Figure 4. Inhibition of viral replication in MDCK cells by receptor analogs.

MDCK cell-based multiple-round replication assay was used to determine the inhibition of ICV (A) and IDV (B) replication by receptor analogs. Aliquotes of IDV (D/bovine/Oklahoma/660/2013) and ICV (C/Johannesburg/1/66) containing 100 TCID₅₀ were respectively pretreated with Neu5,9Ac₂, Neu5Gc9Ac, or Neu5Ac receptor analogs at indicated concentrations for 30 min at 4 °C. MDCK cells were washed once with PBS and then infected with the virus-receptor mixtures for 1 h. Cells were washed 3 times and further incubated in DMEM containing 1 μg/ml of TPCK-trypsin for 6 h, and then lysed for RNA purification. The RNA samples were reverse transcribed using oligo(dT) primer. IDV or ICV NP gene and canine TBP housekeeping gene primers and probes were used in ddPCRs. Non-template controls (NTC) were included in every ddPCR run. The normalized results are shown as NP mRNA copies per million TBP, which represent mean±S.D. of 3 independent experiments performed in triplicate. Note that the different scale (10-fold difference) in Y-axis is used for panel 4A and 4B towards better visualization of influenza C virus replication and the inhibitory effects mediated by receptor analogs.