Abstract
Avian Influenza (AI) is a highly infectious viral disease mainly of birds caused by an orthomyxovirus with broad host range and a wide spectrum of disease manifestations, ranging from asymptomatic to fatal infections. Avian influenza virus (AIV) has been implicated in epizootic and endemic infections in poultry, wild birds as well as accidental infection in human with fatalities. The outbreak of H5N1 highly pathogenic avian influenza started in Bangladesh during 2007. Stamping out and vaccination are the strategies for the control of the disease in Bangladesh. Common sero-monitoring tools for AIV are ELISA and HI test. But the ELISA kits are solely imported from abroad, highly expensive and not readily available. In this context, the present study was conducted for in vitro expression of hemagglutinin protein of H5N1 AIV in Escherichia coli (E. coli) for its downstream application as immunodiagnostic and prophylactic agent. HA1 gene (960bp) of currently circulating clade 2.3.2.1 H5N1 HPAI virus was amplified by RT-PCR and directionally cloned in an expression plasmid vector, PET 161/GW/D-TOPOR. The recombinant vector-insert construct was first cloned into TOP10 E. coli and subsequently transferred to BL21 StarTM (DE3) E. coli. Expression of rH5_HA1 was induced with IPTG. rH5_HA1 was expressed as a fusion protein with tetra Cysteine Lumio tag and 6X Histidine tag. The Lumio tag and Histidine tag were used for fluorescent detection and nickel-agarose column purification of the expressed protein, respectively. The results of SDS-PAGE electrophoresis, Coomassie Brilliant Blue R-250 staining and Lumio Green fluorescent detection revealed that rHA1 fusion protein of ~40 kDa size was successfully expressed in E. coli and best over expression time was found to be 4 hours after induction with IPTG. The E. coli expressed purified rH5_HA1 protein was used as the antigen for the development of an indirect ELISA (ELISA) for the detection of antibody to H5 AIV and validated against HI test as well as a commercial cELISA. On chequer board titration optimum dilutions for antigen and sera were 1:100 (200 ng/well) and 1:500, respectively. The inter-assay coefficient of variation was between 0.09% and 3.13%. The positive-negative threshold baseline as determined from 20 known negative sera (as mean absorbance + 2SD) was 0.324 at 450 nm wavelength. The sensitivity and specificity of the newly developed iELISA calculated from the test results of 96 field sera were 98.55% and 100% as compared to HI test and 100% and 69.23% as compared to a commercial cELISA. The results of iELISA correlated well with that of HI (R2 = 0.885) and cELISA (R2 0.882). In immunogenicity study, rH5_HA1 produced good immune response in chickens on repeated immunization, however, the antibody level was relatively low as compared to a whole virus inactivated vaccine tested simultaneously. The indirect ELISA developed in the present study using E. coli expressed rH5_HA1 fusion protein as antigen is the first in-house development of ELISA in Bangladesh for the detection of antibody to H5AIV in chickens, and this can be used for sero- monitoring of H5AIV in chickens.