Ma 104

Marc145 cells that is a clone of the African green monkey kidney cell line MA-104, were obtained from China Center for type Culture Collection (CCTCC) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 °C in a humidified 5% CO₂ incubator. PRRSV strain WUH3, which was adapted to Marc145 cells at a MOI of 0.5. Virus stocks of PRRSV were prepared in Marc145 cells and infectious virus titer was analyzed in Marc145 cells using the Reed and Muench method [42 ]. For virus infection, cells were initially incubated with PRRSV for 1 h at 37 °C. After 1 h of adsorption, cells were exchanged to the medium containing 5% FBS and cultured for indicated time.

MA104 and BHK-21 cells (obtained from China Center For Type Culture Collection) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) at 37 °C under 5% CO₂. PRV (strain DN30209, isolated in a pig farm, in Heilongjiang province of China) [26 (link)] and vesicular stomatitis virus (VSV, strain Indiana, obtained from prof. Joerg Glende, University of Veterinary Medicine Hannover, Germany) were propagated on the MA104 cells and BHK-21 cells, respectively, as previously described [26 (link), 27 (link)]. In as much as previous reports showed that VSV infection on BHK-21 is not affected by depletion of cholesterol [27 (link)], we used VSV as a negative control for our studies about relationship between lipid rafts and virus infection.
Rabbit polyclonal antibodies to VP4 and VP7 were generated in our laboratory using previously described methods [26 (link), 28 ]. Anti-VSV G-protein polyclonal antibody was purchased from Abcam. An anti-β-actin monoclonal antibody was purchased from Beyotime. The secondary antibodies were purchased from BD Biosciences. Both MβCD and cholesterol were purchased from Sigma and reconstituted in DMEM and alcohol, respectively.