Studies on in vitro interaction of bovine lymphocytes with foot-and-mouth disease virus
Abstract
The present study was conducted to study the in-vitro interaction of bovine lymphocytes with Foot-and-Mouth Disease Virus (FMDV) types O, A and Asia-1. Peripheral blood samples collected from six unvaccinated cow calves and six vaccinated cow calves were used for separation of peripheral blood mononuclear cells (PBMCs). These PBMCs were employed to study the alteration of different T-cell subpopulations i.e. boCD4+, boCD8+ and boWC1+ T-cells, Functional competence of PBMCs using lymphocyte proliferation assay, detection of FMD virus by RT-PCR and expression of 3A-NSP antigen at different hours of post in-vitro infection (hpi) with either FMDV types O or A or Asia-1. The alterations in T-lymphocyte subpopulation were analyzed by cyto-flurometric analysis using T-cell subpopulation specific monoclonal antibodies (mAb) at various hpi. It was demonstrated that FMDV types (O, A, and Asia-1) down-regulate boCD4+ and boCD8+ T cells up to 48 hpi of peripheral blood lymphocytes from unvaccinated cattle calves whereas down regulation of boWC1+ cells was seen up to 48 hpi with FMDV type O only. However, in contrast to unvaccinated animals, lymphocytes from vaccinated animals showed significant up-regulation of boCD4+, boCD8+ and boWC1+ T-cell following exposure to FMD virus serotypes O, A and Asia-1.
Lymphoproliferative test was used to characterize functional competence of PBMCs obtained from unvaccinated and vaccinated animals following in-vitro infection with either FMDV types O or A or Asia-1 using PHA or inactivated FMDV type O, A and Asia-1 antigens. The finding revealed that PHA driven LPR was significantly depressed at 24, 48 and 72 hpi in unvaccinated animals. Homotypic as well as heterotypic FMDV antigen specific LPRs were depressed in PBL cultures of unvaccinated animals
which were infected with either FMDV type O or A or Asia-1 and stimulated with FMDV antigens.
FMDV replication was demonstrated by Reverse transcription-PCR (RT-PCR) and 3A-NSP FMDV antigen expression in PBMCs of unvaccinated and vaccinated cattle calves infected in-vitro with FMDV serotype O, A and Asia-1 at 12, 24, 48 and 72 hpi.. Agarose gel electrophoresis analysis of amplified product, revealed 1D gene specific product of FMD virus serotype O (1301bp product), A (866bp product) and Asia-1 (914bp product) in lymphocytes of unvaccinated calves and vaccinated calves at different hpi with either FMDV type O or A or Asia-1, respectively. The 3A-NSP mAb based flowcytometry revealed significant increase in 3A-NSP antigen expression in lymphocytes of unvaccinated calves and vaccinated calves at different hpi with either FMDV type O or A or Asia-1. The interaction of macrophages with FMDV was demonstrated using MTT dye based assay on macrophages at various hpi (24, 48 and 72hrs). Macrophages obtained from unvaccinated and vaccinated calves when incubated in presence of FMD virus serotype O, A and Asia 1 showed significant depletion of macrophages at 48 and 72 hpi. FMDV cDNA in in-vitro FMDV infected macrophages was detected at 48 and 72 hpi.