4.3. PBMCs infection with BTV

PBMCs were seeded in two six well plates for each species separately. One plate served as a control (uninfected cells) and the other was used for infection. PBMC's were infected with BTV-16 at 105.0 TCID₅₀/ml and incubated at 37 °C in 5% CO₂ incubator for 72 h. Cells were observed for morphological changes like formation of viral inclusion bodies and virus specific tubules at 24 h, 48 h and 72 h p.i. Since non-structural 1 (NS1) gene is conserved in all BTV serotypes [10], PCR and Real time PCR for NS1 gene expression were done to confirm viral infection, at all time points. As the expression of NS1 was observed to be significantly higher at 72 h p.i. in both the species PBMCs at this time point were considered for RNA sequencing.