Markers of Apoptosis in different cell lines by Equine Arteritis Virus

ABEYÁ MM; METZ G. E.; SERENA M. S; PANEI, C:J; ASPITIA C; ECHEVERRÍA MG

Chicago

Congreso; 97th Annual Conference of Researcher Workers on Animal Diseases (CRWAD).; 2016

Markers of Apoptosis in different cell lines by Equine Arteritis Virus Abeya MM, Metz GE, Serena MS, Panei CJ, Aspitia CG, Echeverria MG The induction of apoptosis by Equine Arteritis Virus (EAV) was studied in vitro in three cell lines infected with different EAV strains. In this study we used the Bucyrus strain and infected three mammalian cells lines (Baby Hamster Kidney BHK-21, Rabbit Kidney RK-13 and Vero) to study the apoptosis markers of DNA fragmentation (DNA ladder), caspase activation, and morphology of apoptotic cells. Briefly, a monolayer of each cell line was infected with the Bucyrus strain at a multiplicity of infection (MOI) of 5 and then samples were taken at 24h, 48h, 72h and 96h post infection. To analyze genome fragmentation all samples were treated with phenol/chloroform to purify DNA and then gel electrophoresis was used to visualize.  Activation of caspase 3, 8 and 9 was analyzed in situ in the three cell lines using immunostaining with Amino Ethyl Carbazole (AEC). Staurosporine (1 ug/ml) and non-infected cells were used as positive and negative controls, respectively. The characteristic morphology of apoptotic cells was studied using Ethidium Bromide/Acridine Orange staining. DNA fragmentation was visualized at different times depending on the cell line used. The characteristic DNA ladder was detected at 48h post infection in RK-13 cell line, and at 72h and 96 h post infection in Vero and BHK-21 cell line, respectively. Activation of caspase 3, 8 and 9 was analyzed in the three cell lines at different times post infection. In RK-13 and Vero cell lines was found the activation of the three caspases. Caspase 8 was not detected in the BHK-21 cell line at any of the times analyzed. Using Ethidium Bromide/Acridine Orange staining, it was possible to visualize the apoptotic morphologic features in the three cell lines. In this work it was possible to analyze different apoptosis markers in three cell lines. We concluded that Equine Arteritis Virus induces apoptosis in the three cell lines but that the kinetics of such activation is different depending on the cell line.