Figure 7.

The replication abilities of viruses with different NPs underexpression of eqMx1. (A) MDCK cells expressing HA-tagged eqMx1 were infected with wild type (H3N8_JL89) or mutant viruses (H3N8_JL89-H52N-NP) at an MOI of 0.001 and the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection. Total RNA from the collected supernatants was extracted using the RNeasy plus mini kit (Qiagen) and subjected to one-step real-time quantitative PCR (qPCR) analysis using the AgPath-ID™ One-Step RT-PCR reagents according to manufacturer’s protocol. Relative mRNA expression levels were determined using double-standard curve methods. All the experiments were performed three times and with three replicates with means ± SE shown. (B) MDCK cells expressing eqMx1 were infected with wild type (H3N8_JL89) or mutant viruses (H3N8_JL89-H52N-NP) at an MOI of 0.001 and the supernatants were collected at 0, 12, 24, 36, and 48 hr post-infection. These supernatants were subsequently used to infect MDCK cells at different dilutions (10⁻¹ to 10⁻¹¹) with at least four repeats. 48 hr post-infection, immunofluorescence assays (IFA) were performed using specific antibodies against viral NP (from our lab) and FITC-labelled secondary antibody. Finally the viral titers were calculated using Reed and Munech methodology and results are shown as TCID₅₀/mL. The results of a single experiment performed with four repeats are shown and results were subsequently confirmed in three separate experiments. Error bars indicate standard deviations (SD).