Fig. 1.

Replication of foot and mouth disease virus in mice peritoneal macrophages. (A) Detection of replicative negative-strand viral RNA by strand specific RT-PCR in mice macrophages after experimental infection with FMDV O/ IND/R2/75 at 2 MOI. Macrophages were collected at different time points from 0 to 24 h; total RNA was extracted and cDNA was prepared using a primer specific to the negative-strand RNA of FMDV. PCR revealed an amplicon of 249 bp specific to the virus. (B) Absolute quantitation of FMDV RNA in the peritoneal macrophages by real−time PCR. Total RNA was extracted from FMDV infected and uninfected (0 h) macrophages and quantitated by qRT−PCR using virus specific primers. The copy number of viral RNA in test samples was interpolated from the standards. The experiment was repeated thrice with n=3/time point. Representative data of a single experiment is presented. * indicates a p value < 0.05, ** indicates a p value < 0.01 and ***indicate a p value < 0.001. (C) Immunofluorescence demonstration of FMDV antigen in the peritoneal macrophages post−infection. Macrophage cells grown in 24-well tissue culture plates were (a) mock−infected and infected with FMDV for (b) 4 (c) 12 and (d) 18 h. After that cells were washed and fixed with 4 % paraformaldehyde and permeabilized with triton X100. Cells were incubated with a monoclonal antibody raised against the 3AB protein of FMDV. Anti−rabbit Alexa Fluor−488 conjugated IgG was used as secondary antibody. (D) Demonstration of virus progeny from infected macrophages. BHK−21 cells grown in 12−well tissue culture plates were (a) mock−infected or passaged with (b) cell lysate and (c) supernatant from 12 h FMDV infected macrophages. Appearance of CPE in (b) and (c) indicates the presence of infectious progeny virus in FMDV infected macrophages and supernatant. (E) Virus titre of FMDV infected cell lysate and supernatant at 4, 12 and 18 hpi. Supernatants and cells were collected separately at different time points and serially titrated on BHK−21 monolayers. The CPE was recorded after 2 days of incubation and the titre (TCID50) of infectious progeny virus in cell lysates and supernatants were calculated by Reed and Muench method. The results are from three independent experiments, expressed as the mean ± SD. Values with different lowercase superscripts (a, b) within a line are significantly different (P < 0.05).