Sialic acid and receptor expression on the respiratory tract in normal subjects and H5N1 and non-avian influenza patients.

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The role of influenza virus gene constellation and viral morphology on cytokine induction, pathogenesis, and viral virulence.

Key Messages 1. H5N1 viruses that cause severe disease in humans are potent inducers of proinflammatory cytokines in contrast to seasonal influenza viruses, and this may play a role in the mechanism of H5N1 pathogenesis. 2. H5N1 viruses are predominantly spherical in morphology. Virus morphology does not influence the ability to induce proinflammatory cytokines. 3. The NS1 viral protein may play a role in the potency of proinflammatory induction. 4. The H5N1 haemagglutinin and neuraminidase do not appear to transfer the high cytokine phenotype. 5. The ability to induce cytokines is a polygenic trait, involving a combination of different viral genes.published_or_final_versio

Formation of virus-like particles from human cell lines exclusively expressing Influenza Neuraminidase

The minimal virus requirements for the generation of influenza virus-like particle (VLP) assembly and budding were reassessed. Using neuraminidase (NA) from the H5N1 and H1N1 subtypes, it was found that the expression of NA alone was sufficient to generate and release VLPs. Biochemical and functional characterization of the NA-containing VLPs demonstrated that they were morphologically similar to influenza virions. The NA oligomerization was comparable to that of the live virus, and the enzymic activity, whilst not required for the release of NA-VLPs, was preserved. Together, these findings indicate that NA plays a key role in virus budding and morphogenesis, and demonstrate that NA-VLPs represent a useful tool in influenza research. © 2010 SGM.link_to_OA_fulltex

Occult respiratory viral infections in coronial autopsies: a pilot project.

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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

Proinflammatory cytokine response and viral replication in mouse bone marrow derived macrophages infected with influenza H1N1 and H5N1 viruses

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes.published_or_final_versio

Optimization and evaluation of an influenza A (H5) pseudotyped lentiviral particle-based serological assay

Background: Novel serological methods provide alternative options for sero-diagnosis, sero-epidemiology and for determining evidence of naturally acquired or vaccine induced immunity. Micro-neutralization tests are currently the gold standard for serological studies of highly pathogenic avian influenza in mammalian species but require handling live virus in a biosafety level (BSL) 3 environment. We previously reported the use of H5 pseudotyped lentiviral particles (H5pp) as an alternative to micro-neutralization tests in a BSL-2 setting (Nefkens et al., 2007). Objective: To optimize and evaluate this newly developed H5pp assay on relevant clinical specimens. Study design: We optimise and evaluate the performance of the H5pp assay using well-characterized sera from humans with confirmed H5N1 disease or controls. Results: The H5pp assay is a reliable serological method for the detection and quantification of neutralizing antibody to H5-viruses. Conclusion: H5pp provide a reliable and safe alternative for sero-diagnosis and sero-epidemiology of H5N1 infections in a BSL-2 setting. © 2009 Elsevier B.V. All rights reserved.postprin

International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1) pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE

The microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future

The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles

Copyright @ 2008 American Society for Microbiology.The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.The University of Hong Kong and the French Ministry of Health

Polarity of influenza H5N1 virus infection in respiratory epithelial cells and the impact of basolateral release of cytokines in disease pathogenesis

Poster Presentations: Virus Host Interaction/PathogenesisINTRODUCTION: Highly pathogenic avian influenza virus (H5N1) is the first avian influenza virus that documented to cause respiratory disease and death in human. The biological basis for the severe human disease and high fatality rate remains unclear. We have previously demonstrated that when compared to human H1N1 and H3N2 influenza viruses, infection of influenza H5N1 virus led to the hyper-induction of pro-inflammatory cytokines in human primary macrophages and ...postprin