Development of an ellspot for the detection of antibody secreting cells against the porcine epidemic diarrhoea virus (PEDV) in different tissues

摘要

Porcine Epidemic Diarrhoea Virus (PEDV) is a coronavirus that causes a gastroenteric disease, the porcine epidemic diarrhoea (FED). PEDV has been demonstrated to be a highly prevalent pathogen among the swine herds of a large area of Spain and manyother countries. Little is known about the immunity related to this virus and no effective prophylactic measures have been described. Due to the particular character of the mucosal immune system in swine, the local immunity in the gastrointestinal tractis the major key in the protection against the disease. The aim of this study was to develop an enzyme-linked immunospot assay (ELISPOT) to detect and quantify the antibody secreting cells primed by PEDV and located not only in the blood but in differentorgans like the gut associated lymphoid tissue. This would make it possible to determine the roles of local immunity in the gastrointestinal tract. Infected and fixed cell monolayers. Vero cells grown in 96-well microplates were inoculated with the cellculture adapted strain CV777 of PEDV. Different viral dilutions were tested in order to find the optimal multiplicity of infection. Infection was performed as described by Hofmann and Wyler with an incubation for 24 to 28 hours in a humid incubator at 37 deg C with 5 percent CO_2. After that, the plates were fixed with 80 percent acetone and kept frozen until use. Control plates were mock inoculafed and treated in the same way.