Prokaryotic Expression Of Avian Metapneumovirous F Protein And Establishment Of Indirect ELISA Method

Avian metapneumovirus (aMPV) mainly causes acute respiratory tract infection in turkeys and chickens, such as turkey rhinotracheitis and swollen head syndrome. Avian metapneumoviruses have been classified into four subtypes (A, B, C, and D), subtypes A and B are prevalent in most countries around the world, while subtype C is only present in the USA and to a limited degree in France and Korea. Subtype D infection in turkeys has only been reported in France. Serology investigation showed that aMPV infection in China was mainly caused by subtype A. In order to understand the prevalence of aMPV infection in China and establish a cost-effective method for detection of antibodies against aMPV infection, this study for the first time demonstrated the presence of subtype C aMPV infection in most Chinese local meat-type commercial chicken farms surveyed by typing RT-PCR, expressed the host-protective immunogen fusion (F) protein in prokaryotic expression system, and established an indirect ELISA for aMPV serological survey. The results are shown as followed.Firstly, it was demonstrated that aMPV infection in chickens is mainly caused by subtype C through the detection of collected materials from part of China by RT-PCR method with published universal primers of aMPV.Secondly, truncated F protein lacking partial hydrophobic regions from subtype A was expressed by prokaryotic expression system and shown to be antigen activity by WB test. Then the purified monomer protein was obtained by cutting the tag protein of expression vector with enterokinase.Finally, the purified monomer protein served as an antigen to coat a 96-well ELISA plate, and the best reaction condition was obtained, resulting in the successful establishment of an indirect ELISA method. Compared with the detection results of IDEXX aMPV antibody detection kit (America), the general coincidence rate was 95.73%, which suggested the established indirect ELISA method with well specificity and sensitivity.In conclusion, the presence of aMPV subtype C infection in the commercial meat-type chickens implicated that aMPV-C viruses may be a potential hazard for chickens, and the establishment of the indirect ELISA method based upon expressed F truncated protein will facilitate to effective control for aMPV infection in chickens.