To test the GFP expression, Vero E6 cells were infected with rZHDNSs-GFP using a MOI of 1. At 15 h post-infection, the cells were fixed for 30 min at room temperature with 4% paraformaldehyde in PBS, permeabilized for 10 min with 0.5% Triton X100 in PBS and incubated for 30 min at room temperature in 5% bovine serum albumin (BSA) in PBS. The cells were next incubated for 30 min at 37uC with a mouse anti-N antibody diluted in 5% BSA in PBS (dilution 1:800), washed with 5% BSA in PBS and incubated for 25 min at 37uC with the secondary antibody Alexa Fluor 555 goat anti-mouse (Invitrogen, Paisley, UK) diluted in 5% BSA in PBS (dilution 1:1200). Finally, the cells were washed with 5% BSA in PBS and then in water. The slides were mounted with Fluoromont-G (SouthernBiotech, Birmingham, AL). The cells were observed under an Axioplan 2 Imaging microscope (Zeiss, Le Pecq, France) using excitation and emission filter allowing simultaneous detection of GFP and Alexa Fluor 555.