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United States Department of Agriculture

Agricultural Research Service

Research Project: APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS Title: A heterologous neuraminidase subtype strategy for the differentiation of infected and vaccinated animals (DIVA) for avian influenza virus using an alternative neuraminidase inhibition test

Authors
item Avellaneda, Gloria
item Sylte, Matthew
item Lee, Chang-Won - OHIO STATE UNIVERSITY
item Suarez, David

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 15, 2009
Publication Date: March 1, 2010
Citation: Avellaneda, G.E., Sylte, M.J., Lee, C., Suarez, D.L. 2010. A heterologous neuraminidase subtype strategy for the differentiation of infected and vaccinated animals (DIVA) for avian influenza virus using an alternative neuraminidase inhibition test. Avian Diseases. 54:272-277.

Interpretive Summary: Avian influenza virus can cause a serious disease in poultry and vaccination for control of avian influenza is being more commonly used. The most common vaccine used is the killed whole virus vaccine that produces high levels of protective antibody. However, this antibody is produced to almost all the influenza proteins, including the nucleoprotein which is targeted with the common serologic tests used to detect infection in poultry flocks. Therefore a common tool for tracking avian influenza virus infections in poultry flocks is lost. A proposed alternative is to use a vaccine that has a matched hemagglutinin subtype, but a mismatched neuraminidase protein in the vaccine. The hemagglutinin protein is much more important than the neuraminidase for protection. This approach allows the differentiation of infected from vaccinated and then infected animals (DIVA). This study looks at this DIVA strategy and showed that vaccinated birds when challenged, developed antibody to the mismatched neuraminidase protein in all the birds by 2 weeks after challenge. The vaccines were also completely protective using the mismatched neuraminidase approach and this method appears to be viable as a DIVA method.

Technical Abstract: The option of vaccinating poultry against avian influenza (AI) as a control tool is gaining greater acceptance by governments and the poultry industry world wide. One reservation about vaccination with killed whole virus vaccines is the loss of the ability to use commonly used serologic surveillance diagnostic tests to identify infected flocks. There has been considerable effort to develop a reliable test for the differentiation of infected from vaccinated animals (DIVA). The heterologous neuraminidase (NA) subtype DIVA approach has been used with some success in the field with an accompanying ad-hoc serological test. The traditional NA inhibition (NI) test can be used for all nine NA subtypes, but it is time consuming and it is not designed to screen large numbers of samples nor determine NA antibody titers. In this study, a quantitative NI test using MUN (2’-(4-methylumbelliferyl)-alpha-D-Nacetylneuraminic acid sodium salt hydrate) as a NA substrate was used as an alternative to the traditional NI test in a heterologous neuraminidase DIVA strategy. Serum NI activity was determined in chickens given different vaccines encoding different H5 and NA subtypes and challenged with a highly pathogenic avian influenza (HPAI) H5N2 virus. Prior to challenge, the NI DIVA test clearly differentiated chickens receiving different vaccine antigens (e.g., N8 or N9) from control birds that had no NA antibody. One vaccinated birds became positive one week post challenge and after two weeks post-challenge, 100% of the vaccinated birds had significant levels of N2 NI activity, activity that was not interfered with by the presence of vaccine-induced NI activity against N8 or N9. The N2 antibody levels continued to increase to the last sampling date 4 weeks post challenge showing a robust response. These results indicate that a DIVA strategy based on immune response to NA is a rapid and sensitive approach that effectively discriminates between the vaccinated and HPAI challenged chickens.

Last Modified: 11/24/2014
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