JVI Accepted Manuscript Posted Online 24 February 2016 J. Virol. doi:10.1128/JVI.00013-16 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Manuscript
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Neuraminidase Activity and The Resistance of 2009 Pandemic H1N1 Influenza Virus to
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Antiviral Activity in Bronchoalveolar Fluid
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Kanyarat Ruangrung1, Ornpreya Suptawiwat1, Kittipong Maneechotesuwan2,
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Chompunuch Boonarkart1, Warunya Chakritbudsabong6, Jirawatna Assawabhumi2,
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Parvapan Bhattarakosol5, Mongkol Uiprasertkul3, Pilaipan Puthavathana1, 8,
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Witthawat Wiriyarat6, Anan Jongkaewwattana7, Prasert Auewarakul1, 4*
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Thailand
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Medicine Siriraj Hospital, Mahidol University, Thailand
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Biotechnology, Thailand
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Thailand
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University,
Division of Respiratory Diseases and Tuberculosis, Department of Medicine, Faculty of
Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand Institute of Molecular Biosciences, Mahidol University, Thailand; Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Thailand; Faculty of Veterinary Science, Mahidol University, Thailand and Virology and Cell Technology Laboratory, National Center for Genetic Engineering and
Center of Research and Innovation, Faculty of Medical Technology, Mahidol University,
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*Corresponding author: Prasert Auewarakul, MD, Dr med
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Mailing address: Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol
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University, Bangkok 10700, Thailand
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Tel: +662 4197059
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Fax: +662 4182663
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E-mail:
[email protected] 31 1
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KEYWORDS: neuraminidase/ resistance / oseltamivir/ pandemic H1N1 2009/ influenza A
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virus/ antiviral/ bronchoalveolar fluid
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RUNNING TITLE: The Resistance of 2009 pdmH1N1 Influenza Virus to BALF
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ABSTRACT
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Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be
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a frontline innate defense against the virus. Several anti-viral factors, including surfactant protein
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D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza was previously
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shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza
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strains have different sensitivity to the overall anti-influenza activity of human bronchoalveolar
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fluid was not known. We compared the sensitivity of 2009 pandemic H1N1, seasonal H1N1, and
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seasonal H3N2 influenza strains to an inhibition by human bronchoalveolar lavage (BAL). The
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pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL than the H3N2
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strains. The BAL anti-influenza activity could be enhanced by Oseltamivir, indicating that the
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viral neuraminidase (NA) activity could provide a resistance to the anti-viral defense. In
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accordance with this finding, the BAL anti-influenza activity was found to be sensitive to
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sialidase. The Oseltamivir-resistant mutation, H275Y, rendered the pandemic H1N1 virus but not
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the seasonal H1N1 virus more sensitive to BAL. Since only the seasonal H1N1 but not the
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pandemic H1N1 had compensatory mutations that allowed Oseltamivir-resistant strains to
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maintain NA enzymatic activity and transmission fitness, the resistance to BAL of the drug
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resistant seasonal H1N1 virus might play a role in the viral fitness.
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IMPORTANCE
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Human airway secretion contains anti-influenza activity. Different influenza strains may
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vary in their susceptibility to this antiviral activity. Here we show that the 2009 pandemic and
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seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage than
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H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the
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pandemic virus was determined by its NA gene and that the NA inhibitor resistant mutation,
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H275Y, abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which
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had compensatory mutations that maintained the fitness of drug resistant strains. Therefore, the
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innate respiratory tract defense may be a barrier against NA inhibitor resistant mutants and
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evasion of this defense may play a role in emergence and spreading of drug resistant strains.
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INTRODUCTION
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Despite being regarded as a mild pandemic, the H1N1 influenza virus can cause
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unusually severe diseases, which can rapidly progress to respiratory failure and death (1).
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Comparative studies in the same outbreak season showed that patients infected with the
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pandemic H1N1 virus are more likely to develop severe disease and consequently die than those
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infected with seasonal influenza virus (2). In addition, animal experiments in mice, ferrets and
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macaques have shown that the H1N1 2009 pandemic influenza virus is more virulent and
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replicates more efficiently in lungs than seasonal influenza virus (3, 4). These suggest that the
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pandemic virus may invade lungs more effectively.
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Innate defense plays important roles in susceptibility to viral infection and pathogenesis.
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Soluble anti-viral factors are important components of innate defense in the respiratory tract.
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Several soluble factors with anti-influenza activity have been identified in BAL, including
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surfactant protein (SP)-D, SP-A, scavenger receptor gp340, long pentraxin PTX3, L-ficolin, H-
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ficolin, serum amyloid P (5-12). These factors are present in serum and respiratory secretions.
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Serum influenza inhibitors can be classified as α-, β-, and γ-inhibitors according to their physical
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properties and mechanisms of inhibition (13-15). Because α- and γ-inhibitors are sialylated
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glycoproteins or receptor analogues that compete with sialic acid in the binding of influenza HA,
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they are heat-resistant and Ca++-independent, but can be destroyed by sialidase or receptor
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destroying enzyme (RDE). In contrast, β-inhibitors, such as SP-D, are heat labile, require Ca++
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and can be inhibited by some monosaccharides, but are resistant to RDE. They are lectins that
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bind influenza HA via carbohydrate moiety on glycosylation sites of HA, and inhibit HA
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function probably through steric hindrance. In addition, exosome from human bronchial
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epithelial cells has been shown to exhibit anti-influenza activity (16).
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Among these known anti-influenza factors, SP-D is believed to be the main contributor to
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the BAL anti-influenza activity (6). It was shown that the 2009 H1N1 pandemic virus is resistant
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to SP-D because it had less N-linked glycosylation site on the globular head of hemagglutinin
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(HA) (17, 18). However, whether susceptibility to other innate anti-viral factors, especially α-
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and γ-inhibitors, contributes to the virulence of the 2009 pandemic influenza is not known. The
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antiviral activity of α- and γ-inhibitors can be detected without interference from β-inhibitor by
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performing the assay in a condition without calcium ion. We therefore assessed the sensitivity of
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2009 pandemic influenza strains in comparison with seasonal influenza strains, to human BAL.
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MATERIALS AND METHODS
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Ethics statement. The human study was approved by the Ethics Committee of Faculty of
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Medicine Siriraj Hospital (Siriraj Institutional Review Board), which is in full compliance with
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the Declaration of Helsinki, the Belmont Report, CIOMS Guidelines and the International
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Conference on Harmonization in Good Clinical Practice (ICH-GCP). The study was performed
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under protocol COA No.SI572/2009 ‘‘Sensitivity of Influenza 2009 H1N1 to antiviral factors in
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bronchoalveolar lavage’’. All human subjects provided written informed consent.
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BAL samples. BAL samples were collected from 52 patients aged 30-96 years (mean + SD = 64
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+ 12) with a male-to-female ratio of 1:1.17, who underwent routine bronchoscopy and
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bronchoalveolar lavage for investigation of suspected lung cancer. None of the patients were
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being treated with any anti-viral treatment. Bronchoalveolar lavage with 0.9% normal saline was
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performed at a non-lesional lung segment. The retrieved fluid was collected and immediately
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carried on ice to the laboratory. The BAL samples were centrifuged at 1,500g, 4°C for 5 minutes.
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The supernatant was stored frozen in aliquots at -80ºC.
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Viral strains. The 2009 pandemic H1N1 (pdmH1N1) influenza virus strains were isolated from
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patients with influenza-liked illness in Thailand during 2009 – 2011 (A/Nonthaburi/102/2009,
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A/Thailand/104/2009,
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Seasonal influenza strains used in this study are A/Thailand/Siriraj-03/2006 (sH1N1) (A/New
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Caledonia/20/99-like virus), A/Thailand/Siriraj-12/2006 (sH1N1) (A/New Caledonia/20/99-like
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virus), A/Thailand/SirirajICRC_SEA-3479/2008 (sH1N1) (A/Brisbane/59/2007-like virus),
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A/Thailand/Siriraj-03/2004 (H3N2) (A/Fujian/411/02-like virus) and A/Thailand/Siriraj-04/2003
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(H3N2). All the viruses were isolated and propagated for less than 10 passages in Madin-Darby
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Canine Kidney (MDCK) cells with 1 mML-1-tosylamido-2-phenylethyl chloromethyl ketone
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(TPCK)-treated trypsin (SIGMA-ALDRICH®) in Minimum Essential Medium (MEM)
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(GIBCO®).
A/Thailand/MVCU-013/2009
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and
A/Thailand/ICRC_NSN_1/2011).
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Antiviral activity. The antiviral activity in BAL samples was assessed using hemagglutination
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inhibition (HI) and neutralization (NT) assays as previously described for serum antibody assay
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(19), except that the samples were not treated by sialidase prior to the assay. Briefly, for HI
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assay, BAL was pre-absorbed with 0.5% goose red blood cell (GRBC) suspension to eliminate
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non-specific agglutinins, serially diluted in phosphate buffered saline (PBS) with and without 5
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mM CaCl2, mixed with 4 hemagglutination unit of virus and 0.5% GRBC in a 96-well U-shaped
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microtiter plate, incubated at 4ºC for 30 minutes. HI titers were read as reciprocal of the highest
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dilution with complete hemagglutination. For NT assay, BAL was serially diluted in MEM plus
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1 mM TPCK-treated trypsin (with 1.8 mM CaCl2) and mixed with 25 TCID50 (50% tissue
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culture infectious dose) of virus, incubated at 37°C for an hour and inoculated onto MDCK cells
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in a 96-well tissue culture plate. Briefly, 3x104 cells in 200 μl MEM plus 10% heat-inactivated
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fetal bovine serum (FBS) (GIBCO®) were seeded to each well and incubated at 37°C overnight).
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After an overnight incubation at 37°C, the tissue culture plate was washed and fixed with 80%
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cold acetone in 1X PBS, the level of viral infection was measured using an antiviral
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nucleoprotein monoclonal antibody and a peroxidase-conjugated secondary antibody. Optical
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densities were measured and NT titers with 50% inhibition of specific signals were calculated.
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BAL sample was treated with Receptor Destroying Enzyme (RDE II DENKA SEIKEN) (1:2)
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37°C overnight. RDE was inactivated at 56ºC for 30 minutes before the HI testing. Protease
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treatment was done by adding 30U of Proteinase K (SIGMA-ALDRICH) to 1 ml of BAL
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sample, incubated at 37°C overnight and inactivated at 92ºC for 10 minutes.
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Reverse genetics viruses. The HA and NA genes of selected pdmH1N1 strains were amplified
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from RNA extracted from infected MDCK cells, cloned into the reverse genetic plasmid
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pHw2000 and sequenced. Specific mutations were introduced into the NA gene by PCR site-
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directed mutagenesis with Dpn I digestion. Reverse genetic viruses were produced in HEK293T
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cells by co-transfecting pHw2000 plasmids carrying the cloned HA and NA genes of the 2009
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pandemic virus and the other 6 genes from the A/Puerto Rico/8/1934 (H1N1)(PR8) strain as
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previously described (20).
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IgA and IgG depletion. IgA and IgG in BAL sample were precipitated using a lectin (Jacalin)
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from Artocarpus integrifolia cross-linked with 4% beaded agarose (SIGMA-ALDRICH) (21)
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and Pierce™ protein G agarose (Thermo scientific), respectively. Two hundred and fifty
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microlitre of BAL sample was added with 100 μl of settle agarose bead and mixed thoroughly.
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The mixture was rotated and incubated at 4°C overnight. The agarose beads were precipitated
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from sample by centrifugation at 1,000 rpm for 30 seconds. The supernatant was collected. To
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precipitate total protein in sample, 10 μl of supernatant was added with 250 μl of absolute
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ethanol, mixed thoroughly and centrifuged at 13,000 rpm 4°C for an hour. The supernatant was
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discarded and dried. Twenty microliter of reducing loading dye was added, mixed thoroughly
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and incubated at 70ºC for 10 minutes. The sample was run in sodium dodecyl sulfate-
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polyacrylamide gel electrophoresis (SDS-PAGE) and performed Western blotting. IgA and IgG
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were detected using 1: 2000 of Goat anti-human IgA HRP-labeled antibody (SIGMA-
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ALDRICH) and 1: 2000 of Goat anti-human IgG HRP-labeled antibody (Invitrogen) in 2%
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bovine serum albumin (SIGMA-ALDRICH), respectively. Both IgA and IgG detected antibody
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were developed with DAB (3, 3'-diaminobenzidine) HRP substrate (SIGMA-ALDRICH). One
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hundred microliters of IgA and IgG depleted sample were further measured for HI titer.
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Animal experiment. Ferrets aged 6-18 months were screened for influenza HI antibody with a
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2009 pandemic H1N1 strain. Three animals with no HI titer were randomly assigned into each
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experimental group. The animals were intra-tracheally inoculated with either MVCU-013/2009
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or 104/2009 at the dosage of 106 TCID50 in 1.2 ml volume. The animals were kept separately in
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animal isolators in a BSL-3 laboratory. The animals were observed clinically every day, and at
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day 10 post-infection, they were euthanized and lung tissue was collected and processed for
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pathological study and RNA extraction for viral load measurement. The tissue was cut in small
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pieces in TriZol solution, and RNA was extracted using the manufacture’s protocol. Viral RNA
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was measured by a real time RT-PCR using a primer pair for Influenza A M gene and TaqMan®
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probes (5’6-FAM and 3’BHQ1) (CDC protocol of real time RT-PCR for influenza A (H1N1)
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(22, 23).
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Statistical analysis. HI/NT titers and NT %Inhibition were shown in geometric mean with 95%
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Confidence Interval (CI) and arithmetic mean+SEM, respectively. The comparisons of HI and 7
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NT titers and NT %Inhibition were tested by paired/un-paired t-test (GraphPad Prism version 5.1
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Software).
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RESULTS
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H1N1 influenza viruses were less sensitive to human BAL
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BAL samples from 24 subjects were tested for their NT activity against two H1N1
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pandemic
(A/Nonthaburi/102/2009
and
A/Thailand/104/2009),
two
seasonal
H3N2
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(A/Thailand/Siriraj03/2004 and A/Thailand/Siriraj04/2003), and two seasonal H1N1 strains
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(A/Thailand/Siriraj03/2006 and A/Thailand/Siriraj12/2006). Together as a group, pandemic and
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seasonal H1N1 viruses had lower BAL NT titers as compared to the H3N2 virus (t-test, p