High Humidity Leads to Loss of Infectious Influenza Virus from Simulated Coughs

Background The role of relative humidity in the aerosol transmission of influenza was examined in a simulated examination room containing coughing and breathing manikins. Methods Nebulized influenza was coughed into the examination room and Bioaerosol samplers collected size-fractionated aerosols (\u3c1 µM, 1–4 µM, and \u3e4 µM aerodynamic diameters) adjacent to the breathing manikin’s mouth and also at other locations within the room. At constant temperature, the RH was varied from 7–73% and infectivity was assessed by the viral plaque assay. Results Total virus collected for 60 minutes retained 70.6–77.3% infectivity at relative humidity ≤23% but only 14.6–22.2% at relative humidity ≥43%. Analysis of the individual aerosol fractions showed a similar loss in infectivity among the fractions. Time interval analysis showed that most of the loss in infectivity within each aerosol fraction occurred 0–15 minutes after coughing. Thereafter, losses in infectivity continued up to 5 hours after coughing, however, the rate of decline at 45% relative humidity was not statistically different than that at 20% regardless of the aerosol fraction analyzed. Conclusion At low relative humidity, influenza retains maximal infectivity and inactivation of the virus at higher relative humidity occurs rapidly after coughing. Although virus carried on aerosol particles \u3c4 µM have the potential for remaining suspended in air currents longer and traveling further distances than those on larger particles, their rapid inactivation at high humidity tempers this concern. Maintaining indoor relative humidity \u3e40% will significantly reduce the infectivity of aerosolized virus

Viable Influenza A Virus in Airborne Particles Expelled During Coughs Versus Exhalations

Background To prepare for a possible influenza pandemic, a better understanding of the potential for the airborne transmission of influenza from person to person is needed. Objectives The objective of this study was to directly compare the generation of aerosol particles containing viable influenza virus during coughs and exhalations. Methods Sixty-one adult volunteer outpatients with influenza-like symptoms were asked to cough and exhale three times into a spirometer. Aerosol particles produced during coughing and exhalation were collected into liquid media using aerosol samplers.The samples were tested for the presence of viable influenza virus using a viral replication assay (VRA). Results Fifty-three test subjects tested positive for influenza A virus. Of these, 28 (53%) produced aerosol particles containing viable influenza A virus during coughing, and 22 (42%) produced aerosols with viable virus during exhalation. Thirteen subjects had both cough aerosol and exhalation aerosol samples that contained viable virus, 15 had positive cough aerosol samples but negative exhalation samples, and 9 had positive exhalation samples but negative cough samples. Conclusions Viable influenza A virus was detected more often in cough aerosol particles than in exhalation aerosol particles, but the difference was not large. Because individuals breathe much more often than they cough, these results suggest that breathing may generate more airborne infectious material than coughing over time. However, both respiratory activities could be important in airborne influenza transmission. Our results are also consistent with the theory that much of the aerosol containing viable influenza originates deep in the lung

High Humidity Leads to Loss of Infectious Influenza Virus from Simulated Coughs

Abstract Background: The role of relative humidity in the aerosol transmission of influenza was examined in a simulated examination room containing coughing and breathing manikins

Synthetic Heparan Sulfate Oligosaccharides Inhibit Endothelial Cell Functions Essential for Angiogenesis

Heparan sulfate (HS) is an important regulator of the assembly and activity of various angiogenic signalling complexes. However, the significance of precisely defined HS structures in regulating cytokine-dependent angiogenic cellular functions and signalling through receptors regulating angiogenic responses remains unclear. Understanding such structure-activity relationships is important for the rational design of HS fragments that inhibit HS-dependent angiogenic signalling complexes.We synthesized a series of HS oligosaccharides ranging from 7 to 12 saccharide residues that contained a repeating disaccharide unit consisting of iduronate 2-O-sulfate linked to glucosamine with or without N-sulfate. The ability of oligosaccharides to compete with HS for FGF2 and VEGF165 binding significantly increased with oligosaccharide length and sulfation. Correspondingly, the inhibitory potential of oligosaccharides against FGF2- and VEGF165-induced endothelial cell responses was greater in longer oligosaccharide species that were comprised of disaccharides bearing both 2-O- and N-sulfation (2SNS). FGF2- and VEGF165-induced endothelial cell migration were inhibited by longer 2SNS oligosaccharide species with 2SNS dodecasaccharide activity being comparable to that of receptor tyrosine kinase inhibitors targeting FGFR or VEGFR-2. Moreover, the 2SNS dodecasaccharide ablated FGF2- or VEGF165-induced phosphorylation of FAK and assembly of F-actin in peripheral lamellipodia-like structures. In contrast, FGF2-induced endothelial cell proliferation was only moderately inhibited by longer 2SNS oligosaccharides. Inhibition of FGF2- and VEGF165-dependent endothelial tube formation strongly correlated with oligosaccharide length and sulfation with 10-mer and 12-mer 2SNS oligosaccharides being the most potent species. FGF2- and VEGF165-induced activation of MAPK pathway was inhibited by biologically active oligosaccharides correlating with the specific phosphorylation events in FRS2 and VEGFR-2, respectively.These results demonstrate structure-function relationships for synthetic HS saccharides that suppress endothelial cell migration, tube formation and signalling induced by key angiogenic cytokines

β-Defensin-1 Regulates Influenza Virus Infection in Human Bronchial Epithelial Cells through the STAT3 Signaling Pathway

Understanding the host response to influenza A virus (IAV) infection is vital for developing intervention strategies. The primary barriers for invading respiratory pathogens are the respiratory tract epithelial cells and antimicrobial proteins generated by these cells. The antimicrobial peptide, β-defensin-1, has antiviral activity against both enveloped and non-enveloped viruses. Significant downregulation of β-defensin1 gene (DEFB1) expression was observed when human bronchial epithelial cells (HBEpCs) were exposed to IAV. HBEpCs overexpressing DEFB1 caused a significant reduction in IAV, that was confirmed by IAV matrix gene analysis, plaque assay, and confocal microscopy. DEFB1 expression after transfection with two micro RNAs (miRNAs), hsa-miR-186-5p and hsa-miR-340-5p, provided evidence that DEFB1 expression could be modulated by these miRNAs and hsa-miR-186-5p had a higher binding efficiency with DEFB1. Overexpression of DEFB1 in IAV-infected HBEpCs led to increased NF-κB expression. In a PCR array analysis of 84 transcription factors, either overexpressing DEFB1 or siRNA silencing of DEFB1 expression significantly modulated the expression of signal transducer and activator of transcription 3 (STAT3). In addition, Ingenuity Pathway Analysis (IPA) integrated with PCR array data showed that the JAK1/STAT3 pathway was significantly altered in cells overexpressing DEFB1, suggesting this to be one of the pathways by which defensin regulates IAV replication in HBEpCs. In conclusion, the reduction in IAV copy number in DEFB1 overexpressing cells suggests that β-defensin-1 plays a key role in regulating IAV survival through STAT3 and is a potential target for antiviral drug development

Upregulation of miRNA-4776 in Influenza Virus Infected Bronchial Epithelial Cells Is Associated with Downregulation of NFKBIB and Increased Viral Survival

Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwide. One key transcription factor that is activated upon IAV infection is nuclear factor Kappa B (NF-κB). NF-κB regulation involves the inhibitor proteins NF-κB inhibitor beta (NFKBIB), (also known as IκB β), which form complexes with NF-κB to sequester it in the cytoplasm. In this study, microarray data showed differential expression of several microRNAs (miRNAs) on exposure to IAV. Target scan analysis revealed that miR-4776, miR-4514 and miR-4742 potentially target NFKBIB messenger RNA (mRNA). Time-course analysis of primary bronchial epithelial cells (HBEpCs) showed that miR-4776 expression is increased within 1 h of infection, followed by its downregulation 4 h post-exposure to IAV. NFKBIB upregulation of miR-4776 correlated with a decrease in NFKBIB expression within 1 h of infection and a subsequent increase in NFKBIB expression 4 h post-infection. In addition, miRNA ago-immunoprecipitation studies and the three prime untranslated region (3’ UTR) luciferase assay confirmed that miR-4776 targets NFKBIB mRNA. Furthermore, uninfected HBEpCs transfected with miR-4776 mimic showed decreased expression of NFKBIB mRNA. Overexpression of NFKBIB protein in IAV infected cells led to lower levels of IAV. Taken together, our data suggest that miRNA-4776 modulates IAV production in infected cells through NFKBIB expression, possibly through the modulation of NF-κB

Survival of Staphylococcus Aureus on the Outer Shell of Fire Fighter Turnout Gear After Sanitation in a Commercial Washer/extractor

Background: Methicillin-resistant Staphylococcus aureus contamination on surfaces including turnout gear had been found throughout a number of fire stations. As such, the outer shell barrier of turnout gear jackets may be an indirect transmission source and proper disinfection is essential to reduce the risk of exposure to fire fighters. Cleaning practices vary considerably among fire stations, and a method to assess disinfection of gear washed in commercial washer/extractors is needed. Methods: Swatches (1 in. × 1.5 in.) of the outer shell fabrics, Gemini™, Advance™, and Pioneer™, of turnout gear were inoculated with S. aureus, and washed with an Environmental Protection Agency-registered sanitizer commonly used to wash turnout gear. To initially assess the sanitizer, inoculated swatches were washed in small tubes according to the American Society for Testing Materials E2274 Protocol for evaluating laundry sanitizers. Inoculated swatches were also pinned to turnout gear jackets and washed in a Milnor commercial washer/extractor. Viable S. aureus that remained attached to fabric swatches after washing were recovered and quantified. Scanning Electron Microscopy was used to characterize the stages of S. aureus biofilm formation on the swatches that can result in resistance to disinfection. Results: Disinfection in small tubes for only 10 s reduced the viability of S. aureus on Gemini™, Advance™, and Pioneer™ by 73, 99, and 100%, respectively. In contrast, disinfection of S. aureus-contaminated Gemini™ swatches pinned to turnout gear and washed in the washer/extractor was 99.7% effective. Scanning Electron Microscopy showed that biofilm formation begins as early as 5 h after attachment of S. aureus. Conclusion: This sanitizer and, likely, others containing the anti-microbial agent didecyl dimethyl ammonium chloride, is an effective disinfectant of S. aureus. Inclusion of contaminated outer shell swatches in the wash cycle affords a simple and quantitative method to assess sanitization of gear by commercial gear cleaning facilities. This methodology can be extended to assess for other bacterial contaminants. Sanitizer-resistant strains will continue to pose problems, and biofilm formation may affect the cleanliness of the washed turnout gear. Our methodology for assessing effectiveness of disinfection may help reduce the occupational exposure to fire fighters from bacterial contaminants

Differential Expression of Serum Exosome microRNAs and Cytokines in Influenza A and B Patients Collected in the 2016 and 2017 Influenza Seasons

MicroRNAs (miRNAs) have remarkable stability and are key regulators of mRNA transcripts for several essential proteins required for the survival of cells and replication of the virus. Exosomes are thought to play an essential role in intercellular communications by transporting proteins and miRNAs, making them ideal in the search for biomarkers. Evidence suggests that miRNAs are involved in the regulation of influenza virus replication in many cell types. During the 2016 and 2017 influenza season, we collected blood samples from 54 patients infected with influenza and from 30 healthy volunteers to identify the potential role of circulating serum miRNAs and cytokines in influenza infection. Data comparing the exosomal miRNAs in patients with influenza B to healthy volunteers showed 76 miRNAs that were differentially expressed (p < 0.05). In contrast, 26 miRNAs were differentially expressed between patients with influenza A (p < 0.05) and the controls. Of these miRNAs, 11 were commonly expressed in both the influenza A and B patients. Interferon (IFN)-inducing protein 10 (IP-10), which is involved in IFN synthesis during influenza infection, showed the highest level of expression in both influenza A and B patients. Influenza A patients showed increased expression of IFNα, GM-CSF, interleukin (IL)-13, IL-17A, IL-1β, IL-6 and TNFα, while influenza B induced increased levels of EGF, G-CSF, IL-1α, MIP-1α, and TNF-β. In addition, hsa-miR-326, hsa-miR-15b-5p, hsa-miR-885, hsa-miR-122-5p, hsa-miR-133a-3p, and hsa-miR-150-5p showed high correlations to IL-6, IL-15, IL-17A, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) with both strains of influenza. Next-generation sequencing studies of H1N1-infected human lung small airway epithelial cells also showed similar pattern of expression of miR-375-5p, miR-143-3p, 199a-3p, and miR-199a-5p compared to influenza A patients. In summary, this study provides insights into the miRNA profiling in both influenza A and B virus in circulation and a novel approach to identify the early infections through a combination of cytokines and miRNA expression