Pathogenesis of the novel avian-origin influenza A (H7N9) virus Influenza H7N9 virus in human lower respiratory tract

Poster Session: News and Views from H7N9 OutbreakBackground: As of May 2013, 131 laboratory-confirmed human infections with a novel influenza H7N9 virus had been reported from China. The source of human infection appears to be poultry. There is so far no evidence of sustained human-to-human transmission. Genetic analysis revealed that all eight gene segments of H7N9 were of avian origin; six internal gene segments from avian influenza H7N9 viruses, while hemagglutinin and neuraminidase genes were derived from influenza viruses circulating in ducks and wild ducks, respectively. The emergence of the H7N9 influenza virus catches global attention about whether the new virus could spark another pandemic. The majority of the infected patients were hospitalized and suffered from ARDS, with a fatality rate of about 37%. Our study aimed to determine the mechanism contributing to the pathogenesis of the H7N9 virus. A panel of proinflammatory cytokines and chemokines will be examined upon influenza H7N9 virus infection in alveolar epithelial cells in order to examine if these mediators were induced differentially when compared with the highly pathogenic avian influenza (HPAI) H5N1 and the 2009 pandemic H1N1 virus. Moreover, because cleaved caspase 3 is commonly employed as a marker for the indication of apoptosis, we further examined the extensiveness of cleaved caspase 3 in influenza virus infection in human lung ex vivo cultures. Materials and Methods: Fresh biopsies of human lung tissue were obtained from patients undergoing surgical resection of lung tissues. Lung tissue fragments were cultured with F12K medium incubated at 37°C. For viral infection experiments, influenza viruses A/Shanghai/1/2013 (SH1, H7N9), A/Shanghai/2/2013 (SH2, H7N9), A/Hong Kong/483/97 (H5N1), and A/California/07 (Ca07, H1N1pdm) at a viral titer of 106 TCID50/mL were used for ex vivo lung culture infection. Infected lung tissues were collected in 10% formalin at 24, 48, and 72 hpi for immunohistochemical staining. Costaining of cleaved caspase 3 and influenza virus nucleoprotein was carried out for the detection of apoptosis. Furthermore, primary culture of human alveolar epithelial cells was isolated from human lungs by mincing the lung, followed by filtration and centrifugation. Human alveolar epithelial cells were infected with the novel influenza H7N9, the HPAI H5N1, and the pandemic H1N1 virus. Virus replication was monitored by measuring infectious viral particles using TCID50. mRNA and protein expression of proinflammatory cytokines and chemokines were quantified by real time qPCR and ELISA. Results: We found extensive apoptosis in influenza H7N9 (both SH1 and SH2) and H5N1, but not H1N1pdm infected ex vivo lung tissues, suggesting that both avian influenza viruses can induce apoptosis and cause severe cell death in human lung tissue. Furthermore, unlike HPAI H5N1 which induces dysregulated proinflammatory cytokine responses, the novel influenza H7N9 virus elicited poor proinflammatory cytokine responses, inducing type I and III interferon in ex vivo human lung explant cultures. The novel influenza H7N9 virus is an intrinsically more potent inducer of proinflammatory cytokine than the H1N1pdm virus but less than the H5N1 virus. Conclusions: The proinflammatory cytokine and chemokine responses may contribute modestly to the severity of human H7N9 disease, but it is likely that direct viral cytopathology is probably playing a more important role in pathogenesis of human H7N9 diseases. The recognition of the role of cleaved caspase 3 in severe human infection of avian influenza virus can provide insights on the development of novel therapeutic approaches for the preparedness of the future outbreak of pandemics.published_or_final_versio

Poisson-Boltzmann calculations of ions in charged capillaries

The Poisson-Boltzmann (PB) equation of ions inside an infinitely long charged pore is solved using two different boundary conditions of the electric field due to the pore wall. The results show that the boundary condition generally adopted in the literature leads to a violation of electroneutrality in the ion-pore system. © 1994 American Institute of Physics.published_or_final_versio

Emission characteristics of nonmethane hydrocarbons from private cars and taxis at different driving speeds in Hong Kong

Vehicular emissions are the major sources of a number of air pollutants including nonmethane hydrocarbons (NMHCs) in urban area. The emission composition and emission factors of NMHCs from vehicles are currently lacking in Hong Kong. In this study, speciation and emission factors of NMHCs emitted from gasoline-fuelled private cars and liquefied petroleum gas (LPG)-fuelled taxis at different driving speeds were constructed using a chassis dynamometer. Large variations in the contributions of individual NMHC species to total emission were observed for different private cars at different driving speeds. The variations of individual NMHC emissions were relatively smaller for taxis due to their relatively homogeneous year of manufacture and mileages. Incomplete combustion products like ethane, ethene and propene were the major component of both types of vehicles, while unburned fuel component was also abundant in the exhausts of private cars and taxis (i.e. i-pentane and toluene for private car, and propane and butanes for taxi). Emission factors of major NMHCs emitted from private cars and taxis were estimated. High emission factors of ethane, n-butane, i/n-pentanes, methylpentanes, trimethylpentanes, ethene, propene, i-butene, benzene, toluene and xylenes were found for private cars, whereas propane and i/n-butanes had the highest values for taxis. By evaluating the effect of vehicular emissions on the ozone formation potential (OFP), it was found that the contributions of olefinic and aromatic hydrocarbons to OFP were higher than that from paraffinic hydrocarbons for private car, whereas the contributions of propane and i/n-butanes were the highest for taxis. The total OFP value was higher at lower speeds (≤50 km h-1) for private cars while a minimum value at driving speed of 100 km h-1 was found for taxis. At the steady driving speeds, the total contribution of NMHCs emitted from LPG-fuelled taxis to the OFP was much lower than that from gasoline-fuelled private cars. However, at idling state, the contribution of NMHCs from LPG-fuelled vehicles to OFP was comparable to that from gasoline-fuelled vehicles. The findings obtained in this study can be used to mitigate the air pollution caused by vehicles in highly dense urban areas. © 2011 Elsevier Ltd

Immunomodulatory and anti-viral effects of statins in influenza H5N1 virus infection of human alveolar epithelial cells and peripheral blood–derived macrophages

Poster Session: Novel TherapeuticsBackground: Highly pathogenic avian influenza (HPAI) H5N1 virus panzootic in poultry continues to spread. It causes zoonotic human disease with a high (> 60%) fatality rate and continues to pose a pandemic threat. Based on clinical, animal, and in vitro cell studies, we and others have suggested that differences in viral replication competence, tissue tropism, and cytokine dysregulation between H5N1 and low pathogenic viruses may contribute to disease pathogenesis. Statins as HMG-CoA inhibitors act to reduce cholesterol and have been demonstrated to have anti-inflammatory and immune-modulatory activities. However, there is controversy about the benefits of statin use on influenza infection in mice and humans. In this study, we aimed to evaluate the effects of statin treatment in influenza infection using physiologically relevant in vitro models—human alveolar epithelial cells (AECs) and peripheral blood–derived macrophages (PBDMs). Materials and Methods: Primary human AECs and PBDMs were infected with HPAI H5N1 (A/HK/483/97) and seasonal H1N1 (A/HK/54/98) viruses in the presence or absence of statin (simvastatin and sevastatin) treatment. Virus replication was monitored by measuring infectious viral particles in cell culture supernatants using TCID50. Immuno-modulatory effects of statins were examined by measuring the mRNA and protein expression of cytokines and chemokines using qPCR and ELISA. In order to understand the intervention of statins and influenza infection, the gene expression profile of selected members of the sterol-biosynthesis pathway in influenza virus–infected AECs and PBDMs were also monitored. The responses of a variety of cytokine treatments on the genes of the sterol-biosynthesis pathway were investigated in AECs. Furthermore, the intracellular free cholesterol level was also examined by enzymatic assay in AECs infected with influenza virus. Results: We demonstrated that both simvastatin and mevastatin exhibited a dose-dependent inhibition of influenza virus replication for both HPAI H5N1 and seasonal H1N1 viruses in human AECs and PBDMs. The observed inhibitory effect of simvastatin and mevastatin occurred below the non-specific toxic effects to cells, which were measured by MTT assay. Treatment of simvastatin and mevastatin significantly suppressed H5N1 virus–induced pro-inflammatory cytokines such as TNF-α in PBDMs and chemokines, including IP-10 and MCP-1 secretion in both AECs and PBDMs at 24 hours post-infection. We further showed that human AECs and PBDMs infected with both HPAI H5N1 and seasonal H1N1 viruses had significant down-regulation of sterol pathway gene expression at 24 hours post-infection. AECs and PBDMs treated with IFN-γ or IFN-β but not IL-1β, TNF, or IL-6, showed down-regulation of sterol pathway gene expression. In addition, we found that the free cholesterol level was significantly reduced at 24 and 48 h post-H5N1 virus infection in AECs and in IFN-β–treated AECs. These results further support a specific modulation of the sterol metabolic pathway upon influenza virus infection. Conclusions: Taken together, the controversy about the beneficial effects of statin use in influenza infection and our data suggest that statins possess both the antiviral and immune-regulatory effects in H5N1-infected in vitro cell models. We also demonstrated a highly specific response of AECs and PBDMs through a coordinated negative regulation of multiple sterol pathway members upon influenza virus infection or treatment of interferon. Identification of a reduction in sterol pathway gene expression and cholesterol levels with IFN treatment in human AECs offers new insights on the host-mediated antiviral responses through the sterol metabolism pathway and opens new therapeutic options for human influenza disease.published_or_final_versio

Characteristics of nonmethane hydrocarbons (NMHCs) in industrial, industrial-urban, and industrial-suburban atmospheres of the Pearl River Delta (PRD) region of south China

In a study conducted in late summer 2000, a wide range of volatile organic compounds (VOCs) were measured throughout five target cities in the Pearl River Delta (PRD) region of south China. Twenty-eight nonmethane hydrocarbons (NMHCs; 13 saturated, 9 unsaturated, and 6 aromatic) are discussed. The effect of rapid industrialization was studied for three categories of landuse in the PRD: Industrial, industrial-urban, and industrial-suburban. The highest VOC mixing ratios were observed in industrial areas. Despite its relatively short atmospheric lifetime (2-3 days), toluene, which is largely emitted from industrial solvent use and vehicular emissions, was the most abundant NMHC quantified. Ethane, ethene, ethyne, propane, n-butane, i-pentane, benzene, and m-xylene were the next most abundant VOCs. Direct emissions from industrial activities were found to greatly impact the air quality in nearby neighborhoods. These emissions lead to large concentration variations for many VOCs in the five PRD study cities. Good correlations between isoprene and several short-lived combustion products were found in industrial areas, suggesting that in addition to biogenic sources, anthropogenic emissions may contribute to urban isoprene levels. This study provides a snapshot of industrial, industrial-urban, and industrial-suburban NMHCs in the five most industrially developed cities of the PRD. Increased impact of industrial activities on PRD air quality due to the rapid spread of industry from urban to suburban and rural areas, and the decrease of farmland, is expected to continue until effective emission standards are implemented. Copyright 2006 by the American Geophysical Union

A new Kalman filter-based power spectral density estimation for nonstationary pressure signals

IEEE International Symposium on Circuits and Systems, Island of Kos, Greece, 21-24 May 2006This paper presents a new Kalman filter-based power spectral density estimation (PSD) algorithm for nonstationary pressure signals. The pressure signal is assumed to be an autoregressive (AR) process, and a stochastically perturbed difference equation constraint model is used to describe the dynamics of the AR coefficients. The proposed Kalman filter frame uses variable number of measurements to estimate the time-varying AR coefficients and yield the PSD estimation with better time-frequency resolution. Simulation results show that the proposed algorithm achieves a better time-frequency resolution than conventional algorithms for nonstationary pressure signals. © 2006 IEEE.published_or_final_versio

Photoconductivity and charge transporting properties of metal-containing poly(p-phenylenevinylene)s

A novel type of poly(p-phenylenevinylene)s which contain bis(2.2′:6′,2″-terpyridine) ruthenium (II) complexes has been developed. The absorption of the polymers at 500 nm was strongly enhanced by the metal complexes due to the presence of the metal-ligand charge transfer transition. The charge transportation is dispersive with hole carrier mobilities and activation energy of ∼7 × 10-5 cm2 V-1 s-1 and 0.20 eV. respectively, depending on the concentration of the metal complex. A log μ vs E1/2 plot shows that hole mobilities decrease with increasing field, which suggests the presence of off-diagonal disorder in the hopping sites. © 1997 American Institute of Physics.published_or_final_versio

Approximate QR-based algorithms for recursive nonlinear least squares estiamtion

This paper proposes new approximate QR-based algorithms for recursive nonlinear least squares (NLS) estimation. Two QR decomposition-based recursive algorithms are introduced based on the classical Gauss-Newton (GN) and Levenberg-Marquardt (LM) algorithms in nonlinear unconstrained optimization or least squares problems. Instead of using the matrix inversion formula, recursive QR decomposition is employed, which is known to be numerically more stable in finite wordlength implementation. A family of p-A-QR-LS algorithms is then proposed to solve the LS problem resulting from the linearization of the NLS problem. It achieves different complexity-performance tradeoffs by retaining different number of diagonal plus off-diagonals (denoted by an integer p) of the triangular factor of the augmented data matrix. Simulation results on identifying a nonlinear perceptron are provided to illustrate the principle of the new algorithms. © 2005 IEEE.published_or_final_versio

Tracking down the migration of mouse neural crest cells

During early embryonic development, cell migration is one of the most important morphogenetic processes. Neural crest cells arise from the dorsal part of the neural tube and migrate along different pathways to numerous locations where they differentiate into a variety of tissues. In the mouse, studies of neural crest cell migration have been difficult partly because of the absence of specific markers which can label neural crest cells throughout their migration from their origin to the site of differentiation. Nevertheless, the use of different experimental strategies involving extrinsic, intrinsic or genetic cell markers has already led to a good understanding of this migration. In our studies, extrinsic markers such as wheat germ agglutinin-gold conjugates and DiI and genetic markers including Hoxb2-lacZ and green fluorescent protein have been employed in tracing migrating neural crest cells. The labelling procedures and the strength and weaknesses of the tracing methods are reviewed herein. Copyright © 2003 S. Karger AG, Basel.published_or_final_versio

Influenza B viruses in swine: virus tropism in swine respiratory organ explant cultures

Poster Session: Virology and Viral ReceptorsBackground: Swine has been considered an animal reservoir of pandemic influenza A virus (IAV), for example, the 2009 H1N1 pandemic virus, swine is acting as a “mixing vessel” for the reassortment of swine, human and avian IAVs. Certain influenza B virus (IBV) strains were also found to be readily infecting piglets as early as in 1969. However, tissue tropism of IBV in swine is understudied, at least in 2000s, mainly due to the misconception that IBV causes milder disease than IAV. IBV has in fact circulated in many parts of the world causing regular seasonal epidemics in humans with mortality rates sometimes higher than that in IAV seasons. Here, our research group hypothesizes that swine could be a neglected host of IBV, apart from human and seal, due to the previous infectivity of IBV in this animal, as well as the fact that swine has close contact with human and possesses a similar sialic acid (influenza virus receptor) distribution profile as the human respiratory tract. We aim to examine the characteristics of IBV tissue tropism using swine tracheal and lung explant models, and risk assess swine susceptibility to a panel of IBV strains from both Yamagata and Victoria lineages of different years. Materials and Methods: The tracheal and lung explants were prepared from fresh swine respiratory organs from approximately 6-month-old pigs, and cultured with maximal similarity to the in vivo conditions. A panel of IBV strains, from both Yamagata and Victoria lineages and from different years, were used to infect the tissue explants at 37oC or 39oC according to the original physiological temperature of the tissue. The virus replication efficiencies were evaluated through viral titration and immunohistochemistry of the collected supernatant and formalin-fixed tissue explants respectively at 1, 24, 48 and 72 h postinfection. Seasonal IAVs (H1N1 - A/OK/447/08 and H3N2 - A/OK/370/05) were used as controls. Results: Most of the tested IBVs showed productive replication in the swine lung explants. Swine tracheal explants, on the other hand, supported the replication of limited IBV strains. Most of these IBVs belong to the Victoria lineage, which spread across the years from 2005 to 2011. IBVs that could replicate in swine lung explants reached their maxima at 48 hpi or sometimes later. This is comparatively slower than the replication rates of seasonal IAVs (H1N1 & H3N2) used in the study, which usually showed significant increase at 24 hpi with still increasing virus yields at 48 hpi in some cases. However, the overall increase in titres between the IBVs and seasonal IAVs were similar. In swine tracheal explants, both IBVs and seasonal IAVs showed limited replications with similar trends of having maxima being reached at 24 hpi. Conclusions: The successful replication of IBVs in swine explants cultures indicates the possible susceptibility of swine to IBV and provides the essential basis for further investigation on the likelihood for swine to be an animal reservoir of the virus, as well as the threat it may pose to humans. Continuous studies on the replication kinetics of a greater number of IBVs in swine explant cultures across a wider range of years, countries and lineages will probably be our future target.published_or_final_versio