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Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Respiratory diseases constitute a major health problem for ruminants, resulting in considerable economic losses throughout the world. Parainfluenza type 3 virus (PIV3) is one of the most important respiratory pathogens of ruminants. The pathogenicity and phylogenetic analyses of PIV3 virus have been reported in sheep and goats. However, there are no recent studies of the vaccination of sheep or goats against PIV3. Here, we developed a purified inactivated ovine parainfluenza virus type 3 (OPIV3) vaccine candidate. In addition, we immunized sheep with the inactivated OPIV3 vaccine and evaluated the immune response and pathological outcomes associated with OPIV3 TX01 infection. The vaccinated sheep demonstrated no obvious symptoms of respiratory tract infection, and there were no gross lesions or pathological changes in the lungs. The average body weight gain significantly differed between the vaccinated group and the control group (P < 0.01). The serum neutralization antibody levels rapidly increased in sheep post-vaccination and post-challenge with OPIV3. Furthermore, viral shedding in nasal swabs and viral loads in the lungs were reduced. The results of this study suggest that vaccination with this candidate vaccine induces the production of neutralizing antibodies and provides significant protection against OPIV3 infection. These results may be helpful for further studies on prevention and control strategies for OPIV3 infections., (© 2024. The Author(s).)

Asthma is a chronic disease of childhood, but for unknown reasons, disease activity sometimes subsides as children mature. In this study, we present clinical and animal model evidence suggesting that the age dependency of childhood asthma stems from an evolving host response to respiratory viral infection. Using clinical data, we show that societal suppression of respiratory virus transmission during coronavirus disease 2019 lockdown disrupted the traditional age gradient in pediatric asthma exacerbations, connecting the phenomenon of asthma remission to virus exposure. In mice, we show that asthmatic lung pathology triggered by Sendai virus (SeV) or influenza A virus is highly age-sensitive: robust in juvenile mice (4-6 wk old) but attenuated in mature mice (>3 mo old). Interestingly, allergen induction of the same asthmatic traits was less dependent on chronological age than viruses. Age-specific responses to SeV included a juvenile bias toward type 2 airway inflammation that emerged early in infection, whereas mature mice exhibited a more restricted bronchiolar distribution of infection that produced a distinct type 2 low inflammatory cytokine profile. In the basal state, aging produced changes to lung leukocyte burden, including the number and transcriptional landscape of alveolar macrophages (AMs). Importantly, depleting AMs in mature mice restored post-SeV pathology to juvenile levels. Thus, aging influences chronic outcomes of respiratory viral infection through regulation of the AM compartment and type 2 inflammatory responses to viruses. Our data provide insight into how asthma remission might develop in children., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Respiratory syncytial virus (RSV) infection in infancy is associated with increased risk of asthma, except in those with allergic disease at the time of infection. Using house dust mite allergen, we examined the effect of pre-existing atopy on postviral airway disease using Sendai virus in mice, which models RSV infection in humans. Sendai virus drives postviral airway disease in nonatopic mice; however, pre-existing atopy protected against the development of airway disease. This protection depended upon neutrophils, as depletion of neutrophils at the time of infection restored the susceptibility of atopic mice to postviral airway disease. Associated with development of atopy was an increase in polymorphonuclear neutrophil-dendritic cell hybrid cells that develop in Th2 conditions and demonstrated increased viral uptake. Systemic inhibition of IL-4 reversed atopic protection against postviral airway disease, suggesting that increased virus uptake by neutrophils was IL-4 dependent. Finally, human neutrophils from atopic donors were able to reduce RSV infection of human airway epithelial cells in vitro, suggesting these findings could apply to the human. Collectively our data support the idea that pre-existing atopy derives a protective neutrophil response via potential interaction with IL-4, preventing development of postviral airway disease., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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.

Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex class II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in modest worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited compared to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained antigen presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.

Post-translational modifications, including O -GlcNAcylation, play fundamental roles in modulating cellular events, including transcription, signal transduction, and immune signaling. Several molecular targets of O -GlcNAcylation associated with pathogen-induced innate immune responses have been identified; however, the direct regulatory mechanisms linking O -GlcNAcylation with antiviral RIG-I-like receptor signaling are not fully understood. In this study, we found that cellular levels of O -GlcNAcylation decline in response to infection with Sendai virus. We identified a heavily O -GlcNAcylated serine-rich region between amino acids 249-257 of the mitochondrial antiviral signaling protein (MAVS); modification at this site disrupts MAVS aggregation and prevents MAVS-mediated activation and signaling. O -GlcNAcylation of the serine-rich region of MAVS also suppresses its interaction with TRAF3; this prevents IRF3 activation and production of interferon-β. Taken together, these results suggest that O -GlcNAcylation of MAVS may be a master regulatory event that promotes host defense against RNA viruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Seo, Park, Kweon, Kang, Son, Kim, Seo, Kang, Yi, Lee, Kim, Park, Yang and Cho.)

Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro . These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.

Viral pneumonias remain global health threats, as exemplified in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, requiring novel treatment strategies both early and late in the disease process. We have reported that mice treated before or soon after infection with a combination of inhaled Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are broadly protected against microbial pathogens including respiratory viruses, but the mechanisms remain incompletely understood. The objective of this study was to validate strategies for immune modulation in a preclinical model of viral pneumonia and determine their mechanisms. Mice were challenged with the Sendai paramyxovirus in the presence or absence of Pam2-ODN treatment. Virus burden and host immune responses were assessed to elucidate Pam2-ODN mechanisms of action and to identify additional opportunities for therapeutic intervention. Enhanced survival of Sendai virus pneumonia with Pam2-ODN treatment was associated with reductions in lung virus burden and with virus inactivation before internalization. We noted that mortality in sham-treated mice corresponded with CD8 + T-cell lung inflammation on days 11-12 after virus challenge, after the viral burden had declined. Pam2-ODN blocked this injurious inflammation by minimizing virus burden. As an alternative intervention, depleting CD8 + T cells 8 days after viral challenge also decreased mortality. Stimulation of local innate immunity within the lungs by TLR agonists early in disease or suppression of adaptive immunity by systemic CD8 + T-cell depletion late in disease improves outcomes of viral pneumonia in mice. These data reveal opportunities for targeted immunomodulation to protect susceptible human subjects.

Human Parainfluenza Virus-3 (HPIV3) causes severe respiratory illness in immunocompromised patients and lacks approved anti-viral therapies. A phase I study of adoptively transferred virus-specific T-cells (VSTs) targeting HPIV3 following bone marrow transplantation is underway (NCT03180216). We sought to identify immunodominant epitopes within HPIV3 Matrix protein and their cross-reactivity against related viral proteins. VSTs were generated from peripheral blood of healthy donors by ex-vivo expansion after stimulation with a 15-mer peptide library encompassing HPIV3 matrix protein. Epitope mapping was performed using IFN-γ ELIspot with combinatorial peptide pools. Flow cytometry was used to characterize products with intracellular cytokine staining. In 10 VST products tested, we discovered 12 novel immunodominant epitopes. All products recognized an epitope at the C-terminus. On IFN-γ ELISpot, individual peptides eliciting activity demonstrated mean IFN-γ spot forming units per well (SFU)/1x10 5 cells of 115.5 (range 24.5-247.5). VST products were polyfunctional, releasing IFN-γ and TNF-α in response to identified epitopes, which were primarily HLA Class II restricted. Peptides from Human Parainfluenza Virus-1 corresponding to the HPIV3 epitopes showed cross-reactivity for HPIV1 in 11 of 12 tested epitopes (mean cross reactivity index: 1.19). Characterization of HPIV3 epitopes may enable development of third-party VSTs to treat immune suppressed patients with HPIV infection., (Copyright © 2020 Harris, Horn, Grant, Lang, Sani, Jensen-Wachspress, Kankate, Datar, Lazarski, Bollard and Keller.)

The virus-induced signaling adaptor (VISA) complex plays a critical role in the innate immune response to RNA viruses. However, the mechanism of VISA complex formation remains unclear. Here, we demonstrate that thioredoxin 2 (TRX2) interacts with VISA at mitochondria both in vivo and in vitro Knockdown and knockout of TRX2 enhanced the formation of the VISA-associated complex, as well as virus-triggered activation of interferon regulatory factor 3 (IRF3) and transcription of the interferon beta 1 ( IFNB1 ) gene. TRX2 inhibits the formation of VISA aggregates by repressing reactive oxygen species (ROS) production, thereby disrupting the assembly of the VISA complex. Furthermore, our data suggest that the C93 residue of TRX2 is essential for inhibition of VISA aggregation, whereas the C283 residue of VISA is required for VISA aggregation. Collectively, these findings uncover a novel mechanism of TRX2 that negatively regulates VISA complex formation. IMPORTANCE The VISA-associated complex plays pivotal roles in inducing type I interferons (IFNs) and eliciting the innate antiviral response. Many host proteins are identified as VISA-associated-complex proteins, but how VISA complex formation is regulated by host proteins remains enigmatic. We identified the TRX2 protein as an important regulator of VISA complex formation. Knockout of TRX2 increases virus- or poly(I·C)-triggered induction of type I IFNs at the VISA level. Mechanistically, TRX2 inhibits the production of ROS at its C93 site, which impairs VISA aggregates at its C283 site, and subsequently impedes the assembly of the VISA complex. Our findings suggest that TRX2 plays an important role in the regulation of VISA complex assembly., (Copyright © 2020 American Society for Microbiology.)

MicroRNAs (miRNAs) are small noncoding RNAs that suppress the expression of multiple genes and are involved in numerous biologic functions and disorders, including human diseases. Here, we report that two miRNAs, miR-302b and miR-372, target mitochondrial-mediated antiviral innate immunity by regulating mitochondrial dynamics and metabolic demand. Using human cell lines transfected with the synthetic analog of viral dsRNA, poly(I-C), or challenged with Sendai virus, we found that both miRNAs are up-regulated in the cells late after viral infection and ultimately terminate the production of type I interferons and inflammatory cytokines. We found that miR-302b and miR-372 are involved in dynamin-related protein 1 (DRP1)-dependent mitochondrial fragmentation and disrupt mitochondrial metabolism by attenuating solute carrier family 25 member 12 (SLC25A12), a member of the SLC25 family. Neutralizing the effects of the two miRNAs through specific inhibitors re-established the mitochondrial dynamics and the antiviral responses. We found that SLC25A12 contributes to regulating the antiviral response by inducing mitochondrial-related metabolite changes in the organelle. Structure-function analysis indicated that SLC25A12, as part of a prohibitin complex, associates with the mitochondrial antiviral-signaling protein in mitochondria, providing structural insight into the regulation of the mitochondrial-mediated antiviral response. Our results contribute to the understanding of how miRNAs modulate the innate immune response by altering mitochondrial dynamics and metabolic demand. Manipulating the activities of miR-302b and miR-372 may be a potential therapeutic approach to target RNA viruses., (© 2020 Yasukawa et al.)

DEAD-box helicase 3, X-linked (DDX3X) regulates the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated antiviral response, but can also be a host factor contributing to the replication of viruses of significance to human health, such as human immunodeficiency virus type 1 (HIV-1). These roles are mediated in part through its ability to actively shuttle between the nucleus and the cytoplasm to modulate gene expression, although the trafficking mechanisms, and impact thereof on immune signaling and viral infection, are incompletely defined. We confirm that DDX3X nuclear export is mediated by the nuclear transporter exportin-1/CRM1, dependent on an N-terminal, leucine-rich nuclear export signal (NES) and the monomeric guanine nucleotide binding protein Ran in activated GTP-bound form. Transcriptome profiling and ELISA show that exportin-1-dependent export of DDX3X to the cytoplasm strongly impacts IFN-β production and the upregulation of immune genes in response to infection. That this is key to DDX3X's antiviral role was indicated by enhanced infection by human parainfluenza virus-3 (hPIV-3)/elevated virus production when the DDX3X NES was inactivated. Our results highlight a link between nucleocytoplasmic distribution of DDX3X and its role in antiviral immunity, with strong relevance to hPIV-3, as well as other viruses such as HIV-1.

Caprine parainfluenza virus type 3 (CPIV3) is one of the important viral respiratory tract agents in goats. The pathogenicity of CPIV3 has been examined in goats but it has not been explored in other laboratory animals. In the present study, an experimental infection of guinea pigs with CPIV3 was performed. The virus-inoculated guinea pigs displayed clinical signs related to the respiratory disease at 2-12 days post inoculation (dpi). Five infected guinea pigs died during 2 and 7 dpi. Apparent gross pneumonic lesions including consolidation and congestion in one or more lung lobes were observed in necropsied and dead animals. Histo-pathological changes in lungs including expansions of the alveolar interstitium, congestion, macrophage infiltration and compensatory emphysema were also observed. Virus was detectable at 2-10 dpi, 2-10 dpi and 2-7 dpi, as detected by virus isolation, real-time RT-PCR and immunohistochemistry staining, respectively. Viremia was also confirmed after CPIV3 infection during 3-7 dpi. The severe pathological lesions and highest viral load were observed before 7 dpi. Viral specific hemagglutination inhibition and neutralizing antibodies were produced from 7 dpi and 10 dpi, respectively, which related to the clearance of virus. The results present here indicated that guinea pig could be an ideal laboratory animal model for CPIV3 studies in the future., (Copyright © 2019. Published by Elsevier Ltd.)

Transcription factor IRF3-mediated type I interferon induction plays a role in antiviral innate immunity. However, mechanisms for the control and regulation of IRF3 nuclear import remain largely unknown. We have identified DEAD box polypeptide 56 (DDX56) as a negative regulator of virus-triggered IFN-β induction. Overexpression of DDX56 suppressed nuclear translocation of IRF3 via disrupting the IRF3-IOP5 interaction, whereas knockdown or knockout of DDX56 had the opposite effect. In addition, the interaction between DDX56 and IRF3 increased during viral infection. We further found that the D166 site of DDX56 was essential for inhibiting IRF3 import into the nucleus. Our findings suggest that DDX56 regulates antiviral innate immunity by inhibiting the nuclear translocation of IRF3, revealing a novel mechanism of the DDX56-mediated innate antiviral response.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell capacity for IFN signal transduction is presumed to protect against respiratory viral infection. However, it has been difficult to fully test these concepts given the absence of tools to analyze IFN signaling specific to airway epithelial cells in vivo. To address these issues, we generated a new line of transgenic mice with Cre -driver genes ( Foxj1 and Scgb1a1 ) for a floxed- Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1 f/f mice) to target the master IFN signal regulator STAT1 in airway epithelial cells and tested these mice for control of infection because of mouse parainfluenza (Sendai) virus and human enterovirus D68 (EV-D68). Indeed, both types of infections showed increases in viral titers and severity of acute illness in Foxj1-Scgb1a1-Cre-Stat1 f/f mice and conventional Stat1 -/- mice compared with wild-type mice. In concert, the chronic lung disease that develops after Sendai virus infection was also increased in Foxj1-Scgb1a1-Cre-Stat1 f/f and Stat1 -/ - mice, marked by airway and adjacent parenchymal immune cell infiltration and mucus production for at least 7 wk postinfection. Unexpectedly, relatively mild EV-D68 infection also progressed to chronic lung disease in Foxj1-Scgb1a1-Cre-Stat1 f/f and Stat1 -/- mice but was limited (like viral replication) to airways. The results thereby provide proof-of-concept for a critical role of barrier epithelial cells in protection from acute illness and chronic disease after viral infection and suggest a specific role for airway epithelial cells given the limitation of EV-D68 replication and acute and chronic manifestations of disease primarily to airway tissue., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Human parainfluenza virus 3 (PIV3) and respiratory syncytial virus (RSV) are major causative agents of serious respiratory tract illness in newborns and infants. Maternal vaccination could be a promising approach to provide immediate protection against severe PIV3 and RSV infection in young infants. Previously, we demonstrated that maternal immunization with a subunit vaccine consisting of the RSV fusion (F) protein formulated with TriAdj, an adjuvant consisting of poly(I:C), immune defense regulatory peptide and polyphosphazene, protects newborn lambs from RSV. In the present study we evaluated the protective efficacy of a novel bivalent RSV-PIV3 vaccine candidate, F RipSc HN/TriAdj, as a maternal vaccine against PIV3 infection in a neonatal lamb model. This vaccine consists of the pre-fusion form of the RSV F protein linked to the haemagglutinin-neuraminidase (HN) of PIV3, formulated with TriAdj. First, we successfully established PIV3 infection in neonatal lambs. Lambs infected with human PIV3 showed gross pathology, bronchointerstitial pneumonia and viral replication in the lungs. Subsequently, ewes were immunized with F RipSc HN/TriAdj. RSV F RipSc - and PIV3 HN-specific antibodies with virus-neutralizing activity were detected in both the serum and the colostrum of the vaccinated ewes. The newborn lambs had RSV- and PIV3- neutralizing antibodies in their serum, which demonstrates that maternal antibodies were transferred to the neonates. At three days of age, the newborn lambs received an intrapulmonary challenge with PIV3. The lung pathology and virus production were significantly reduced in lambs that had received PIV3-specific maternal antibodies compared to lambs born to non-vaccinated ewes. These results suggest that maternal vaccination with a bivalent F RipSc HN/TriAdj vaccine might be an effective method to provide protection against both PIV3 and RSV in neonates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The response of eosinophils (Eos) to respiratory virus has emerged as an important link between pulmonary infection and allergic asthmatic exacerbations. Eos activate innate immune responses through TLR signaling. In this study, using mouse and human Eos and mice lacking the prolyl isomerase Pin1 selectively in Eos, we show that Pin1 is indispensable for eosinophilopoiesis in the bone marrow (BM) and mature cell function in the presence of TLR7 activation. Unbiased in vivo analysis of mouse models of allergic airway inflammation revealed that TLR7 activation in knockout mice resulted in systemic loss of Eos, reduced IFN production, and an inability to clear respiratory viruses. Consistent with this finding, BM mouse Eos progenitors lacking Pin1 showed markedly reduced cell proliferation and survival after TLR7 activation. Mechanistically, unlike wild-type cells, Pin1 null mouse Eos were defective in the activation of the endoplasmic reticulum stress-induced unfolded protein response. We observed significant reductions in the expression of unfolded protein response components and target genes, aberrant TLR7 cleavage and trafficking, and reduced granule protein production in knockout Eos. Our data strongly suggest that Pin1 is required for BM Eos generation and function during concurrent allergen challenge and viral infection., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization., Competing Interests: The authors declare no conflict of interest.

To suppress virus multiplication, infected macrophages produce NO. However, it remains unclear how infecting viruses then overcome NO challenge. In the present study, we report the effects of accessory protein C from Sendai virus (SeV), a prototypical paramyxovirus, on NO output. We found that in RAW264.7 murine macrophages, a mutant SeV without C protein (4C(-)) significantly enhanced inducible NO synthase (iNOS) expression and subsequent NO production compared to wild type SeV (wtSeV). SeV 4C(-) infection caused marked production of IFN-β, which is involved in induction of iNOS expression via the JAK-STAT pathway. Addition of anti-IFN-β Ab, however, resulted in only marginal suppression of NO production. In contrast, NF-κB, a primarily important factor for transcription of the iNOS gene, was also activated by 4C(-) infection but not wtSeV infection. Induction of NO production and iNOS expression by 4C(-) was significantly suppressed in cells constitutively expressing influenza virus NS1 protein that can sequester double-stranded (ds)RNA, which triggers activation of signaling pathways leading to activation of NF-κB and IRF3. Therefore, C protein appears to suppress NF-κB activation to inhibit iNOS expression and subsequent NO production, possibly by limiting dsRNA generation in the context of viral infection.

Background: Viral respiratory tract infections increase the risk of development and exacerbation of atopic disease. Previously, we demonstrated the requirement for a neutrophil (PMN) subset expressing CD49d to drive development of postviral atopic airway disease in mice., Objective: We sought to determine whether human CD49d + PMNs are present in the nasal mucosa during acute viral respiratory tract infections and further characterize this PMN subset in human subjects and mice., Methods: Sixty subjects (5-50 years old) were enrolled within 4 days of acute onset of upper respiratory symptoms. Nasal lavage for flow cytometry and nasal swabs for viral PCR were performed at enrollment and during convalescence. The Sendai virus mouse model was used to investigate the phenotype and functional relevance of CD49d + PMNs., Results: CD49d + PMN frequency was significantly higher in nasal lavage fluid during acute respiratory symptoms in all subjects (2.9% vs 1.0%, n = 42, P < .001). In mice CD49d + PMNs represented a "proatopic" neutrophil subset that expressed cysteinyl leukotriene receptor 1 (CysLTR1) and produced TNF, CCL2, and CCL5. Inhibition of CysLTR1 signaling in the first days of a viral respiratory tract infection was sufficient to reduce accumulation of CD49d + PMNs in the lungs and development of postviral atopic airway disease. Similar to the mouse, human CD49d + PMNs isolated from nasal lavage fluid during a viral respiratory tract infection expressed CysLTR1., Conclusion: CD49d and CysLTR1-coexpressing PMNs are present during symptoms of an acute viral respiratory tract infection in human subjects. Further study is needed to examine selective targeting of proatopic neutrophils as a potential therapeutic strategy to prevent development of postviral atopic airway disease., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) are major causes of serious lower respiratory tract disease in infants. Currently there is no licensed vaccine against RSV or PIV3. To make an effective bivalent subunit vaccine, a chimeric truncated F R HN protein containing the N-terminal ectodomain of the RSV fusion (F) protein linked to the C-terminal ectodomain of the PIV3 haemagglutinin-neuraminidase (HN) protein was produced in HEK293T cells. Mice, cotton rats and hamsters were immunized intramuscularly (IM) with both RSV F and PIV3 HN (F R +HN) or F R HN, formulated with TriAdj, which consists of poly(I:C), innate defense regulator peptide and poly[di(sodium carboxylatoethylphenoxy)]-phosphazene. Both formulations were immunogenic and elicited full protection from RSV; however, animals vaccinated with F R HN/TriAdj were significantly better protected from PIV3 than animals vaccinated with F R +HN/TriAdj. To develop a potentially more effective subunit vaccine, a chimeric glycoprotein (F RipSc HN), encoding the RSV F ectodomain stabilized in the pre-fusion form linked to PIV3 HN was generated. Intramuscular vaccination with F RipSc HN/TriAdj induced virus neutralizing antibodies followed by complete protection from RSV and PIV3 replication in the lungs of challenged cotton rats. Furthermore, intranasal vaccination with F RipSc HN/TriAdj significantly reduced both RSV and PIV3 replication in cotton rats. Mucosal immunization with F RipSc HN/TriAdj also elicited strong antigen-specific mucosal and systemic immune responses in a lamb model. In conclusion, the chimeric F RipSc HN protein combined with TriAdj has potential for development of a safe, effective, bivalent vaccine against both RSV and PIV3., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Host recognition of intracellular viral RNA and subsequent induction of cytokine signaling are tightly regulated at the cellular level and are a target for manipulation by viruses and therapeutics alike. Here, we characterize chromosome 6 ORF 106 (C6orf106) as an evolutionarily conserved inhibitor of the innate antiviral response. C6orf106 suppresses the synthesis of interferon (IFN)-α/β and proinflammatory tumor necrosis factor (TNF) α in response to the dsRNA mimic poly(I:C) and to Sendai virus infection. Unlike canonical inhibitors of antiviral signaling, C6orf106 blocks interferon-regulatory factor 3 (IRF3) and, to a lesser extent, NF-κB activity without modulating their activation, nuclear translocation, cellular expression, or degradation. Instead, C6orf106 interacts with IRF3 and inhibits IRF3 recruitment to type I IFN promoter sequences while also reducing the nuclear levels of the coactivator proteins p300 and CREB-binding protein (CBP). In summary, we have defined C6orf106 as a negative regulator of antiviral immunity that blocks IRF3-dependent cytokine production via a noncanonical and poorly defined mechanism. This work presents intriguing implications for antiviral immunity, autoimmune disorders, and cancer., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Although much is known about the mechanisms by which pathogen recognition drives the initiation of T cell responses, including those to respiratory viruses, the role of pathogen recognition in fate decisions of T cells once they have become effectors remains poorly defined. Here, we review our recent studies that suggest that the generation of CD4 T cell memory is determined by recognition of virus at an effector "checkpoint." We propose this is also true of more highly differentiated tissue-restricted effector cells, including cytotoxic "ThCTL" in the site of infection and T FH in secondary lymphoid organs. We point out that ThCTL are key contributors to direct viral clearance and T FH to effective Ab response, suggesting that the most protective immunity to influenza, and by analogy to other respiratory viruses, requires prolonged exposure to antigen and to infection-associated signals. We point out that many vaccines used today do not provide such prolonged signals and suggest this contributes to their limited effectiveness. We also discuss how aging impacts effective CD4 T cell responses and how new insights about the response of aged naive CD4 T cells and B cells might hold implications for effective vaccine design for both the young and aged against respiratory viruses.

Background: Alloimmune-mediated lung syndromes (allo-LSs) are life-threatening complications after hematopoietic cell transplantation (HCT). Respiratory virus (RV) has been suggested to play a role in the pathogenesis., Objective: We studied the relation between RV DNA/RNA detection in the upper/lower airways before HCT and the occurrence of allo-LSs., Methods: We retrospectively analyzed all HCT recipients between 2004 and 2014, in whom real-time PCR for RV was performed in nasopharyngeal aspirates (NPAs) and bronchoalveolar lavage (BAL) fluid before HCT. The main outcome of interest was the presence of an allo-LS, which was defined as idiopathic pneumonia syndrome or bronchiolitis obliterans syndrome. Other outcomes were overall survival and treatment-related mortality. We used Cox proportional hazard models, logistic regression models, and Fine-Gray competing risk regression for analyses., Results: One hundred seventy-nine children (median age, 6.8 years) were included. RVs were found in 61% (41% in BAL fluid/NPAs and 20% in NPAs only). Rhinovirus was the most frequently detected RV (42%). Allo-LSs occurred in 13%. RV positivity in BAL fluid was a predictor for allo-LSs (hazard ratio, 3.8; 95% CI, 1.4-10.7; P = .01), whereas RV positivity in NPAs only was not. No other predictors were found. Grade II to IV acute graft-versus-host disease related to steroid treatment shows a trend toward a protective effect (odds ratio, 0.16; 95% CI, 0.0-1.3; P = .08). Allo-LSs significantly increased treatment-related mortality (52% ± 10% in allo-LSs and 20% ± 4% in non-allo-LSs, P = .007)., Conclusions: These results show that pre-HCT BAL fluid RV positivity was a predictor for allo-LSs. Screening for RVs before HCT might identify patients at risk for allo-LSs. This could have implications for prevention and treatment and might subsequently influence the outcomes of HCT., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

TBK1, STING, and MDA5 are important players within the antiviral innate immune response network. We mapped the interactome of endogenous TBK1, STING, and MDA5 by affinity enrichment MS in virally infected or uninfected THP-1 cells. Based on quantitative data of more than 2000 proteins and stringent statistical analysis, 58 proteins were identified as high-confidence interactors for at least one of three bait proteins. Our data indicated that TBK1 and MDA5 mostly interacted within preexisting protein networks, while STING interacted with different proteins with different viral infections. Functional analysis was performed on 17 interactors, and six were found to have functions in innate immune responses. We identified TTC4 as a TBK1 interactor and positive regulator of sendai virus-induced innate immunity., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Patients with asthma experience circadian variations in their symptoms. However it remains unclear how specific aspects of this common airway disease relate to clock genes, which are critical to the generation of circadian rhythms in mammals. Here, we used a viral model of acute and chronic airway disease to examine how circadian clock disruption affects asthmatic lung phenotypes. Deletion of the core clock gene bmal1 or environmental disruption of circadian function by jet lag exacerbated acute viral bronchiolitis caused by Sendai virus (SeV) and influenza A virus in mice. Post-natal deletion of bmal1 was sufficient to trigger increased SeV susceptibility and correlated with impaired control of viral replication. Importantly, bmal1 -/- mice developed much more extensive asthma-like airway changes post infection, including mucus production and increased airway resistance. In human airway samples from two asthma cohorts, we observed altered expression patterns of multiple clock genes. Our results suggest a role for bmal1 in the development of asthmatic airway disease via the regulation of lung antiviral responses to common viral triggers of asthma.

Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus-infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1 Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions., (Copyright © 2017 by The American Association of Immunologists, Inc.)

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.

Parainfluenza virus type 3 (PIV3) infections are a major cause of morbidity and mortality in immunocompromised individuals, with no approved therapies. Our group has demonstrated the safety and efficacy of adoptively transferred virus-specific T cells for the prevention and treatment of a broad range of viral infections including BK virus, cytomegalovirus, adenovirus, human herpesvirus 6, and Epstein-Barr virus. However, this approach is restricted to well-characterized viruses with known immunogenic/protective T-cell target antigens, precluding extension to PIV3. We now characterize the cellular immune response to all 7 PIV3-encoded antigens in 17 healthy donors and define a hierarchy of immunogenicity based on the frequency of responding donors and the magnitude of specific cells. We show that reactive populations of both CD4+ and CD8+ T cells are capable of producing Th1-polarized effector cytokines and killing PIV3-expressing targets. Furthermore, we confirm the clinical relevance of these cells by demonstrating a direct correlation between the presence of PIV3-specific T cells and viral control in allogeneic hematopoietic stem cell transplant recipients. Taken together, our findings support the clinical use of PIV3-specific T cells produced with our Good Manufacturing Practice-compliant manufacturing process, in immunocompromised patients with uncontrolled infections., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Introduction: Asthma is major health burden throughout the world, and there are no therapies that have been shown to be able to prevent the development of disease. A severe respiratory paramyxoviral infection early in life has been demonstrated to greatly increase the risk of developing asthma. We have a mouse model of a severe respiratory paramyxoviral infection (Sendai virus, SeV) that mimics human disease, and requires early expression of the cytokine CCL28 to drive the development of post-viral airway disease. The known receptors for CCL28 are CCR3 and CCR10. However, it is not known if blockade of these receptors will prevent the development of post-viral airway disease. The objective of this study was to determine if treatment with a protein epitope mimetic antagonist of CCR10, POL7085, will provide sufficient protection against the development of post-viral airway disease., Methods: C57BL6 mice were inoculated with SeV or UV inactivated SeV. From day 3-19 post inoculation (PI), mice were subcutaneously administered daily POL7085 or saline, or every other day anti-CCL28 mAb. On days 8, 10, and 12 PI bronchoalveolar cytokines, serum IgE, and lung cellular constituents were measured. At day 21 PI airway hyper-reactivity to methacholine and mucous cell metaplasia was measured., Results: Treatment with either anti-CCL28 or POL7085 significantly reduced development of airway hyper-reactivity and mucous cell metaplasia following SeV infection. The prevention of post-viral airway disease was associated with early reductions in innate immune cells, but did not appear to be due to a reduction in IL-13 or IgE., Conclusions: Blockade of CCL28 or CCR10 during an acute severe respiratory paramyxoviral infection is sufficient to prevent the development of post-viral airway disease. However, the mechanism of action is unclear and requires further exploration., (© 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

BMAL1 (brain and muscle ARNT-like protein 1, also known as MOP3 or ARNT3) belongs to the family of the basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors, and is a key component of the molecular oscillator that generates circadian rhythms. Here, we report that BMAL1-deficient cells are significantly more susceptible to infection by two major respiratory viruses of the Paramyxoviridae family, namely RSV and PIV3. Embryonic fibroblasts from Bmal1 -/- mice produced nearly 10-fold more progeny virus than their wild type controls. These results were supported by animal studies whereby pulmonary infection of RSV produced a more severe disease and morbidity in Bmal1 -/- mice. These results show that BMAL1 can regulate cellular innate immunity against specific RNA viruses.

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.

The aim of this study was to assess the immune response to parainfluenza virus type 3 (PIV3), rhinovirus 1B (RV1B) and intracellular Toll-like receptors (TLR) agonists in nasal epithelial cells (NECs) from patients with allergic rhinitis and healthy controls. NECs were obtained from eight patients with allergic rhinitis (AR) and 11 non-atopic healthy controls (HC) by nasal scraping, grown to confluence and exposed to PIV3, RV1B infection or TLR-3 and TLR-7/8 agonists. Interferon (IFN)-λ1, IFN-α, IFN-β and regulated on activation, normal T expressed and secreted (RANTES) release into the cell culture supernatants was assessed at 8, 24 and 48 h upon infection or 8 and 24 h after stimulation with poly(I:C) and R848. mRNA levels of IFNs, RANTES, interferon regulatory transcription factor (IRF)3, IRF7 and viral gene copy number were determined using real-time polymerase chain reaction (RT-PCR). PIV3 but not RV1B replication 48 h after infection was significantly lower (P < 0·01) in NECs from AR patients compared to HC. PIV3 infection induced significantly less IFN-λ1 (both protein and mRNA) in NECs from AR compared to HC. IFN-β mRNA expression and RANTES protein release and mRNA expression tended to be smaller in AR compared HC cells in response to both viruses. Stimulation with TLR-3 agonist [poly (I:C)] induced similar IFN-λ1 and RANTES generation in AR and HC subjects. Viral infections in NECs induced IRF7 expression, which correlated with IFN and RANTES expression. These data suggest that virus proliferation rates and the immune response profile are different in nasal epithelial cells from patients with allergic rhinitis compared to healthy individuals., (© 2016 British Society for Immunology.)

How tissue-specific anatomical distribution and phenotypic specialization are linked to protective efficacy of memory T cells against reinfection is unclear. Here, we show that lung environmental cues program recently recruited central-like memory cells with migratory potentials for their tissue-specific functions during lethal respiratory virus infection. After entering the lung, some central-like cells retain their original CD27 hi CXCR3 hi phenotype, enabling them to localize near the infected bronchiolar epithelium and airway lumen to function as the first line of defense against pathogen encounter. Others, in response to local cytokine triggers, undergo a secondary program of differentiation that leads to the loss of CXCR3, migration arrest, and clustering within peribronchoarterial areas and in interalveolar septa. Here, the immune system adapts its response to prevent systemic viral dissemination and mortality. These results reveal the striking and unexpected spatial organization of central- versus effector-like memory cells within the lung and how cooperation between these two subsets contributes to host defense., (© 2016 Abboud et al.)

Background Aims: Human parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients and currently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients but requires determination of T-cell antigens on targeted viruses., Methods: HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by interferon (IFN)-γ ELISpot, intracellular cytokine staining and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51 Cr release and CD107a mobilization assays. Virus-specific T cells targeting six viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T cells was determined by immunomagnetic sorting for IFN-γ-producing cells., Results: HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T cells showed a CD4 + predominance (mean CD4:CD8 ratio 2.89) and demonstrated specificity for multiple HPIV3 antigens. The expanded T cells were polyfunctional based on cytokine production but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3 + /CD45RA - /CCR7 - ) in responder cells., Discussion: HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4 + T cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of six-virus T cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune-compromised patients., Competing Interests: COI Statement: The authors have no relevant conflicts of interest., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Sendai virus (SeV), a murine paramyxovirus, has been used to study the induction of type I interferon (IFN) subtypes in robust quantities. Few studies have measured whether the IFN that SeV induces actually fulfills its intended purpose of interfering with virus-mediated effects in the cells in which it is produced. We determined the effects of IFN on SeV-mediated cytopathic effects (CPE) and the ability of IFN to protect against virus infection. SeV-induced biologically active IFN resulted in Jak/STAT activation and the production of a number of interferon-stimulated genes (ISGs). However, these responses did not inhibit SeV replication or CPE. This observation was not due to SeV effects on canonical IFN signaling. Furthermore, pretreating cells with type I IFN and establishing an antiviral state before infection did not mediate SeV effects. Therefore, the induction of canonical IFN signaling pathways and ISGs does not always confer protection against the IFN-inducing virus. Because type I IFNs are approved to treat various infections, our findings suggest that typical markers of IFN activity may not be indicative of a protective antiviral response and should not be used alone to determine whether an antiviral state against a particular virus is achieved., Competing Interests: Author Disclosure Statement No competing financial interests exist.

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.

Recognition of viral dsRNA by the retinoic acid-inducible gene-1-like receptors (RLRs) triggers signaling cascades that lead to activation of the TBK1 kinase and transcription factor IFN regulatory factor 3, induction of downstream antiviral genes, and innate antiviral responses. In this study, we identified muscle segment homeobox1 (MSX1) as an important modulator of RLR-mediated signaling pathways. Knockdown or knockout of MSX1 significantly impaired Sendai virus-triggered activation of TBK1 and IFN regulatory factor 3, induction of downstream antiviral genes, and cellular antiviral responses. Interestingly, MSX1 was translocated from the nucleus to cytoplasm, particularly mitochondria upon infection of Sendai virus. Biochemcially, MSX1 was important for assembly of TBK1/IKK-related kinase-associated protein 1/TNFR-associated factor-associated NF-κB activator complexes. Our results suggest that MSX1 is an important component of RLR-mediated signaling and reveal mechanisms on innate immune responses against RNA viruses., (Copyright © 2016 by The American Association of Immunologists, Inc.)

More than one third of patients with chronic lung disease undergoing lung transplantation have pre-existing Abs against lung-restricted self-Ags, collagen type V (ColV), and k-α1 tubulin (KAT). These Abs can also develop de novo after lung transplantation and mediate allograft rejection. However, the mechanisms leading to lung-restricted autoimmunity remain unknown. Because these self-Ags are normally sequestered, tissue injury is required to expose them to the immune system. We previously showed that respiratory viruses can induce apoptosis in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), the key mediators of self-tolerance. Therefore, we hypothesized that lung-tissue injury can lead to lung-restricted immunity if it occurs in a setting when Tregs are impaired. We found that human lung recipients who suffer respiratory viral infections experienced a decrease in peripheral Tregs. Pre-existing lung allograft injury from donor-directed Abs or gastroesophageal reflux led to new ColV and KAT Abs post respiratory viral infection. Similarly, murine parainfluenza (Sendai) respiratory viral infection caused a decrease in Tregs. Intratracheal instillation of anti-MHC class I Abs, but not isotype control, followed by murine Sendai virus infection led to development of Abs against ColV and KAT, but not collagen type II (ColII), a cartilaginous protein. This was associated with expansion of IFN-γ-producing CD4(+) T cells specific to ColV and KAT, but not ColII. Intratracheal anti-MHC class I Abs or hydrochloric acid in Foxp3-DTR mice induced ColV and KAT, but not ColII, immunity, only if Tregs were depleted using diphtheria toxin. We conclude that tissue injury combined with loss of Tregs can lead to lung-tissue-restricted immunity., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Long noncoding RNAs (lncRNAs) can regulate gene expression in a cell-specific fashion during development. Here, we identify a novel lncRNA from the X chromosome that we named lncRHOXF1 and which is abundantly expressed in trophectoderm and primitive endoderm cells of human blastocyst-stage embryos. lncRHOXF1 is a spliced and polyadenylated lncRNA about 1 kb in length that is found in both the nuclear and cytoplasmic compartments of in vitro differentiated human trophectoderm progenitor cells. Gain-of-function experiments in human embryonic stem cells, which normally lack lncRHOXF1 RNA, revealed that lncRHOXF1 reduced proliferation and favored cellular differentiation. lncRHOXF1 knockdown using small interfering RNAs (siRNAs) in human trophectoderm progenitors increased expression of viral response genes, including type I interferon. Sendai virus infection of human trophectoderm progenitor cells increased lncRHOXF1 RNA levels, and siRNA-mediated disruption of lncRHOXF1 during infection reduced the expression of viral response genes leading to higher virus replication. Thus, lncRHOXF1 RNA is the first example of a lncRNA that regulates the host response to viral infections in human placental progenitor cells, and we propose that it functions as a repressor of the viral response during early human development., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The World Health Organization (WHO) estimates that 250 million children under the age of five suffer from vitamin A deficiencies (VAD). Individuals with VAD experience higher rates of mortality and increased morbidity during enteric and respiratory infections compared with those who are vitamin A sufficient. Previously, our laboratory has demonstrated that VAD mice have significantly impaired virus-specific IgA and CD8(+) T-cell responses in the airways. Here, we demonstrate that VAD mice experience enhanced cytokine/chemokine gene expression and release in the respiratory tract 10 days following virus infection compared with control vitamin A sufficient animals. Cytokines/chemokines that are reproducibly up-regulated at the gene expression and protein levels include IFNγ and IL-6. Despite previous indications that cytokine dysregulation in VAD animals might reflect low forkhead box P3 (FoxP3)-positive regulatory T-cell frequencies, we found no reduction in FoxP3(+) T cells in VAD respiratory tissues. As an alternative explanation for the high cytokine levels, we found that the extent of virus infection and the persistence of viral antigens were increased on day 10 post-infection in VAD animals compared with controls, and consequently that respiratory tract tissues had an increased potential to activate virus-specific T cells. Results encourage cautious management of viral infections in patients with VAD, as efforts to enhance FoxP3(+) T cell frequencies and quell immune effectors could potentially exacerbate disease if the virus has not been cleared., (© The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-β production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.

The mitochondrial anti-viral signaling protein (MAVS) plays an important role in the type I IFN response. In this study, two feline MAVS transcripts were cloned. Both transcripts have the same open reading frame encoding 523 amino acids. The putative protein shares 76.6 % similarity with canine and exhibits similarity to human, mouse, rat, bovine and porcine, ranging from 46.1 to 65.8 %. Deletion mutant analysis indicated that the transmembrane (TM) domain is necessary for localization in the mitochondrial membrane, and both the caspase activation and recruitment domain and TM domain are indispensible for activating the IFN-β response. Additionally, Sendai virus-induced IFN-β promoter activation was significantly inhibited by siRNA targeting MAVS. Finally, miniMAVS, a second protein encoded by MAVS mRNA, was identified, which interfered with the IFN-β response via the inhibition of NF-κB activation. The identification of MAVS will promote the understanding and control of feline infectious diseases.

Unlabelled: Type I interferons (IFNs) are induced upon viral infection and important mediators of innate immunity. While there is 1 beta interferon (IFN-β) protein, there are 12 different IFN-α subtypes. It has been reported extensively that different viruses induce distinct patterns of IFN subtypes, but it has not been previously shown how the viral multiplicity of infection (MOI) can affect IFN induction. In this study, we discovered the novel finding that human U937 cells infected with 2 different concentrations of Sendai virus (SeV) induce 2 distinct type I IFN subtype profiles. Cells infected at the lower MOI induced more subtypes than cells infected at the higher MOI. We found that this was due to the extent of signaling through the IFN receptor (IFNAR). The cells infected at the lower viral MOI induced the IFNAR2-dependent IFN-α subtypes 4, 6, 7, 10, and 17, which were not induced in cells infected at higher virus concentrations. IFN-β and IFN-α1, -2, and -8 were induced in an IFNAR-independent manner in cells infected at both virus concentrations. IFN-α5, -14, -16, and -21 were induced in an IFNAR-dependent manner in cells infected at lower virus concentrations and in an IFNAR-independent manner in cells infected at higher virus concentrations. These differences in IFN subtype profiles in the 2 virus concentrations also resulted in distinct interferon-stimulated gene induction. These results present the novel finding that different viral MOIs differentially activate JAK/STAT signaling through the IFNAR, which greatly affects the profile of IFN subtypes that are induced., Importance: Type I IFNs are pleiotropic cytokines that are instrumental in combating viral diseases. Understanding how the individual subtypes are induced is important in developing strategies to block viral replication. Many studies have reported that different viruses induce distinct type I IFN subtype profiles due to differences in the way viruses are sensed in different cell types. However, we report in our study the novel finding that the amount of virus used to infect a system can also affect which type I IFN subtypes are induced due to the extent of activation of certain signaling pathways. These distinct IFN subtype profiles in cells infected at different MOIs are correlated with differences in interferon-stimulated gene induction, indicating that the same virus can induce distinct antiviral responses depending on the MOI. Because type I IFNs are used as therapeutic agents to treat viral diseases, understanding their antiviral mechanisms can enhance clinical treatments., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The effective recognition of viral infection and subsequent type I IFN production is essential for the host antiviral innate immune responses. The phosphorylation and activation of kinase TANK-binding kinase 1 (TBK1) plays crucial roles in the production of type I IFN mediated by TLR and retinoic acid-inducible gene I-like receptors. Type I IFN expression must be tightly regulated to prevent the development of immunopathological disorders. However, how the activated TBK1 is negatively regulated by phosphatases remains poorly understood. In this study, we identified a previously unknown role of protein phosphatase (PP)4 by acting as a TBK1 phosphatase. PP4 expression was upregulated in macrophages infected with RNA virus, vesicular stomatitis virus, and Sendai virus in vitro and in vivo. Knockdown of PP4C, the catalytic subunit of PP4, significantly increased type I IFN production in macrophages and dentritic cells triggered by TLR3/4 ligands, vesicular stomatitis virus, and Sendai virus, and thus inhibited virus replication. Similar results were also found in peritoneal macrophages with PP4C silencing in vivo and i.p. infection of RNA virus. Accordingly, ectopic expression of PP4C inhibited virus-induced type I IFN production and promoted virus replication. However, overexpression of a phosphatase-dead PP4C mutant abolished the inhibitory effects of wild-type PP4C on type I IFN production. Mechanistically, PP4 directly bound TBK1 upon virus infection, then dephosphorylated TBK1 at Ser(172) and inhibited TBK1 activation, and subsequently restrained IFN regulatory factor 3 activation, resulting in suppressed production of type I IFN and IFN-stimulated genes. Thus, serine/threonine phosphatase PP4 functions as a novel feedback negative regulator of RNA virus-triggered innate immunity., (Copyright © 2015 by The American Association of Immunologists, Inc.)