Differentiation of Infected and Vaccinated Animals (DIVA) Using the NS1 Protein of Avian Influenza Virus Gloria Avellaneda, A Egbert Mundt, B Chang-Won Lee, C Samadhan Jadhao, A and David L. Suarez AD A Southeast Poultry Research Laboratory, Agriculture Research Service, 934 College Station Road, USDA-ARS, Athens, GA 30605 B Department of Infectious Diseases, School of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA 30605 C Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691 Received 5 February 2009; Accepted and published ahead of print 13 April 2009 SUMMARY. Vaccination against avian influenza (AI) virus, a powerful tool for control of the disease, may result in issues related to surveillance programs and international trade of poultry and poultry products. The use of AI vaccination in poultry would have greater worldwide acceptance if a reliable test were available that clearly discriminated between naturally infected and vaccinated-only animals (DIVA). Because the nonstructural protein (NS1) is expressed in influenza virus–infected cells, and it is not packaged in the virion, it is an attractive candidate for a DIVA differential diagnostic test. The aim of this work was to determine the onset of the antibody response to the NS1 protein in chickens infected with low pathogenic avian influenza (LPAI) virus, and to evaluate the diagnostic potential of a baculovirus-expressed purified NS1 protein in an indirect ELISA-based DIVA strategy. An antibody response against NS1 was first detected 3 wk after infection, but the antibody levels were decreasing rapidly by 5 wk after infection. However, most chickens did not have detectable antibodies in spite of high hemagglutination inhibition (HI) antibody titers in one group. In birds vaccinated with inactivated oil-emulsion vaccines, antibodies against NS1 were not detected before virulent challenge, and only a small percentage of birds seroconverted after homologous LPAI virus challenge. Vaccinated birds challenged with highly pathogenic AI showed a higher NS1 antibody response, but at most only 40% of birds seroconverted against NS1 protein by 3 wk after challenge. Because of the variability of seroconversion and the duration of the antibody response in chickens, the NS1 protein DIVA strategy did not perform as well as expected, and if this strategy were to be used, it would require sampling a higher number of birds to compensate for the lower seroconversion rate. RESUMEN. Diferenciacio ´n de animales infectados y vacunados (DIVA) utilizando la proteı ´na NS1 del virus de la influenza aviar. La vacunacio ´n contra la influenza aviar, que es una herramienta poderosa para el control de la enfermedad, puede resultar en problemas relacionados con los programas de vigilancia y comercio internacional de aves y productos avı ´colas. El uso de la vacunacio ´n contra la influenza aviar tendrı ´a mayor aceptacio ´n mundialmente si hubiera disponible una prueba que discriminara claramente entre animales infectados de manera natural y animales solamente vacunados (DIVA). Debido a que la proteı ´na no- estructural NS1 se expresa en la ce ´lulas infectadas con el virus de la influenza, y no se encuentra como parte del virio ´n, es una excelente candidata para una prueba de diagno ´stico diferencial basada en el concepto DIVA. El objetivo de este trabajo fue determinar el comienzo de la respuesta por anticuerpos hacia la proteı ´na NS1 en pollos infectados con un virus de influenza de baja patogenicidad y evaluar el potencial diagno ´stico de la proteı ´na purificada NS1 expresada en baculovirus dentro de una estrategia DIVA basada en una prueba de ELISA indirecta. La respuesta de anticuerpos contra NS1 fue detectada tres semanas despue ´s de la infeccio ´n, pero los niveles de anticuerpos decrecieron ra ´pidamente alrededor de las cinco semanas despue ´s de la infeccio ´n. Sin embargo, la mayorı ´a de los pollos no tenı ´an anticuerpos detectables a pesar de los tı ´tulos altos detectados con la prueba de inhibicio ´n de la hemaglutinacio ´n en un grupo. En las aves inmunizadas con vacunas inactivadas emulsionadas en aceite, los anticuerpos contra la proteı ´na NS1 no fueron detectados antes del desafı ´o virulento y solo un pequen ˜o porcentaje de las aves mostraron seroconversio ´n despue ´s del desafı ´o con un virus de la influenza de baja patogenicidad homo ´logo. Las aves vacunadas y desafiadas con influenza aviar de alta patogenicidad mostraron una respuesta de anticuerpos alta contra la proteı ´na NS1, pero solamente se observo ´ como ma ´ximo un 40% de seroconversio ´n contra la proteı ´na NS1 alrededor de las tres semanas despue ´s del desafı ´o. Debido a la variabilidad de la seroconversio ´n y a la duracio ´n de la respuesta de anticuerpos en pollos, la estrategia DIVA utilizando la proteı ´na NS1 no funciono ´ como se esperaba y si esta estrategia se utilizara, se requerirı ´a muestrear un nu ´mero mayor de aves para compensar el bajo nivel de seroconversio ´n. Key words: influenza, DIVA, NS1, ELISA, poultry Abbreviations: A/CK/California 5 A/CK/California/K0301417Ct/03 (H6N2); A/CK/Queretaro 5 A/CK/Queretaro/14588- 19/95 (H5N2); AI 5 avian influenza; AIV 5 avian influenza virus; A/TK/Wisconsin/68 5 A/TK/Wisconsin/68 (H5N9); BPL 5 b- propiolactone; DIVA 5 differentiating infected from vaccinated animals; ELISA 5 enzyme-linked immunosorbent assay; HPAI 5 highly pathogenic avian influenza; LPAI 5 low pathogenic avian influenza Influenza viruses are segmented, negative-strand RNA viruses belonging to the family Orthomyxoviridae, and they are divided in three types, A, B, and C (35). All three types of influenza are found in humans, but only the type A viruses are found in birds and are referred to as avian influenza (AI) viruses. Type A influenza viruses are divided into subtypes based on the antigenic properties of the major surface glycoproteins: the hemagglutinin (HA) and neur- aminidase (NA) proteins. In total, 16 HA subtypes (H1–H16) and nine neuraminidase subtypes (N1–N9) are described currently (35). AI viruses can be further divided into two distinct virulence groups based on the severity of the disease that they cause in chickens in standard pathotyping studies (35). Highly pathogenic avian influenza (HPAI) results in mortality of 75% or greater in chickens after intravenous inoculation and, in the field, typically results in flock mortality in susceptible species that may be as high as 100% D Corresponding author. E-mail: David.Suarez@ars.usda.gov AVIAN DISEASES 54:278–286, 2010 278