Contains protocols for virus detection, isolation and propagation, and virus characterization Includes cellular immunological methods and immunohistochemistry with a focus on avian hosts Covers reverse genetics with avian influenza virus Details wild bird monitoring, including sampling methods and virus detection With the growing global fear of a major pandemic, avian influenza virus research has increased greatly in importance during this young century. In Avian Influenza Virus, an expert team of researchers and diagnosticians examine the fundamental yet essential virological methods for AI virus research and diagnostics as well as some of the newest molecular procedures currently used for basic and applied research. Exciting, cutting-edge new methods focus on studying the virus itself and work with avian hosts, an area greatly lacking in research. Following the format of the highly successful Methods in Molecular Biology? series, each chapter provides readily reproducible laboratory protocols offering step-by-step instruction and lists of the necessary equipment for the task. Comprehensive and timely, Avian Influenza Virus equips diagnosticians and researchers with the current tools and information they need to learn more about this high impact disease.

Preface v Contributors ix A Brief Introduction to the Avian Influenza Virus Erica Spackman 1 Avian Influenza Virus Sample Types, Collection, and Handling Mary Lea Killian 7 Avian Influenza Virus RNA Extraction from Tissue and Swab Material Erica Spackman David L. Suarez 13 Type A Influenza Virus Detection and Quantitation by Real-Time RT-PCR Erica Spackman David L. Suarez 19 Detection and Identification of the H5 Hemagglutinin Subtype by Real-Time RT-PCR Erica Spackman David L. Suarez 27 Avian Influenza Virus Isolation and Propagation in Chicken Eggs Peter R. Woolcock 35 Hemagglutination Assay for the Avian Influenza Virus Mary Lea Killian 47 Hemagglutination-Inhibition Test for Avian Influenza Virus Subtype Identification and the Detection and Quantitation of Serum Antibodies to the Avian Influenza Virus Janice C. Pedersen 53 Neuraminidase-Inhibition Assay for the Identification of Influenza A Virus Neuraminidase Subtype or Neuraminidase Antibody Specificity Janice C. Pedersen 67 Immunohistochemical Staining for the Detection of the Avian Influenza Virus in Tissues Mary J. Pantin-Jackwood 77 Wild Bird Surveillance for the Avian Influenza Virus Justin D. Brown David E. Stallknecht 85 Reverse Genetics of the Avian Influenza Virus Chang-Won Lee David L. Suarez 99 Evaluating the Cell-Mediated Immune Response of Avian Species to Avian Influenza Viruses Darrell R. Kapczynski 113 Measurement of Avian Cytokines with Real-Time RT-PCR Following Infection with the Avian Influenza Virus Darrell R. Kapczynski Michael H. Kogut 127 Index 135