Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses.

Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.

Non-respiratory particles emitted by guinea pigs in airborne disease transmission experiments.

Animal models are often used to assess the airborne transmissibility of various pathogens, which are typically assumed to be carried by expiratory droplets emitted directly from the respiratory tract of the infected animal. We recently established that influenza virus is also transmissible via "aerosolized fomites," micron-scale dust particulates released from virus-contaminated surfaces (Asadi et al. in Nat Commun 11(1):4062, 2020). Here we expand on this observation, by counting and characterizing the particles emitted from guinea pig cages using an Aerodynamic Particle Sizer (APS) and an Interferometric Mie Imaging (IMI) system. Of over 9000 airborne particles emitted from guinea pig cages and directly imaged with IMI, none had an interference pattern indicative of a liquid droplet. Separate measurements of the particle count using the APS indicate that particle concentrations spike upwards immediately following animal motion, then decay exponentially with a time constant commensurate with the air exchange rate in the cage. Taken together, the results presented here raise the possibility that a non-negligible fraction of airborne influenza transmission events between guinea pigs occurs via aerosolized fomites rather than respiratory droplets, though the relative frequencies of these two routes have yet to be definitively determined.

Key Amino Acids of M1-41 and M2-27 Determine Growth and Pathogenicity of Chimeric H17 Bat Influenza Virus in Cells and in Mice.

Based on our previous studies, we show that the M gene is critical for the replication and pathogenicity of the chimeric H17 bat influenza virus (Bat09:mH1mN1) by replacing the bat M gene with those from human and swine influenza A viruses. However, the key amino acids of the M1 and/or M2 proteins that are responsible for virus replication and pathogenicity remain unknown. In this study, replacement of the PR8 M gene with the Eurasian avian-like M gene from the A/California/04/2009 pandemic H1N1 virus significantly decreased viral replication in both mammalian and avian cells in the background of the chimeric H17 bat influenza virus. Further studies revealed that M1 was more crucial for viral growth and pathogenicity than M2 and that the amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins identified in this study might be important for influenza virus surveillance and could be used to produce live attenuated vaccines in the future. IMPORTANCE The M1 and M2 proteins influence the morphology, replication, virulence, and transmissibility of influenza viruses. Although a few key residues in the M1 and M2 proteins have been identified, whether other residues of the M1 and M2 proteins are involved in viral replication and pathogenicity remains to be discovered. In the background of the chimeric H17 bat influenza virus, the Eurasian avian-like M gene from the A/California/04/2009 virus significantly decreased viral growth in mammalian and avian cells. Further study showed that M1 was implicated more than M2 in viral growth and pathogenicity in vitro and in vivo and that the key amino acid residues M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of the M1 and M2 proteins could be used for influenza virus surveillance and live attenuated vaccine applications in the future. These findings provide important contributions to knowledge of the genetic basis of the virulence of influenza viruses.

CACO-2 cells: A continuous cell line with sensitive and broad-spectrum utility for respiratory virus culture.

BACKGROUND: Primary rhesus monkey kidney cells (RhMK) can be used for the detection of respiratory viruses, including influenza and parainfluenza. The human colon adeno-carcinoma cell line, CACO-2, has been previously used for the growth of multiple influenza viruses, including seasonal, novel and avian lineages. OBJECTIVE: We compared CACO-2, Madin-Darby Canine Kidney (MDCK), and RhMK cells for the isolation of viruses from patients presenting with influenza like-illness (ILI). STUDY DESIGN: Nasopharyngeal specimens from patients with ILI in primary care settings were processed for conventional viral culture in MDCK, RhMK, and CACO-2. Cells were examined microscopically for cytopathic effect (CPE) and confirmatory testing included immunofluorescent antigen (IFA) detection and real-time RT-PCR. Additionally, 16 specimens positive for respiratory syncytial virus (RSV) by PCR were inoculated on CACO-2 cells. Statistical analysis was done using Chi-square test with IBM Statistical Program. RESULTS: Of 1031 respiratory specimens inoculated, viruses were isolated and confirmed from 331 (32.1 %) in MDCK cells, 304 (29.5 %) in RhMk cells, and 433 (42.0 %) in CACO-2 cells. These included influenza A/(H1N1)pdm09, influenza A(H3N2), influenza B, parainfluenza virus (PIV) types 1, 2, and 3, human coronavirus 229E (CoV-229E), human adenovirus (HAdV), herpes simplex virus 1 (HSV 1), and enterovirus (EV). Influenza A viruses grew best in the CACO-2 cell line. Time to observation of CPE was similar for all three cell types but unlike RhMK and MDCK cells, virus-specific morphological changes were indistinguishable in CACO-2 cells. None of the 16 specimens positive for RSV by PCR grew on CACO-2 cells. CONCLUSIONS: The overall respiratory virus culture isolation rate in CACO-2 cells was significantly higher than that in RhMK or MDCK cells (p < 0.05). CACO-2 cells also supported the growth of some viruses that did not grow in either RhMK or MDCK cells. Except for RSV, CACO-2 cells provide a worthwhile addition to culture algorithms for respiratory specimens.

Functional design of glycan-conjugated molecules using a chemoenzymatic approach.

Carbohydrates play important and diverse roles in the fundamental processes of life. We have established a method for accurately and a large-scale synthesis of functional carbohydrates with diverse properties using a unique enzymatic method. Furthermore, various artificial glycan-conjugated molecules have been developed by adding these synthetic carbohydrates to macromolecules and to middle- and low-molecular-weight molecules with different properties. These glycan-conjugated molecules have biological activities comparable to or higher than those of natural compounds and present unique functions. In this review, several synthetic glycan-conjugated molecules are taken as examples to show design, synthesis, and function.

Growth capability of epidemic influenza viruses in Japan since the 2009 H1N1 pandemic.

The correlation of viral growth capability (n = 156) with the viral load in nasopharyngeal swabs (n = 76) was assessed. Epidemic influenza A/H1N1, A/H3N2, and B viruses showed a wide range of growth capability (104-1011 copies/mL) in Madin-Darby canine kidney cells. The growth was correlated with the nasopharyngeal viral load (r = 0.53). Six selected strains showed growth-dependent cell death (r = 0.96) in a growth kinetics assay. Epidemic influenza viruses exhibit a wide range of growth capability. Growth capability should be considered one of the key factors in disease prognosis.

The Two Sides of the Same Coin-Influenza Virus and Intracellular Signal Transduction.

Cells respond to extracellular agents by activation of intracellular signaling pathways. Viruses can be regarded as such agents, leading to a firework of signaling inside the cell, primarily induced by pathogen-associated molecular patterns (PAMPs) that provoke safeguard mechanisms to defend from the invader. In the constant arms race between pathogen and cellular defense, viruses not only have evolved mechanisms to suppress or misuse supposedly antiviral signaling processes for their own benefit but also actively induce signaling to promote replication. This creates viral dependencies that may be exploited for novel strategies of antiviral intervention. Here, we will summarize the current knowledge of activation and function of influenza virus-induced signaling pathways with a focus on nuclear factor (NF)-κB signaling, mitogen-activated protein kinase cascades, and the phosphatidylinositol-3-kinase pathway. We will discuss the opportunities and drawbacks of targeting these signaling pathways for antiviral intervention.

Lower respiratory tract infection in the community: associations between viral aetiology and illness course.

OBJECTIVES: This study determined associations between respiratory viruses and subsequent illness course in primary care adult patients presenting with acute cough and/or suspected lower respiratory tract infection. METHODS: A prospective European primary care study recruited adults with symptoms of lower respiratory tract infection between November 2007 and April 2010. Real-time in-house polymerase chain reaction (PCR) was performed to test for six common respiratory viruses. In this secondary analysis, symptom severity (scored 1 = no problem, 2 = mild, 3 = moderate, 4 = severe) and symptom duration were compared between groups with different viral aetiologies using regression and Cox proportional hazard models, respectively. Additionally, associations between baseline viral load (cycle threshold (Ct) value) and illness course were assessed. RESULTS: The PCR tested positive for a common respiratory virus in 1354 of the 2957 (45.8%) included patients. The overall mean symptom score at presentation was 2.09 (95% confidence interval (CI) 2.07-2.11) and the median duration until resolution of moderately bad or severe symptoms was 8.70 days (interquartile range 4.50-11.00). Patients with influenza virus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), coronavirus (CoV) or rhinovirus had a significantly higher symptom score than patients with no virus isolated (0.07-0.25 points or 2.3-8.3% higher symptom score). Time to symptom resolution was longer in RSV infections (adjusted hazard ratio (AHR) 0.80, 95% CI 0.65-0.96) and hMPV infections (AHR 0.77, 95% CI 0.62-0.94) than in infections with no virus isolated. Overall, baseline viral load was associated with symptom severity (difference 0.11, 95% CI 0.06-0.16 per 10 cycles decrease in Ct value), but not with symptom duration. CONCLUSIONS: In healthy, working adults from the general community presenting at the general practitioner with acute cough and/or suspected lower respiratory tract infection other than influenza impose an illness burden comparable to influenza. Hence, the public health focus for viral respiratory tract infections should be broadened.

The past, present and future of RNA respiratory viruses: influenza and coronaviruses.

Influenza virus and coronaviruses continue to cause pandemics across the globe. We now have a greater understanding of their functions. Unfortunately, the number of drugs in our armory to defend us against them is inadequate. This may require us to think about what mechanisms to address. Here, we review the biological properties of these viruses, their genetic evolution and antiviral therapies that can be used or have been attempted. We will describe several classes of drugs such as serine protease inhibitors, heparin, heparan sulfate receptor inhibitors, chelating agents, immunomodulators and many others. We also briefly describe some of the drug repurposing efforts that have taken place in an effort to rapidly identify molecules to treat patients with COVID-19. While we put a heavy emphasis on the past and present efforts, we also provide some thoughts about what we need to do to prepare for respiratory viral threats in the future.

Oseltamivir analogs with potent anti-influenza virus activity.

Influenza A and B viruses cause seasonal worldwide influenza epidemics each winter, and are a major public health concern and cause of morbidity and mortality. A substantial reduction in influenza-related deaths can be attributed to both vaccination and administration of oseltamivir (OS), which is approved for oral administration and inhibits viral neuraminidase (NA), a transmembrane protein. OS carboxylate (OSC), the active form of OS, is formed by the action of endogenous esterase, which targets NA and is shown to significantly reduce influenza-related deaths. However, the development of resistance in various viral variants, including H3N2 and H5N1, has raised concern about the effectiveness of OS. This comprehensive review covers a range of OS analogs shown to be effective against influenza virus, comparing different types of substituent group that contribute to the activity and bioavailability of these compounds.

Carryover effects of baloxavir acid in human nasopharyngeal/pharyngeal swabs on infectious titer testing of influenza virus.

Baloxavir marboxil (BXM) demonstrated a rapid and profound decline in infectious viral titer 1 day after BXM administration. Rapid reduction in virus titer is a characteristic of BXM. There may be a possibility that drug carryover effects have impacts on the observed antiviral effects due to the poor correlation that was observed between viral titer reduction and alleviation of influenza symptoms. Here, we report possible carryover effects of baloxavir acid (BXA), an active form of BXM, on infectious titer testing. Our findings indicate that there is little impact of BXA carryover on the infectious titer testing.

Comparison of suspension MDCK cells, adherent MDCK cells, and LLC-MK2 cells for selective isolation of influenza viruses to be used as vaccine seeds.

BACKGROUND: Cell-based influenza vaccines can solve the problem of the frequent occurrence of egg adaptation-associated antigenic changes observed in egg-based vaccines. Seed viruses for cell-based vaccines can be prepared from clinical specimens by cell culture; however, clinical samples risk harboring respiratory viruses other than influenza virus. Therefore, it is necessary to investigate the patterns of co-infection in clinical samples and explore whether cell culture technology can selectively propagate influenza viruses from samples containing other respiratory viruses. METHODS: A total of 341 clinical specimens were collected from patients with influenza or influenza-like illness and analyzed by ResPlex II assay to detect 18 respiratory viruses. The patterns of co-infection were statistically analyzed with Fisher's exact test. The samples with double or triple infections were passaged in suspension MDCK cells (MDCK-S), adherent MDCK cells (MDCK-A), and LLC-MK2D cells. Cell-passaged samples were analyzed by ResPlex II assay again to investigate whether each cell line could amplify influenza viruses and eliminate other respiratory viruses. RESULTS: Double infections were detected in 8.5% and triple infections in 0.9% of the collected clinical specimens. We identified four pairs of viruses with significant correlation. For all samples with double and triple infection, MDCK-S and MDCK-A could selectively propagate influenza viruses, while eliminating all contaminating viruses. In contrast, LLC-MK2D showed lower isolation efficiency for influenza virus and higher isolation efficiency for coxsackievirus/echovirus than MDCK-S and MDCK-A. CONCLUSIONS: Both MDCK-S and MDCK-A are considered suitable for the preparation of influenza vaccine seed viruses without adventitious agents or egg-adaptation mutations.

Mutations in the Neuraminidase-Like Protein of Bat Influenza H18N11 Virus Enhance Virus Replication in Mammalian Cells, Mice, and Ferrets.

To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets.IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals.

Changes in RNA secondary structure affect NS1 protein expression during early stage influenza virus infection.

RNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82-148) NS RNA region influence NS1 protein expression.

Generation of DelNS1 Influenza Viruses: a Strategy for Optimizing Live Attenuated Influenza Vaccines.

Nonstructural protein 1 (NS1) of influenza virus is a key virulence element with multifunctional roles in virus replication and a potent antagonist of host immune response. Deletion of NS1 (DelNS1) would create a safer and more extensively immunogenic live attenuated influenza virus (LAIV) vaccine. However, DelNS1 viruses are very difficult to grow in regular vaccine-producing systems, which has hampered the application of DelNS1 LAIV vaccines in humans. We have developed two master backbones of deleted-NS1 (DelNS1) viral genomes from influenza A or B viruses which contain novel adaptive mutations to support DelNS1-LAIV replication. These DelNS1-LAIVs are highly attenuated in human cells in vitro and nonpathogenic in mice but replicate well in vaccine-producing cells. Both influenza A and influenza B DelNS1 LAIVs grow better at 33°C than at 37 to 39°C. Vaccination with DelNS1 LAIV performed once is enough to provide potent protection against lethal challenge with homologous virus and strong long-lasting cross protection against heterosubtypic or antigenically distantly related influenza viruses in mice. Mechanistic investigations revealed that DelNS1-LAIVs induce cross protective neutralizing antibody and CD8+ and CD4+ T cell immunities. Importantly, it has been shown that DelNS1-LAIV can be used to enhance specific anti-influenza immunity through expression of additional antigens from the deleted-NS1 site. Generation of DelNS1 viruses which are nonpathogenic and able to grow in vaccine-producing systems is an important strategy for making highly immunogenic LAIV vaccines that induce broad cross protective immunity against seasonal and emerging influenza.IMPORTANCE Current seasonal influenza vaccines are suboptimal and low in immunogenicity and do not provide long-lasting immunity and cross protection against influenza virus strains that have antigenically drifted. More-effective influenza vaccines which can induce both humoral immunity and T cell immunity are needed. The NS1 protein of influenza virus is a virulence element and the critical factor for regulation of the host immune response during virus infection. Deletion of the NS1 protein is a strategy to make an optimal LAIV vaccine. However, DelNS1 viruses are very difficult to grow in regular vaccine-producing systems, hampering the application of DelNS1 LAIV vaccines in humans. We have generated a panel of both influenza A and influenza B DelNS1 LAIVs which are able to grow in regular vaccine-producing cells. These DelNS1 LAIV vaccines are completely nonpathogenic, exhibit potent and long-lasting immunity, and can be used to express extra viral antigen to induce cross protective immunity against seasonal and emerging influenza.

Defective interferon priming and impaired antiviral responses in a patient with an IRF7 variant and severe influenza.

Influenza infection is common worldwide with many individuals affected each year during epidemics and occasionally pandemics. Previous studies in animal models and a few human cases have established an important role of innate type I and III interferon (IFN) for viral elimination and mounting of antiviral responses. However, genetic and immunological determinants of very severe disseminated influenza virus infection in humans remain incompletely understood. Here, we describe an adult patient with severe influenza virus A (IAV) infection, in whom we identified a rare variant E331V in IFN regulatory factor (IRF)7 by whole-exome sequencing. Examination of patient cells demonstrated a cellular phenotype suggesting functional IRF7 impairment, since priming with IFN was almost abolished and IFN responses to IAV were significantly impaired in patient cells. Moreover, IAV replication was significantly higher in patient cells than in controls. Finally, expression of IRF7 E331V in HEK293 cells demonstrated significantly reduced activation of both IFNA7 and IFNB promoters in a luciferase reporter gene expression assay compared to IRF7 wild type. These findings provide further support for the essential role of IRF7 in amplifying antiviral IFN responses to ensure potent and sustained IFN responses during influenza virus infection in humans.

Transportan-derived cell-penetrating peptide delivers siRNA to inhibit replication of influenza virus in vivo.

INTRODUCTION: In this study, we report on the development of an effective delivery system for siRNAs; a novel cell-penetrating peptide (CPP), T9(dR), obtained from transportan (TP), was used for in vivo and in vitro testing. METHODS: In this study, toxicity of T9(dR) and TP and efficient delivery of siRNA were tested in 293T, MDCK, RAW, and A549 cells. Furthermore, T9(dR)- and TP-delivered siRNAs against nucleoprotein (NP) gene segment of influenza virus (siNP) were studied in both cell lines and mice. RESULTS: Gel retardation showed that T9(dR) effectively condensed siRNA into nanoparticles sized between 350 and 550 nm when the mole ratio of T9(dR) to siRNA was ≥4:1. In vitro studies demonstrated that T9(dR) successfully delivered siRNA with low cellular toxicity into several cell lines. It was also observed that T9(dR)-delivered siRNAs inhibited replication of influenza virus more efficiently as compared to that delivered by TP into the MDCK and A549 cells. It was also noticed that when given a combined tail vein injection of siNP and T9(dR) or TP, all mice in the 50 nmol siNP group infected with PR8 influenza virus survived and showed weight recovery at 2 weeks post-infection. CONCLUSION: This study indicates that T9(dR) is a promising siRNA delivery tool with potential application for nucleotide drug delivery.

Targeting Peroxisome Proliferator-Activated Receptor-Gamma Decreases Host Mortality After Influenza Infection in Obese Mice.

Obesity is an independent risk factor for severe influenza infection. However, the underlying cellular and molecular mechanisms are still incompletely understood. In this study, we have utilized a murine influenza infection model in genetic-induced obese (db/db) mice to explore the mechanisms by which obesity increases host susceptibility to influenza infection. We find that db/db mice have enhanced viral replication, exaggerated inflammatory responses, and dysregulated lung repair process after influenza infection, and consequently increased host mortality. Furthermore, we demonstrate that the transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-γ), an important inflammation regulator, was downregulated in the lung macrophages of db/db mice after influenza infection. Strikingly, the treatment of 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), a PPAR-γ agonist, largely rescued the survival of db/db mice after influenza infection. Interestingly, macrophage PPAR-γ-deficient mice exhibited enhanced mortality after influenza infection and 15d-PGJ2 fails to rescue host mortality in macrophage PPAR-γ-deficient mice, suggesting that PPAR-γ expression in macrophages is critical for the action of 15d-PGJ2. These data indicate that obesity attenuates lung antiviral immunity and hampers host recovery through the modulation of macrophage PPAR-γ expression. Furthermore, modalities targeting macrophage PPAR-γ expression and/or function may serve as promising therapeutics to treat severe influenza infection in obese patients.

Sphingolipids in early viral replication and innate immune activation.

In this review, we summarize the mechanisms by which sphingolipids modulate virus multiplication and the host innate immune response, using a number of host-virus systems as illustrative models. Sphingolipids exert diverse functions, both at the level of the viral life cycle and in the regulation of antiviral immune responses. Sphingolipids may influence viral replication in three ways: by serving as (co)receptors during viral entry, by modulating virus replication, and by shaping the antiviral immune response. Several studies have demonstrated that sphingosine kinases (SphK) and their product, sphingosine-1-phosphate (S1P), enhance the replication of influenza, measles, and hepatitis B virus (HBV). In contrast, ceramides, particularly S1P and SphK1, influence the expression of type I interferon (IFN-I) by modulating upstream antiviral signaling and enhancing dendritic cell maturation, differentiation, and positioning in tissue. The synthetic molecule α-galactosylceramide has also been shown to stimulate natural killer cell activation and interferon (IFN)-γ secretion. However, to date, clinical trials have failed to demonstrate any clinical benefit for sphingolipids in the treatment of cancer or HBV infection. Taken together, these findings show that sphingolipids play an important and underappreciated role in the control of virus replication and the innate immune response.

Comparison of Porcine Airway and Intestinal Epithelial Cell Lines for the Susceptibility and Expression of Pattern Recognition Receptors upon Influenza Virus Infection.

Influenza viruses infect the epithelial cells of the swine respiratory tract. Cell lines derived from the respiratory tract of pigs could serve as an excellent in vitro model for studying the pathogenesis of influenza viruses. In this study, we examined the replication of influenza viruses in the MK1-OSU cell line, which was clonally derived from pig airway epithelium. MK1-OSU cells expressed both cytokeratin and vimentin proteins and displayed several sugar moieties on the cell membrane. These cells also expressed both Sial2-3Gal and Sial2-6Gal receptors and were susceptible to swine influenza A, but not to human B and C viruses. Interestingly, these cells were also permissive to infection by influenza D virus that utilized 9-O-acetylated glycans. To study the differences in the expression of pattern recognition receptors (PRRs) upon influenza virus infection in the respiratory and digestive tract, we compared the protein expression of various PRRs in MK1-OSU cells with that in the SD-PJEC cell line, a clonally derived cell line from the porcine jejunal epithelium. Toll-like receptor 7 (TLR-7) and melanoma differentiation-associated protein 5 (MDA5) receptors showed decreased expression in influenza A infected MK1-OSU cells, while only TLR-7 expression decreased in SD-PJEC cells. Further research is warranted to study the mechanism behind the virus-mediated suppression of these proteins. Overall, this study shows that the porcine respiratory epithelial cell line, MK1-OSU, could serve as an in-vitro model for studying the pathogenesis and innate immune responses to porcine influenza viruses.