to suppress growth of the organism, maintain lung function and reduce the frequency of pulmonary exacerbations., Areas Covered: Herein, the authors discuss the currently available inhaled antibiotics for management of lung infections in CF patients. They also describe the recent results from clinical trials of agents under development, with a focus on liposomal amikacin for inhalation (LAI, Arikace™). The article also provides a summary of the results from relevant articles published in the English., Expert Opinion: LAI is a unique formulation of amikacin that enhances drug delivery and retention in CF airways via incorporation into neutral liposomes. Results of a recent Phase II trial suggest that LAI, with the capacity for once-daily dosing and prolonged off-drug periods, may be an attractive choice of inhaled antibiotic to manage PsA lung infections in CF patients. Further data from Phase III studies assessing the efficacy and safety of LAI should better elucidate its potential.

Background: In 2001, Cincinnati Children's Hospital embarked on a journey to improve healthcare delivery to patients with cystic fibrosis (CF). Data from the Cystic Fibrosis Foundation National Patient Registry revealed our below-average clinical outcomes, prompting us to initiate improvement interventions., Objective: To improve clinical outcomes for patients with CF through a comprehensive quality-improvement approach directed at increasing patient centredness and improving healthcare delivery., Interventions: In 2001, we shared our below-average outcomes with patients, families and care providers. We instituted a quality-improvement steering committee with parental and hospital leadership, and our data-management support was restructured to provide real-time clinical data to monitor our progress. In 2002, our weekly chart conference changed to a prospective planning session and individualised daily schedules were created for inpatients. In 2003, an influenza vaccination campaign was initiated and our infection-control practices were redesigned. In 2005, best-practice guidelines were developed for airway-clearance therapy. In 2007, evidence-based clinical algorithms were designed and implemented and key care-team members were added., Measurements: Primary outcome measures were median forced expiratory volume in 1 s per cent predicted (age range 6-17 years) and median body mass index percentile (age range 2-20 years)., Results: From 2000 to 2010, median forced expiratory volume in 1 s increased from 81.7% to 100.1% predicted and median body mass index increased from the 35th to the 55th centile., Discussion: By focusing on specific outcomes, empowering families and patients, effectively using data, and standardising care processes, we transformed the culture and delivery of care for our patients with CF and learned valuable lessons potentially translatable to other chronic-care providers.

Infectious entry of the nonenveloped rotavirus virion requires proteolysis of the spike protein VP4 to mediate conformational changes associated with membrane penetration. We sequenced and characterized an isolate that was cultured in the absence of trypsin and found that it is more resistant to proteolysis than WT virus. A substitution mutation abrogates one of the defined trypsin-cleavage sites, suggesting that blocking proteolysis at this site reduces the overall kinetics of proteolysis. Kinetic analysis of the membrane penetration-associated conformational change indicated that the 'fold-back' of the mutant spike protein is slower than that of WT. Despite these apparent biochemical defects, the mutant virus replicates in an identical manner to the WT virus. These findings enhance an understanding of VP4 functions and establish new strategies to interrogate rotavirus cell entry.

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.)

Current microscopy-based approaches for immunofluorescence detection of viral infectivity are time consuming and labor intensive and can yield variable results subject to observer bias. To circumvent these problems, we developed a rapid and automated infrared immunofluorescence imager-based infectivity assay for both rotavirus and reovirus that can be used to quantify viral infectivity and infectivity inhibition. For rotavirus, monolayers of MA104 cells were infected with simian strain SA-11 or SA-11 preincubated with rotavirus-specific human IgA. For reovirus, monolayers of either HeLa S3 cells or L929 cells were infected with strains type 1 Lang (T1L), type 3 Dearing (T3D), or either virus preincubated with a serotype-specific neutralizing monoclonal antibody (mAb). Infected cells were fixed and incubated with virus-specific polyclonal antiserum, followed by an infrared fluorescence-conjugated secondary antibody. Well-to-well variation in cell number was normalized using fluorescent reagents that stain fixed cells. Virus-infected cells were detected by scanning plates using an infrared imager, and results were obtained as a percent response of fluorescence intensity relative to a virus-specific standard. An expected dose-dependent inhibition of both SA-11 infectivity with rotavirus-specific human IgA and reovirus infectivity with T1L-specific mAb 5C6 and T3D-specific mAb 9BG5 was observed, confirming the utility of this assay for quantification of viral infectivity and infectivity blockade. The imager-based viral infectivity assay fully automates data collection and provides an important advance in technology for applications such as screening for novel modulators of viral infectivity. This basic platform can be adapted for use with multiple viruses and cell types., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Following attachment and internalization, mammalian reoviruses undergo intracellular proteolytic disassembly followed by viral penetration into the cytoplasm. The initiating event in reovirus disassembly is the cathepsin-mediated proteolytic degradation of viral outer capsid protein σ3. A single tyrosine-to-histidine mutation at amino acid 354 (Y354H) of strain type 3 Dearing (T3D) σ3 enhances reovirus disassembly and confers resistance to protease inhibitors such as E64. The σ3 amino acid sequence of strain type 3 Abney (T3A) differs from that of T3D at eight positions including Y354H. However, T3A displays disassembly kinetics and protease sensitivity comparable with T3D. We hypothesize that one or more additional σ3 polymorphisms suppress the Y354H phenotype and restore T3D disassembly characteristics. To test this hypothesis, we engineered a panel of reovirus variants with T3A σ3 polymorphisms introduced individually into T3D-σ3Y354H. We evaluated E64 resistance and in vitro cathepsin L susceptibility of these viruses and found that one containing a glycine-to-glutamate substitution at position 198 (G198E) displayed disassembly kinetics and E64 sensitivity similar to those properties of T3A and T3D. Additionally, viruses containing changes at positions 233 and 347 (S233L and I347T) developed de novo compensatory mutations at position 198, strengthening the conclusion that residue 198 is a key determinant of σ3 proteolytic susceptibility. Variants with Y354H in σ3 lost infectivity more rapidly than T3A or T3D following heat treatment, an effect abrogated by G198E. These results identify a regulatory network of residues that control σ3 cleavage and capsid stability, thus providing insight into the regulation of nonenveloped virus disassembly.

The cathepsin family of endosomal proteases is required for proteolytic processing of several viruses during entry into host cells. Mammalian reoviruses utilize cathepsins B (Ctsb), L (Ctsl), and S (Ctss) for disassembly of the virus outer capsid and activation of the membrane penetration machinery. To determine whether cathepsins contribute to reovirus tropism, spread, and disease outcome, we infected 3-day-old wild-type (wt), Ctsb(-/-), Ctsl(-/-), and Ctss(-/-) mice with the virulent reovirus strain T3SA+. The survival rate of Ctsb(-/-) mice was enhanced in comparison to that of wt mice, whereas the survival rates of Ctsl(-/-) and Ctss(-/-) mice were diminished. Peak titers at sites of secondary replication in all strains of cathepsin-deficient mice were lower than those in wt mice. Clearance of the virus was delayed in Ctsl(-/-) and Ctss(-/-) mice in comparison to the levels for wt and Ctsb(-/-) mice, consistent with a defect in cell-mediated immunity in mice lacking cathepsin L or S. Cathepsin expression was dispensable for establishment of viremia, but cathepsin L was required for maximal reovirus growth in the brain. Treatment of wt mice with an inhibitor of cathepsin L led to amelioration of reovirus infection. Collectively, these data indicate that cathepsins B, L, and S influence reovirus pathogenesis and suggest that pharmacologic modulation of cathepsin activity diminishes reovirus disease severity.

Mammalian reoviruses infect respiratory and gastrointestinal epithelia and cause disease in neonates. Junctional adhesion molecule-A (JAM-A) is a serotype-independent receptor for reovirus. JAM-A localizes to tight junctions and contributes to paracellular permeability in polarized epithelia. To investigate the mechanisms of reovirus infection of polarized epithelial cells, we assessed reovirus replication, release, and spread after apical and basolateral adsorption to primary human airway epithelial cultures. Reovirus infection of human airway epithelia was more efficient after adsorption to the basolateral surface than after adsorption to the apical surface, and it was dependent on JAM-A. Reovirus binding to carbohydrate coreceptor sialic acid inhibited apical infection, which was partially ameliorated by treatment of the cultures with neuraminidase. Despite the preference for basolateral infection, reovirus was released from the apical surface of respiratory epithelia and did not disrupt tight junctions. These results establish the existence of an infectious circuit for reovirus in polarized human respiratory epithelial cells.

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.

Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.

We previously described a filament-based antibody recognition assay (FARA) that generates ELISA-like sandwich structures immobilized on a filament. FARA allows the coupling of antibodies to precise locations along a filament, on-line fluorescence detection of captured pathogen, and feedback-directed filament motion. These properties suggest that this approach might be useful as an automated means to rapidly classify unknown pathogens. In this report, we describe validation of the novel decision tree aspect of this technology using mammalian reovirus. Based on available antibodies, we developed a decision tree algorithm to detect virus with increasing specificity at each level of the tree. Using three strains of reovirus and a bacteriophage control, our system correctly classified the reovirus strains at a concentration of 2 x 10(12) virions ml(-1) and M13K07 phage at 3 x 10(11) virions ml(-1). Classification of reovirus strain type 3 Dearing (T3D) required three levels of testing: general reovirus classification in level 1, serotype 3 classification in level 2, and final T3D strain classification in level 3. Strain T3SA + also required three levels of testing before a final classification was returned in level 3. Classification of strain type 1 Lang (T1L) required two levels of testing. M13K07 phage detection required only one level of testing for classification. These results indicate that automated pathogen classification using FARA is feasible. Furthermore, the simplicity of the design could be exploited for development of more complex sub-classification networks with additional levels and branches.

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.

Mice infected with reovirus develop abnormalities in glucose homeostasis. Reovirus strain type 3 Abney (T3A) was capable of systemic infection of nonobese diabetic (NOD) mice, an experimental model of autoimmune diabetes. Reovirus antigen was detected in pancreatic islets of T3A-infected mice, and primary cultures of pancreatic islets from NOD mice supported T3A growth. Significantly fewer T3A-infected animals compared to uninfected controls developed diabetes. However, despite the alteration in diabetes penetrance, insulitis was evident in T3A-infected mice. These results suggest that viral infection of NOD mice alters autoimmune responses to beta-cell antigens and thereby delays development of diabetes.

Mammalian reoviruses are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis resulting in removal of the sigma3 protein and proteolytic cleavage of the mu1/mu1C protein. Ammonium chloride (AC) is a weak base that blocks disassembly of reovirus virions by inhibiting acidification of intracellular vacuoles. To identify domains in reovirus proteins that influence pH-sensitive steps in viral disassembly, we adapted strain type 3 Dearing (T3D) to growth in murine L929 cells treated with AC. In comparison to wild-type (wt) T3D, AC-adapted (ACA-D) variant viruses exhibited increased yields in AC-treated cells. AC resistance of reassortant viruses generated from a cross of wt type 1 Lang and ACA-D variant ACA-D1 segregated with the sigma3-encoding S4 gene. The deduced sigma3 amino acid sequences of six independently derived ACA-D variants contain one or two mutations each, affecting a total of six residues. Four of these mutations, I180T, A246G, I347S, and Y354H, cluster in the virion-distal lobe of sigma3. Linkage of these mutations to AC resistance was confirmed in experiments using reovirus disassembly intermediates recoated with wt or mutant sigma3 proteins. In comparison to wt virions, ACA-D viruses displayed enhanced susceptibility to proteolysis by endocytic protease cathepsin L. Image reconstructions of cryoelectron micrographs of three ACA-D viruses that each contain a single mutation in the virion-distal lobe of sigma3 demonstrated native capsid protein organization and minimal alterations in sigma3 structure. These results suggest that mutations in sigma3 that confer resistance to inhibitors of vacuolar acidification identify a specific domain that regulates proteolytic disassembly.

Reovirus induces apoptosis in cultured cells and in vivo. In cell culture models, apoptosis is contingent upon a mechanism involving reovirus-induced activation of transcription factor NF-kappaB complexes containing p50 and p65/RelA subunits. To explore the in vivo role of NF-kappaB in this process, we tested the capacity of reovirus to induce apoptosis in mice lacking a functional nfkb1/p50 gene. The genetic defect had no apparent effect on reovirus replication in the intestine or dissemination to secondary sites of infection. In comparison to what was observed in wild-type controls, apoptosis was significantly diminished in the CNS of p50-null mice following reovirus infection. In sharp contrast, the loss of p50 was associated with massive reovirus-induced apoptosis and uncontrolled reovirus replication in the heart. Levels of IFN-beta mRNA were markedly increased in the hearts of wild-type animals but not p50-null animals infected with reovirus. Treatment of p50-null mice with IFN-beta substantially diminished reovirus replication and apoptosis, which suggests that IFN-beta induction by NF-kappaB protects against reovirus-induced myocarditis. These findings reveal an organ-specific role for NF-kappaB in the regulation of reovirus-induced apoptosis, which modulates encephalitis and myocarditis associated with reovirus infection.

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.

We explored the role of Peyer's patch (PP) dendritic cell (DC) populations in the induction of immune responses to reovirus strain type 1 Lang (T1L). Immunofluorescence staining revealed the presence of T1L structural (sigma1) and nonstructural (sigmaNS) proteins in PPs of T1L-infected mice. Cells in the follicle-associated epithelium contained both sigma1 and sigmaNS, indicating productive viral replication. In contrast, sigma1, but not sigmaNS, was detected in the subepithelial dome (SED) in association with CD11c(+)/CD8alpha(-)/CD11b(lo) DCs, suggesting antigen uptake by these DCs in the absence of infection. Consistent with this possibility, PP DCs purified from infected mice contained sigma1, but not sigmaNS, and PP DCs from uninfected mice could not be productively infected in vitro. Furthermore, sigma1 protein in the SED was associated with fragmented DNA by terminal deoxy-UTP nick-end labeling staining, activated caspase-3, and the epithelial cell protein cytokeratin, suggesting that DCs capture T1L antigen from infected apoptotic epithelial cells. Finally, PP DCs from infected mice activated T1L-primed CD4(+) T cells in vitro. These studies show that CD8alpha(-)/CD11b(lo) DCs in the PP SED process T1L antigen from infected apoptotic epithelial cells for presentation to CD4(+) T cells, and therefore demonstrate the cross-presentation of virally infected cells by DCs in vivo during a natural viral infection.

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

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

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein sigma 3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in sigma 3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the sigma 3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant sigma 3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced sigma 3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in sigma3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant sigma 3 proteins revealed structural alterations in sigma 3 that correlate with the Y354H mutation. These results indicate that a single mutation in sigma 3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of sigma 3 cleavage during reovirus disassembly.

A rapid, reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of reovirus RNA in cell culture is described. Total nucleic acids are extracted from a small volume of cell culture supernatant and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR. The PCR primers correspond to sequences conserved between prototype reovirus strains type 1 Lang, type 2 Jones, and type 3 Dearing, as well as those of several reovirus field-isolate strains. Reactions are analyzed by agarose gel electrophoresis, and samples showing a band of the appropriate size in the first and second amplification, or in the second amplification alone, are designated as positive. This protocol allows for the rapid and sensitive detection of reovirus in cell culture. The RT-PCR methods described below can easily be adapted to the amplification of reovirus from other media, including preserved tissues, clinical specimens, and water.

Reoviruses infect virtually all mammalian species, and infection of humans is associated with mild gastrointestinal or upper respiratory illnesses. To improve reovirus detection strategies, we developed a reverse transcription-PCR technique to amplify a fragment of the reovirus L1 gene segment. This assay was capable of detecting 44 of 44 reovirus field isolate strains and was sufficiently sensitive to detect nearly a single viral particle (1.16 +/- 0.13) per PCR of prototype strain type 3 Dearing. Pairwise comparisons of the 44 partial L1 gene sequences revealed that nucleotide variability ranged from 0 to 24.7%, with most of the nucleotide polymorphism occurring at synonymous positions. Phylogenetic trees generated from amplified L1 gene sequences suggest that multiple alleles of the L1 gene cocirculate in nature and that genetic diversity of the L1 gene is largely independent of the host species, geographic locale, or date of isolation. Phylogenetic trees constructed from the L1 gene sequences are distinct from those constructed from the four reovirus S-class gene segments, which supports the hypothesis that reovirus gene segments reassort in nature. This study establishes a new sensitive and specific technique for the identification of mammalian reoviruses and enhances our understanding of reovirus evolution.

Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein sigma1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering sigma1-sigma3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 microg per ml, whereas that of Fabs was >200 microg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of sigma3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins sigma3 and mu1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.

Reovirus virions are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis leading to degradation of sigma3 protein and proteolytic cleavage of micro1/micro1C protein. E64 is a specific inhibitor of cysteine-containing proteases that blocks disassembly of reovirus virions. To identify domains in reovirus proteins that influence susceptibility to E64-mediated inhibition of disassembly, we selected variant viruses by serial passage of strain type 3 Dearing (T3D) in murine L929 cells treated with E64. E64-adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yields than T3D did after infection of cells treated with 100 microM E64. Viral genes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses of E64-sensitive strain type 1 Lang and two prototype D-EA viruses. Growth of reassortant viruses in the presence of E64 segregated with the S4 gene, which encodes outer-capsid protein sigma3. Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a common tyrosine-to-histidine mutation at amino acid 354 in the deduced amino acid sequence of sigma3. Proteolysis of D-EA virions by endocytic protease cathepsin L occurred with faster kinetics than proteolysis of wild-type T3D virions. Treatment of D-EA virions, but not T3D virions, with cathepsin D resulted in proteolysis of sigma3, a property that also was found to segregate with the D-EA S4 gene. These results indicate that a region in sigma3 protein containing amino acid 354 influences susceptibility of sigma3 to proteolysis during reovirus disassembly.

To better understand mechanisms of persistent rotavirus infections of cultured cells, we established independent, persistently infected cultures of MA104 cells, using rotavirus strain SA11. The cultures were either passaged when the cells reached confluence or supplemented with fresh medium every 7 days. Viral titers in culture lysates varied from 10(4) to 10(7) PFU per ml during 350 days of culture maintenance. Trypan blue staining indicated that 72 to 100% of cells in the cultures were viable, and immunocytochemical staining using a monoclonal antibody directed against viral protein VP6 demonstrated that 38 to 63% of the cells contained rotavirus antigen. We tested the capacity of rotaviruses isolated from the persistently infected cultures (PI viruses) to infect cells cured of persistent infection. Although wild-type (wt) and PI viruses produced equivalent yields in parental MA104 cells, PI viruses produced greater yields than wt virus in cured cells, which indicates that viruses and cells coevolve during persistent rotavirus infections of MA104 cells. To determine whether mutations in viruses and cells selected during these persistent infections affect viral entry, we tested the effect of trypsin treatment of the viral inoculum on growth of wt and PI viruses. Trypsin pretreatment is required for postattachment penetration of rotavirus virions into cells. In contrast to the case with wt virus, PI viruses produced equivalent yields with and without trypsin pretreatment in parental MA104 cells. However, PI viruses required trypsin pretreatment for efficient growth in cured cells. These results indicate that mutant viruses and cells are selected during maintenance of persistent rotavirus infections of MA104 cells and suggest that mutations in each affect trypsin-dependent steps in rotavirus entry.

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.

During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein sigma1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1 mutations in each S1 gene resulted in changes in the deduced amino acid sequence of sigma1 protein. Using electrophoresis conditions that favor migration of sigma1 oligomers, we found that sigma1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas sigma1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in sigma1 protein affecting stability of sigma1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 sigma1 protein, we used T1L X PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein sigma3. This finding suggests that in some cases, mutations in sigma3 protein in the absence of sigma1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L X PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the sigma1-encoding S1 gene. Given that the sigma1 and sigma3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.

Mutants of mammalian reoviruses, enteric double-stranded-RNA-containing viruses that spread systemically after primary replication in intestinal tissue, have been extensively studied as models of viral pathogenesis. While reovirus serotype 3 strain Dearing (T3D) causes acute encephalitis in newborn mice, adult severe combined immunodeficient (SCID) mice develop chronic infection with T3D, with some mice living more than 100 days after infection (B. L. Haller, M. L. Barkon, G. P. Vogler, and H. W. Virgin IV, J. Virol. 69:357-364, 1995). To determine whether organ-specific reovirus variants are selected during chronic infection, we characterized the pathogenetic properties of two variants of T3D isolated 87 days after intraperitoneal infection of adult SCID mice. A brain-specific variant (T3DvBr) (i) grew to a higher titer than T3D in SCID mouse brain (but not intestine) after intraperitoneal inoculation, (ii) killed adult SCID mice faster than T3D, and (iii) grew well in neonatal NIH Swiss [NIH(s)] mouse brain tissue after intramuscular but not peroral inoculation. An intestine-specific variant (T3DvInt) (i) grew to a higher titer than T3D in SCID mouse intestine (but not brain) after intraperitoneal inoculation, (ii) killed SCID mice with kinetics equivalent to those of T3D, (iii) was much less virulent than T3D in neonatal NIH(s) mice, (iv) grew better than T3D in intestines after intramuscular or peroral inoculation into neonatal NIH(s) mice, and (v) grew poorly in brain tissue of neonatal NIH(s) mice after intramuscular inoculation. During prolonged infection of SCID mice, organ-specific variants of T3D, which are more efficient than wild-type T3D at one specific stage in reovirus pathogenesis, are selected.

Previous studies demonstrated that both cellular and viral mutants are selected during maintenance of persistent infections established in murine L cells with high-passage stocks of mammalian reoviruses. In particular, when one culture was cured of persistent infection, the resulting cells were found to support the growth of viruses isolated from persistently infected cultures (termed PI viruses here) better than that of wild-type (wt) viruses (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25:325-332, 1981). To address the nature of cellular and viral mutations selected during maintenance of persistent reovirus infections, we established independent, persistently infected cultures with L cells and high-passage stocks of wt reovirus. These cultures served as sources of new PI viruses and cured cells for study. We found that although wt viruses grew poorly in cured cells when infection was initiated with intact virions, they grew well in cured cells when infection was initiated with infectious subvirion particles generated from virions by in vitro treatment with chymotrypsin. This finding indicates that the block to growth of wt viruses in cured cells involves an early step that is unique to infection by virions, such as proteolytic processing in an endocytic compartment. We also found that PI viruses grew better than wt viruses in L cells treated with ammonium chloride, a weak base that inhibits the pH decrease in endosomes and lysosomes. Because ammonium chloride blocks an early step in infection by intact virions, probably the proteolytic processing of viral outer capsid proteins by acid-dependent cellular proteases in late endosomes or lysosomes, this finding indicates that PI viruses differ from wt viruses with respect to viral entry into cells. Therefore, these results indicate that both cells and viruses evolve mutations that affect one or more early steps in the viral growth cycle during maintenance of L-cell cultures persistently infected with reoviruses.

The recognition of cellular receptors by the mammalian reoviruses is an important determinant of cell and tissue tropism exhibited by reovirus strains of different serotypes. To extend our knowledge of the role of reovirus-receptor interactions in reovirus tropism, we determined whether type 1 and type 3 reovirus strains can infect cells derived from erythrocyte precursors. We found that reovirus type 3 Dearing (T3D), but not type 1 Lang, can grow in murine erythroleukemia (MEL) cells. This difference in growth was investigated by using reassortant viruses and we found that the capacity of T3D to infect MEL cells is determined by the viral cell-attachment protein, sigma 1. In experiments using murine monoclonal antibodies (mAbs) that bind to different sigma 1 regions, we show that T3D binding to MEL cells is inhibited by a mAb that identifies a domain important for hemagglutination (HA). We also determined that type 3 strains that can infect murine L cells but do not produce HA do not infect MEL cells. These results suggest that type 3 reovirus binds to and infects erythrocyte precursor cells via a sigma 1 domain important for HA. Moreover, this study suggests that different domains of some viral cell-attachment proteins are used to initiate productive infections of different types of cells.