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Title: Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus

item Avellaneda, Gloria
item Suarez, David

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2009
Publication Date: 3/1/2010
Citation: Avellaneda, G.E., Mundt, E., Lee, C., Jadhao, S., Suarez, D.L. 2010. Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus. Avian Diseases. 54:278-286.

Interpretive Summary: Avian influenza virus can cause serious disease outbreaks in poultry and vaccination is commonly used to control the disease. However, vaccination with killed whole virus vaccines, the most commonly used type of vaccine, resulting in antibodies being produced to all proteins found in the virus particle. Unfortunately this results in the inability to easily distinguish between vaccinated and infected animals by standard serologic tests. It was proposed that if an influenza gene that was not found in the virus particle, but was produced in infected cells, was used in a serologic test, it might be possible to distinguish vaccinated from infected animals. Several previous studies supported this idea, and we tried to improve the process by developing a serologic test using the NS1 protein made from in a insect cell virus system. For birds infected with a low pathogenic strain of avian influenza, some but not all of the birds developed an antibody response to the NS1 protein. In vaccinated and then infected birds, a similar response was seen with only a few birds responding to the NS1 protein. Vaccinated only birds did not have an antibody response to the NS1 protein. The NS1 protein approach could be used to differentiate infected and vaccinated only birds, but the sensitivity of the test was lower than expected.

Technical Abstract: 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 world-wide acceptance if a reliable test that clearly discriminates naturally infected from vaccinated only animals (DIVA) was available. Because the non-structural protein (NS1) is expressed in influenza virus infected cells, and 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 three weeks after infection, but the antibody levels were decreasing rapidly by 5 weeks 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 three weeks 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 was used it would require sampling a higher number of birds to compensate for the lower seroconversion rate.