540. Novel Pyrosequencing Assay for Genetic Characterization of Influenza A(H3N2) Viruses during 2014-15 Influenza Season
Session: Poster Abstract Session: Respiratory Viruses
Thursday, October 8, 2015
Room: Poster Hall
  • ID Week 2015_Spencer_10 1 2015_FINAL.pdf (397.6 kB)
  • Background:

    During the 2014-15 influenza season, the majority of circulating US influenza A(H3N2) viruses did not grow to sufficient hemagglutination titers for antigenic characterization by hemagglutination inhibition (HI). We evaluated a novel, high throughput assay to genetically characterize the hemagglutinin gene of viruses from patients enrolled in the US Flu Vaccine Effectiveness (VE) Network.


    Five Network sites enrolled outpatients with acute respiratory illness. The hemagglutinin gene from influenza A(H3N2) positive specimens was sequenced by MiSeq and/or pyrosequencing. Full length or pyrosequencing data was used to characterize the genetic group. Virus isolation was performed on a subset of specimens and viruses grown to sufficient titers were characterized by HI. Viruses were classified as vaccine-like if HI titer differed by ≤4-fold from the vaccine virus and as low reactors otherwise.


    Genetic group was determined by pyrosequencing for 1394 H3-positive specimens submitted to CDC from participating US Flu VE Network laboratories. 1131 (81%) belonged to genetic group 3C.2a, 159 (11%) to group 3C.3b, 48 (3%) to group 3C.3 and 56 (4%) to group 3C.3a. Both MiSeq and pyrosequencing were performed on 104 specimens, with 100% concordance between genetic groups determined by both methods. HI was performed on 59 influenza viruses; 16 (28%) were characterized as vaccine-like (including 10 viruses belonging to genetic group 3C.3b, 2 belonging to group 3C.3 and 4 3C.2a viruses) and 43 (72%) were classified as low-reactors to A/Texas/50/2012 (including 31 [74%] belonging to group 3C.2a). Among 26 specimens from which virus was not recovered and 76 H3N2 viruses with insufficient titer for HI that were genetically characterized, 11 (42%) and 68 (89%) belonged to genetic group 3C.2a.


    The new pyrosequencing assay improved genetic characterization of H3 viruses during 2014-15. The pyrosequencing assay provides results that are in agreement with those from full-length sequencing with the added advantage of a higher throughput.  

    Sarah Spencer, PhD1, Vasiliy Mishin, PhD1, Anton Chesnokov, MS1, Rebecca Garten, PhD2, Wendy Sessions, MPH1, Angela Foust, MS3, Pedro Piedra, MD4, Manjusha Gaglani, MD (MBBS)5, Rachel Cross, MPH6, Emileigh Johnson, BS7, Charles Rinaldo, PhD8, Richard K. Zimmerman, MD MPH, FIDSA9, Jennifer Meece, PhD10, Huong Q. Mclean, MPH, PhD11, Michael L. Jackson, PhD, MPH12, John R. Barnes, PhD1, Xiyan Xu, PhD1, Jessie Clippard, MPH13, Swathi Thaker, PhD14, Brendan Flannery, PhD3, Larisa Gubareva, PhD1 and Alicia M. Fry, MD, MPH1, (1)Centers for Disease Control and Prevention, Atlanta, GA, (2)Centers for Diesease Control and Prevention, Atlanta, GA, (3)Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, (4)Baylor College of Medicine, Houston, TX, (5)Baylor Scott & White Health, Texas A&M HSC COM, Temple, TX, (6)Department of Epidemiology, University of Michigan School of Public Health, Ann Arbor, MI, (7)University of Michigan School of Public Health, Ann Arbor, MI, (8)University of Pittsburgh Schools of the Health Sciences and UPMC, Pittsburgh, PA, (9)Family Medicine, University of Pittsburgh, Pittsburgh, PA, (10)Integrated Research and Diagnositic Laboratory, Marshfield Clinic Research Foundation, Marshfield, WI, (11)Center for Clinical Epidemiology and Population Health, Marshfield Clinic Research Foundation, Marshfield, WI, (12)Group Health Research Institute, Seattle, WA, (13)Atlanta Research and Education Foundation, Atlanta, GA, (14)Battelle Memorial Institute, Atlanta, GA


    S. Spencer, None

    V. Mishin, None

    A. Chesnokov, None

    R. Garten, None

    W. Sessions, None

    A. Foust, None

    P. Piedra, None

    M. Gaglani, MedImmune: Investigator , Research support

    R. Cross, None

    E. Johnson, None

    C. Rinaldo, None

    R. K. Zimmerman, Merck & Co.: Grant Investigator , Research grant
    Pfizer: Grant Investigator , Research grant
    Sanofi Pasteur: Grant Investigator , Research grant

    J. Meece, None

    H. Q. Mclean, MedImmune: Investigator , Research support

    M. L. Jackson, None

    J. R. Barnes, None

    X. Xu, None

    J. Clippard, None

    S. Thaker, None

    B. Flannery, None

    L. Gubareva, None

    A. M. Fry, None

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