Targeted disruption of influenza A virus hemagglutinin in genetically modified. mice reduces viral replication and improves disease outcome
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1 Supplementary Information Targeted disruption of influenza A virus hemagglutinin in genetically modified mice reduces viral replication and improves disease outcome Song Wang 1#, Chao Chen 1#, Zhou Yang 1, Xiaojuan Chi 1, Jing Zhang 2, and Ji-Long Chen 1,2* 1 College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou , Fujian, China 2 Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing , China *Corresponding author: Ji-Long Chen. chenjl@im.ac.cn; Tel: ; Fax: # These authors contributed equally to this work.
2 Supplementary Figure S1 a b WSN 0h 16h PR8 0h 8h 16h HA HA HA1 HA1 NP NP β-actin β-actin Figure S1. HA and NP expressions in HA knockdown A549 cells. (a, b) A549 cells stably expressing shrnas targeting either HA (sh-ha-1) or luciferase control (sh-luc) were infected with WSN virus (a) or PR8 virus (b) at an MOI of 0.5 and harvested at the indicated times, followed by Western blotting using the indicated antibodies.
3 Supplementary Figure S WT WSN+ * Virus titer (10 3 PFU/ml) TG WSN+ * * Days post-infection Figure S2. Viral loads in the lungs of WT and TG mice. WT and TG mice intranasally infected with WSN virus were sacrificed at the indicated times, and the viral loads in the mice lungs were measured by plaque assay. *P= (WSN infection for 2 days); *P= (WSN infection for 3 days); P= (WSN infection for 4 days).
4 Supplementary Figure S3 a b WSN+ WT TG WT TG WSN- PR8- PR8+ WT TG WT TG Lung Lung Spleen Spleen Thymus Thymus Figure S3. Representative lung, spleen and thymus images of WT and TG mice infected with WSN or PR8 virus. WT and TG mice were mock infected or infected intranasally with WSN or PR8 virus for 4 days. Then mice were sacrificed, and the lungs, spleens and thymuses were collected. (a) and (b) are representative images from three independent experiments.
5 Supplementary Figure S4 a b WT TG WSN- WT TG PR8- WSN+ PR c d WT TG WT TG WSNPR8- WSN+ PR Figure S4. The pathological changes in spleens and thymuses of WT and TG mice after influenza virus infection. (a, b) WT and TG mice were intranasally infected with WSN (a) or PR8 (b) virus. Shown are representative micrographs (magnification, 100) of the mouse spleens stained with hematoxylin and eosin (HE). (c, d) WT and TG mice were intranasally infected with WSN (c) or PR8 (d) virus as described in (a) and (b). Shown are representative micrographs (magnification, 100) of the mouse thymuses stained with HE.
6 Supplementary Figure S5 a b Body Weight (%) TG in contact WT in contact Body Weight (%) TG in contact WT in contact Days post-infection Days post-infection Figure S5. The onward transmission of influenza virus in contact with influenza virus infected WT or TG mice. (a) Two groups of six unchallenged WT mice were housed with three WSN-infected WT mice (WT in contact) or TG mice (TG in contact) for 5 days. Shown is the average body weight changes of groups of six unchallenged WT mice. (b) Two groups of six unchallenged WT mice were housed with three PR8-infected WT mice (WT in contact) or TG mice (TG in contact) for 5 days. Shown is the average body weight changes of groups of six unchallenged WT mice.
7 Supplementary Table S1 Representative influenza A virus strains sharing the conserved sequences in the globular region of hemagglutinin located in HA1 domain. Influenza virus strains Sequence A/swine/Iowa/A /2015(H1N2) AGGTCAAACAG # GGAGGATGAACTATTACTGGACAATAGT A/Oklahoma/06/2014(H1N1) A/LongYan/SWL35/2013(H1N1) AGATCAAGAAGGGAGGATGAACTATTACTGGACACTAGT A/swine/Mie/R02/2012(H1N2) AGATCAGGCAGGGAGGATGAACTATTACTGGACATTGAT A/Beijing/38/2012(H1N1) A/swine/Tochigi/2/2011(H1N2) AGATCAGGCAGGGAGGATGAACTATTACTGGACATTGAT A/Hawaii/05/2011(H1N1) A/swine/Korea/4941/2010(H1N2) GGACCAAGAAGGGAGGATGAACTATTACTGGACACTAGT A/swine/Manitoba/02947/2010(H1N1) AGATCAAGCAGGGAGGATGAACTATTACTGGACATTAGT A/Kansas/21/2009(H1N1) A/swine/4/Mexico/2009(H1N1) GRRTCRAGAAGGGAGGATGAACTATTACTGGACACTAGT A/Hangzhou/4/2009(H1N1) A/swine/Saskatchewan/01974/2008(H1N1) AGATCAAGCAGGGAGGATGAACTATTACTGGACATTAGT A/swine/Manitoba/01781/2007(H1N1) AGATCAAGCAGGGAGGATGAACTATTACTGGACATTAGT A/swine/Alberta/SG1415/2006(H1N1) AGATCAAGCAGGGAGGATGAACTATTACTGGACATTAGT A/swine/Hong Kong/729/2005(H1N2) GGACCAAGCAGGGAGGATGAACTATTACTGGACACTAGT A/swine/Niigata/729/2004(H1N2) AAATCAAACAGGGAGGATGAACTATTACTGGACACTAAT A/swine/Changhua/199-3/2000(H1N1) AGGTCAGGCAGGGAGGATGAACTATTACTGGACATTACT A/swine/Chiba/1/1991(H1N2) AGGTCAAGCAGGGAGGATGAACTATTACTGGACACTAAT A/swine/Ehime/1/1980(H1N2) AGGTCAAGCAGGGAGGATGAACTATTACTGGACACTAAT A/swine/Tennessee/9/1978(H1N1) AGGTCAAGCAGGGAGGATGAACTATTACTGGACACTAAT A/swine/Hong Kong/26/1977(H1N1) AGGTCAAGCAGGGAGGATGAACTATTACTGGACACTAAT A/swine/Wisconsin/11/1976(H1N1) AGGTCAAGCAGGGAGGATGAACTATTACTGGACACTAAT A/New Jersey/8/1976(H1N1) AGGTCAAGCAGGGAGGATGAACTATTACTGGACACTAAT A/swine/Wisconsin/1/1968(H1N1) AGGTCAGGCAGGGAGGATGAACTATTACTGGACATTAAT A/swine/Wisconsin/1/1957(H1N1) AGGTCAAGCAGGGAGGATGAACTATTACTGGACATTAAT A/Hemsbury/1948(H1N1) AGATCAAGCAGGGAGGATGAACTATTACTGGACCCTGCT A/Puerto Rico/8/1934(H1N1) AGATCAAGCTGGGAGGATGAACTATTACTGGACCTTGCT A/WSN/1933(H1N1) AGATCAACATGGGAGGATGAACTATTACTGGACCTTGCT A/swine/JY2/1931(H1N1) AGGTCAGGCAGGGAGGATGAACTATTACTGGACATTACT A/South Carolina/1/1918 (H1N1) AGATCAAGCTGGGAGGATGAACTATTACTGGACATTACT A/duck/Hong Kong/91/1976(H9N2) TGGTCAGCAGGGGAGGATT * GACTATTATTGGTCGGTACT A/duck/Malaysia/2001(H9N2) TGGCCAGCAGGGGAGGATTGACTATTATTGGTCAGTACT A/duck/Jiangxi/3292/2009(H7N9) TGGCCAATCTGGAAGGATTGACTTTCATTGGCTGATGCT A/wild duck/korea/mhc39-26/2011(h7n9) TGGCCAATCTGGAAGGATTGACTTTCATTGGCTGATACT # The nucleotides in red font are the same with the target sequences. * The nucleotides in blue font are different from the target sequences.
8 Supplementary Table S2 Representative influenza A virus strains sharing the conserved sequences in the stem region of hemagglutinin located in HA2 domain. Influenza virus strains A/Xinjiang/1/2009(H5N1) A/Vietnam/UT /2008(H5N1) A/quail/Mingalardone/866/2007(H5N1) A/chicken/Bac Giang/07-74/2007(H5N1) A/duck/New York/484680/2007(H5N2) A/parrot/Guangdong/258/2004(H5N2) A/chicken/Texas/298313/2004(H5N2) A/chicken/Guatemala/ /2003(H5N2) A/swine/Gent/177/2002(H1N2) A/duck/NY/ /2002(H5N2) A/mallard/Maryland/786/2002(H5N2) A/chicken/TX/ /2002(H5N3) A/duck/NY/ /2002(H5N8) A/avian/New York/Sg-00372/2001(H5N2) A/mallard/Maryland/252/2001(H5N2) A/Avian/NY/53726/2000(H5N2) A/chukar/MN/ /1998(H5N2) A/mallard/MN/133/1998(H5N2) A/Mongolia/111/1991 (H1N1) A/Tokyo/3/1967(H1) A/BH/JY2/1935(H1N1) A/Puerto Rico/8/1934(H1N1) A/WSN/1933(H1N1) A/South Carolina/1/1918 (H1N1) A/chicken/Bangladesh/23527/2014(H9N2) A/chicken/Heilongjiang/u/1998(H9N2) A/Kansas/04/2015(H3N2) A/Hong Kong/485197/2014(H3N2) Sequence ATAATGAATGCATGGAAAGTGTAAGAAATGGAACGTATG ATAATGAATGCATGGAAAGTGTAAGAAATGGAACGTATG ATAATGAATGCATGGAAAGTGTAAGAAATGGAACGTATG ATAATGAATGCATGGAAAGTGTAAGAAATGGAACGTATG ACAATGAGTGCATGGAAAGTGTAAGAAATGGAACGTATG ACAATGAATGCATGGAAAGTGTAAGAAATGGAACATATA ACAATGAATGCATGGAAAGTGTAAGAAATGGAACTTATG ACAATGAGTGCATGGAAAGTGTAAGAAATGGAACTTATG ACAATGAGTGCATGGAAAGTGTAAGAAATGGAACGTATG ACAATGAATGCATGGAAAGTGTAAGAAATGGAACATATA ACAATGAGTGCATGGAAAGTGTAAGAAATGGAACGTATG ACAATGAGTGCATGGAAAGTGTAAGAAATGGAACGTATG ACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATG ACAATAAATGCATGGAAAGTGTAAGAAATGGGACTTATG ACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATG ACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATG ACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATG ACGATGCATGCATGGAAAGTGTAAGAAATGGGACTTATG ATGACCAATGCATGGAAACAATTCGAAATGGGACCTATA ATGATCAATGCATGGAAGCAATCAGAAATGGAACCTACG ACAATGCCTGCATAGGATCAATAAGAAATGGAACTTATG ACAATGCCTGCATAGGATCAATAAGAAATGGAACTTATG
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