Research project initiated and funded by the Central Veterinary Institute (sister-institute within Wageningen University and Research Centre), dr. Ruth Bouwstra, project leader and senior scientist and Sandra Venema, BSc.
Avian influenza (AI) outbreaks in poultry cause huge economic losses and are a major threat to public health worldwide. Migratory water birds form a natural reservoir of many different AI virus subtypes and pose a risk for introduction of these viruses to poultry. Low pathogenicity avian influenza (LPAI) viruses of the H5 and H7 subtypes, can evolve to high pathogenicity (HPAI) viruses and therefore are notifiable. Measures to control these subtypes have been included in European Union (EU) legislation. In the Netherlands a part of the surveillance programme is based on subtyping monitoring, where each commercial poultry flock is tested with ELISA for the presence of antibodies against nucleoprotein.The subtypes of the AI virus are characterised by two envelope proteins, i.e., haemagglutinin and neuraminidase. Although for monitoring purposes detection of only H5 and H7 subtypes is required, it is important from a public health point of view to determine all subtypes (for instance H6N1, H9N2 and H10N8). Therefore, gene constructs representing all H and N subtypes were produced using an in vitro expression system. The proteins produced are used to develop an influenza microarray. A first initiative of a similar microarray detecting some human influenza viruses was described in a paper by Koopmans et al. [Koopmans et al., 2012]. In the present study, we present an extension of this platform that also includes the avian haemagglutinin and neuraminidase subtypes (H1-H16 and N1-N9).
A protein microarray consisting of a set of 16 H-type antigens (45 in total) and 9 N-type antigens in a 14x14 microarray is printed on 16-pad nitrocellulose (NC) slides (FAST16) using a non-contact piezo-dispensing system (Scienion S3). Of every antigen a triplicate is printed, as well as a general Nucleoprotein (H1N1) and a positive control (chicken IgY). Each pad is incubated with selected serum samples according to ref. . Secondary antibody labeled with alkaline phosphatase is used to generate signals following a brief incubation with AP substrate. Precipitated dye on the spots is digitized to calculate the results. As a cut-off value the mean of the background spots + 3-times the standard deviation is taken (Signal-to-Noise-Ratio (SNR) = 1). Sometimes very faint positive results (SNR < 2) can be detected, most probably due to cross-reactivities.