ATUL KUMAR PATERIYA, REKHA KHANDIA, SANDEEP BHATIA, FATEH SINGH, PIYUSH SHARMA, DIVYANSHI GUPTA, H.V. MURUGKAR AND ANIKET SANYAL
Abstract
Avian influenza poses a significant global threat due to its zoonotic potential and the associated economic losses in the poultry industry. Inactivated vaccines targeting various viral subtypes encounter challenges, particularly in distinguishing between infected and vaccinated animals (DIVA). The nonstructural 1 (NS1) protein of avian influenza is conserved across all subtypes (H1-H16) and is only translated during viral replication, making it a suitable candidate for DIVA strategies. In this study, NS1 gene (801 bp) of avian influenza H5N1 subtype was amplified in RT-PCR and cloned in pET 28b (+) vector. The recombinant expression of NS1 (rNS1) was achieved in E.coli BL21(DE3) cells with a size of 36 kDa. The rNS1 was purified by Ni-NTA 6x HIS affinity tag and characterized with specific band in western blot using positive reference sera. The immunogenicity of rNS1 was evaluated by immunizing 6-8 week-old chickens with 10 ?g of rNS1 formulated with aluminum hydroxide Al(OH)3 adjuvant. The immune response was monitored through an optimized indirect ELISA using rNS1 protein, viral antigen and a virus neutralization test (VNT). A significant antibody response was detected 14 days post-immunization and remained stable until day 28. However, administration of a booster dose on day 21 failed in a substantial increase in antibody titers. Furthermore, sera collected on 14th 21st and 28th day failed to neutralize the virus in VNT assays. These results indicate that the NS1 protein of avian influenza exhibits low immunogenicity, but it has potential utility in developing DIVA strategies for use with purified inactivated avian influenza vaccines.