Search

Human parainfluenza virus type 1 (hPIV1) and 3 (hPIV3) cause seasonal epidemics, but little is known about their interaction with human airway cells. In this study, we determined cytopathology, replication, and progeny virion release from human airway cells during long-term infection in vitro. Both viruses readily established persistent infection without causing significant cytopathic effects. However, assembly and release of hPIV1 rapidly declined in sharp contrast to hPIV3 due to impaired viral ribonucleocapsid (vRNP) trafficking and virus assembly. Transcriptomic analysis revealed that both viruses induced similar levels of type I and III IFNs. However, hPIV1 induced specific ISGs stronger than hPIV3, such as MX2, which bound to hPIV1 vRNPs in infected cells. In addition, hPIV1 but not hPIV3 suppressed genes involved in lipid biogenesis and hPIV1 infection resulted in ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate limiting enzyme in cholesterol biosynthesis. Consequently, formation of cholesterol-rich lipid rafts was impaired in hPIV1 infected cells. These results indicate that hPIV1 is capable of regulating cholesterol biogenesis, which likely together with ISGs contributes to establishment of a quiescent infection., Competing Interests: The authors have declared that no competing interests exist.

Sensing of viral RNA by RIG-I-like receptors initiates innate antiviral response, which is mediated by the central adaptor VISA. How the RIG-I-VISA-mediated antiviral response is terminated at the late phase of infection is enigmatic. Here we identified the protein kinase A catalytic (PKAC) subunits α and β as negative regulators of RNA virus-triggered signaling in a redundant manner. Viral infection up-regulated cellular cAMP levels and activated PKACs, which then phosphorylated VISA at T54. This phosphorylation abrogated virus-induced aggregation of VISA and primed it for K48-linked polyubiquitination and degradation by the E3 ligase MARCH5, leading to attenuation of virus-triggered induction of downstream antiviral genes. PKACs-deficiency or inactivation by the inhibitor H89 potentiated innate immunity to RNA viruses in cells and mice. Our findings reveal a critical mechanism of attenuating innate immune response to avoid host damage at the late phase of viral infection by the house-keeping PKA kinase.

Asthma exacerbation can be a life-threatening condition, and is most often triggered by common respiratory viruses. Poor asthma control and worsening of respiratory function is associated with increased airway inflammation, including eosinophilia. Prevention of asthma exacerbation relies on treatment with corticosteroids, which preferentially inhibit allergic inflammation like eosinophils. Human studies demonstrate that inactivated virus can trigger eosinophil activation in vitro through antigen presentation and memory CD4+ lymphocytes. We hypothesized that animals with immunologic memory to a respiratory virus would also develop airway hyperresponsiveness in response to a UV-inactivated form of the virus if they have pre-existing allergic airway inflammation. Guinea pigs were ovalbumin-sensitized, infected with live parainfluenza virus (PIV), aerosol-challenged with ovalbumin, and then re-inoculated 60 days later with live or UV-inactivated PIV. Some animals were either treated with dexamethasone prior to the second viral exposure. Lymphocytes were isolated from parabronchial lymph nodes to confirm immunologic memory to the virus. Airway reactivity was measured and inflammation was assessed using bronchoalveolar lavage and lung histology. The induction of viral immunologic memory was confirmed in infected animals. Allergen sensitized and challenged animals developed airway hyperreactivity with eosinophilic airway inflammation when re-exposed to UV-inactivated PIV, while non-sensitized animals did not. Airway hyperreactivity in the sensitized animals was inhibited by pre-treatment with dexamethasone. We suggest that the response of allergic inflammation to virus antigen is a significant factor causing asthma exacerbation. We propose that this is one mechanism explaining how corticosteroids prevent virus-induced asthma attack.

Respiratory paramyxoviruses are important causes of morbidity and mortality, particularly of infants and the elderly. In humans, a T helper (Th)2-biased immune response to these infections is associated with increased disease severity; however, little is known about the endogenous regulators of these responses that may be manipulated to ameliorate pathology. IL-27, a cytokine that regulates Th2 responses, is produced in the lungs during parainfluenza infection, but its role in disease pathogenesis is unknown. To determine whether IL-27 limits the development of pathogenic Th2 responses during paramyxovirus infection, IL-27-deficient or control mice were infected with the murine parainfluenza virus Sendai virus (SeV). Infected IL-27-deficient mice experienced increased weight loss, more severe lung lesions, and decreased survival compared to controls. IL-27 deficiency led to increased pulmonary eosinophils, alternatively activated macrophages (AAMs), and the emergence of Th2 responses. In control mice, IL-27 induced a population of IFN-γ+/IL-10+ CD4+ T cells that was replaced by IFN-γ+/IL-17+ and IFN-γ+/IL-13+ CD4+ T cells in IL-27-deficient mice. CD4+ T cell depletion in IL-27-deficient mice attenuated weight loss and decreased AAMs. Elimination of STAT6 signaling in IL-27-deficient mice reduced Th2 responses and decreased disease severity. These data indicate that endogenous IL-27 limits pathology during parainfluenza virus infection by regulating the quality of CD4+ T cell responses and therefore may have therapeutic potential in paramyxovirus infections., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: CAH is a consultant for the group Surface Oncology and holds patents related to IL-27 and immune regulation. MME is employed by a commercial company, Janssen Research and Development.

In immunocompromised patients, parainfluenza virus (PIV) infections have an increased potential to spread to the lower respiratory tract (LRT), resulting in increased morbidity and mortality. Understanding the immunologic defects that facilitate viral spread to the LRT will help in developing better management protocols. In this study, we immunosuppressed mice with dexamethasone and/or cyclophosphamide then monitored the spread of viral infection into the LRT by using a noninvasive bioluminescence imaging system and a reporter Sendai virus (murine PIV type 1). Our results show that immunosuppression led to delayed viral clearance and increased viral loads in the lungs. After cessation of cyclophosphamide treatment, viral clearance occurred before the generation of Sendai-specific antibody responses and coincided with rebounds in neutrophils, T lymphocytes, and natural killer (NK) cells. Neutrophil suppression using anti-Ly6G antibody had no effect on infection clearance, NK-cell suppression using anti-NK antibody delayed clearance, and T-cell suppression using anti-CD3 antibody resulted in no clearance (chronic infection). Therapeutic use of hematopoietic growth factors G-CSF and GM-CSF had no effect on clearance of infection. In contrast, treatment with Sendai virus-specific polysera or a monoclonal antibody limited viral spread into the lungs and accelerated clearance. Overall, noninvasive bioluminescence was shown to be a useful tool to study respiratory viral progression, revealing roles for NK and T cells, but not neutrophils, in Sendai virus clearance after treatment with dexamethasone and cyclophosphamide. Virus-specific antibodies appear to have therapeutic potential., Competing Interests: The authors have declared that no competing interests exist.

RIG-I-like receptors detect viral RNA in infected cells and promote oligomerization of the outer mitochondrial membrane protein MAVS to induce innate immunity to viral infection through type I interferon production. Mitochondrial reactive oxygen species (mROS) have been shown to enhance anti-viral MAVS signalling, but the mechanisms have remained obscure. Using a biochemical oligomerization-reporter fused to the transmembrane domain of MAVS, we found that mROS inducers promoted lipid-dependent MAVS transmembrane domain oligomerization in the plane of the outer mitochondrial membrane. These events were mirrored by Sendai virus infection, which similarly induced lipid peroxidation and promoted lipid-dependent MAVS transmembrane domain oligomerization. Our observations point to a role for mROS-induced changes in lipid bilayer properties in modulating antiviral innate signalling by favouring the oligomerization of MAVS transmembrane domain in the outer-mitochondrial membrane.

Early life respiratory viral infections and atopic characteristics are significant risk factors for the development of childhood asthma. It is hypothesized that repeated respiratory viral infections might induce structural remodeling by interfering with the normal process of lung maturation; however, the specific molecular processes that underlie these pathological changes are not understood. To investigate the molecular basis for these changes, we used an established Sendai virus infection model in weanling rats to compare the post-infection transcriptomes of an atopic asthma susceptible strain, Brown Norway, and a non-atopic asthma resistant strain, Fischer 344. Specific to this weanling infection model and not described in adult infection models, Sendai virus in the susceptible, but not the resistant strain, results in morphological abnormalities in distal airways that persist into adulthood. Gene expression data from infected and control lungs across five time points indicated that specific features of the immune response following viral infection were heightened and prolonged in lungs from Brown Norway rats compared with Fischer 344 rats. These features included an increase in macrophage cell number and related gene expression, which then transitioned to an increase in mast cell number and related gene expression. In contrast, infected Fischer F344 lungs exhibited more efficient restoration of the airway epithelial morphology, with transient appearance of basal cell pods near distal airways. Together, these findings indicate that the pronounced macrophage and mast cell responses and abnormal re-epithelialization precede the structural defects that developed and persisted in Brown Norway, but not Fischer 344 lungs.

Viral infection induces innate immunity and apoptosis. Apoptosis is an effective means to sacrifice virus-infected host cells and therefore restrict the spread of pathogens. However, the underlying mechanisms of this process are still poorly understood. Here, we show that the mitochondrial antiviral signaling protein (MAVS/VISA/Cardif/IPS-1) is critical for SeV (Sendai virus)-induced apoptosis. MAVS specifically activates c-Jun N-terminal kinase 2 (JNK2) but not other MAP kinases. Jnk2-/- cells, but not Jnk1-/- cells, are unable to initiate virus-induced apoptosis and SeV further fails to trigger apoptosis in MAPK kinase 7 (MKK7) knockout (Mkk7-/-) cells. Mechanistically, MAVS recruits MKK7 onto mitochondria via its 3D domain, which subsequently phosphorylates JNK2 and thus activates the apoptosis pathway. Consistently, Jnk2-/- mice, but not Jnk1-/- mice, display marked inflammatory injury in lung and liver after viral challenge. Collectively, we have identified a novel signaling pathway, involving MAVS-MKK7-JNK2, which mediates virus-induced apoptosis and highlights the indispensable role of mitochondrial outer membrane in host defenses.

The innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. Here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors IRF3 and NF-κB and the production type I IFNs through a mechanism independent of IFN signaling. We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. Remarkably, the hallmark antiviral cytokine IFNβ is only expressed in lung epithelial cells containing DVGs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. Together, our data indicate that DVGs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection.

Little is known about how the mode of respiratory virus transmission determines the dynamics of primary infection and protection from reinfection. Using non-invasive imaging of murine parainfluenza virus 1 (Sendai virus) in living mice, we determined the frequency, timing, dynamics, and virulence of primary infection after contact and airborne transmission, as well as the tropism and magnitude of reinfection after subsequent challenge. Contact transmission of Sendai virus was 100% efficient, phenotypically uniform, initiated and grew to robust levels in the upper respiratory tract (URT), later spread to the lungs, grew to a lower level in the lungs than the URT, and protected from reinfection completely in the URT yet only partially in the lungs. Airborne transmission through 7.6-cm and 15.2-cm separations between donor and recipient mice was 86%-100% efficient. The dynamics of primary infection after airborne transmission varied between individual mice and included the following categories: (a) non-productive transmission, (b) tracheal dominant, (c) tracheal initiated yet respiratory disseminated, and (d) nasopharyngeal initiated yet respiratory disseminated. Any previous exposure to Sendai virus infection protected from mortality and severe morbidity after lethal challenge. Furthermore, a higher level of primary infection in a given respiratory tissue (nasopharynx, trachea, or lungs) was inversely correlated with the level of reinfection in that same tissue. Overall, the mode of transmission determined the dynamics and tropism of primary infection, which in turn governed the level of seroconversion and protection from reinfection. These data are the first description of the dynamics of respiratory virus infection and protection from reinfection throughout the respiratory tracts of living animals after airborne transmission. This work provides a basis for understanding parainfluenza virus transmission and protective immunity and for developing novel vaccines and non-pharmaceutical interventions.

To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-β (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of β-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.

A burst in the production of pro-inflammatory molecules characterizes the beginning of the host response to infection. Cytokines, chemokines, and growth factors work in concert to control pathogen replication and activate innate and adaptive immune responses. Granulocyte colony-stimulating factor (G-CSF) mobilizes and activates hematopoietic cells from the bone marrow, and it has been shown to mediate the generation of effective immunity against bacterial and fungal infections. G-CSF is produced at high levels in the lungs during infection with influenza and parainfluenza viruses, but its role during these infections is unknown. Here we show that during infection of mice with a non-lethal dose of influenza or Sendai virus, G-CSF promotes the accumulation of activated Ly6G+ granulocytes that control the extent of the lung pro-inflammatory response. Remarkably, these G-CSF-mediated effects facilitate viral clearance and sustain mouse survival.

The parainfluenza viruses (PIVs) are highly contagious respiratory paramyxoviruses and a leading cause of lower respiratory tract (LRT) disease. Since no vaccines or antivirals exist, non-pharmaceutical interventions are the only means of control for these pathogens. Here we used bioluminescence imaging to visualize the spatial and temporal progression of murine PIV1 (Sendai virus) infection in living mice after intranasal inoculation or exposure by contact. A non-attenuated luciferase reporter virus (rSeV-luc(M-F*)) that expressed high levels of luciferase yet was phenotypically similar to wild-type Sendai virus in vitro and in vivo was generated to allow visualization. After direct intranasal inoculation, we unexpectedly observed that the upper respiratory tract (URT) and trachea supported robust infection under conditions that result in little infection or pathology in the lungs including a low inoculum of virus, an attenuated virus, and strains of mice genetically resistant to lung infection. The high permissivity of the URT and trachea to infection resulted in 100% transmission to naïve contact recipients, even after low-dose (70 PFU) inoculation of genetically resistant BALB/c donor mice. The timing of transmission was consistent with the timing of high viral titers in the URT and trachea of donor animals but was independent of the levels of infection in the lungs of donors. The data therefore reveals a disconnect between transmissibility, which is associated with infection in the URT, and pathogenesis, which arises from infection in the lungs and the immune response. Natural infection after transmission was universally robust in the URT and trachea yet limited in the lungs, inducing protective immunity without weight loss even in genetically susceptible 129/SvJ mice. Overall, these results reveal a dichotomy between PIV infection in the URT and trachea versus the lungs and define a new model for studies of pathogenesis, development of live virus vaccines, and testing of antiviral therapies.

The innate immune response is essential to the host defense against viruses, through restriction of virus replication and coordination of the adaptive immune response. Induction of antiviral genes is a tightly regulated process initiated mainly through sensing of invading virus nucleic acids in the cytoplasm by RIG-I like helicases, RIG-I or Mda5, which transmit the signal through a common mitochondria-associated adaptor, MAVS. Although major breakthroughs have recently been made, much remains unknown about the mechanisms that translate virus recognition into antiviral genes expression. Beside the reputed detrimental role, reactive oxygen species (ROS) act as modulators of cellular signaling and gene regulation. NADPH oxidase (NOX) enzymes are a main source of deliberate cellular ROS production. Here, we found that NOX2 and ROS are required for the host cell to trigger an efficient RIG-I-mediated IRF-3 activation and downstream antiviral IFNbeta and IFIT1 gene expression. Additionally, we provide evidence that NOX2 is critical for the expression of the central mitochondria-associated adaptor MAVS. Taken together these data reveal a new facet to the regulation of the innate host defense against viruses through the identification of an unrecognized role of NOX2 and ROS.

One of the accessory proteins of Sendai virus (SeV), C, translated from an alternate reading frame of P/V mRNA has been shown to function at multiple stages of infection in cell cultures as well as in mice. C protein has been reported to counteract signal transduction by interferon (IFN), inhibit apoptosis induced by the infection, enhance the efficiency of budding of viral particles, and regulate the polarity of viral genome-length RNA synthesis to maximize production of infectious particles. In this study, we have generated a series of SeV recombinants containing substitutions of highly conserved, charged residues within the C protein, and characterized them together with previously-reported C'/C(-), 4C(-), and F170S recombinant viruses in infected cell cultures in terms of viral replication, cytopathogenicity, and antagonizing effects on host innate immunity. Unexpectedly, the amino acid substitutions had no or minimal effect on viral growth and viral RNA synthesis. However, all the substitutions of charged amino acids resulted in the loss of a counteracting effect against the establishment of an IFN-alpha-mediated anti-viral state. Infection by the virus (Cm2') containing mutations at K77 and D80 induced significant IFN-beta production, severe cytopathic effects, and detectable amounts of viral dsRNA production. In addition to the Cm2' virus, the virus containing mutations at E114 and E115 did not inhibit the poly(I:C)-triggered translocation of cellular IRF-3 to the nucleus. These results suggest that the C protein play important roles in viral escape from induction of IFN-beta and cell death triggered by infection by means of counteracting the pathway leading to activation of IRF-3 as well as of minimizing viral dsRNA production.

The early host response to pathogens is mediated by several distinct pattern recognition receptors. Cytoplasmic RNA helicases including RIG-I and MDA5 have been shown to respond to viral RNA by inducing interferon (IFN) production. Previous in vitro studies have demonstrated a direct role for MDA5 in the response to members of the Picornaviridae, Flaviviridae and Caliciviridae virus families ((+) ssRNA viruses) but not to Paramyxoviridae or Orthomyxoviridae ((-) ssRNA viruses). Contrary to these findings, we now show that MDA5 responds critically to infections caused by Paramyxoviridae in vivo. Using an established model of natural Sendai virus (SeV) infection, we demonstrate that MDA5(-/-) mice exhibit increased morbidity and mortality as well as severe histopathological changes in the lower airways in response to SeV. Moreover, analysis of viral propagation in the lungs of MDA5(-/-) mice reveals enhanced replication and a distinct distribution involving the interstitium. Though the levels of antiviral cytokines were comparable early during SeV infection, type I, II, and III IFN mRNA expression profiles were significantly decreased in MDA5(-/-) mice by day 5 post infection. Taken together, these findings indicate that MDA5 is indispensable for sustained expression of IFN in response to paramyxovirus infection and provide the first evidence of MDA5-dependent containment of in vivo infections caused by (-) sense RNA viruses.