Abstract: Objective To study inhibitory effect of unmethylated synthetic CpG-containing oligodeoxynucleotide(CpG-ODN) motif on influenza virus replication in vitro.Methods After the exposure to CpG-ODN,the dynamic expression of Toll-like receptor 9(TLR9)and cytokines in Hep-2 cells were then determined with real-time PCR.After Hep-2 cells were pre-incubated with CpG-ODN and exposed to seasonal influenza A H1N1(sH1N1) virus,the copies of intracellular influenza virus were measured with real-time PCR to assess the effects of CpG exposure of different dose and time on influenza virus replication.Results TLR9 mRNA expression reached the peak at 72 hours post CpG-ODN treatment,which was 18.6±3.6 times of that of the control group(P<0.05)and the expressions of interleukin-6(IL-6),interferon-β(IFN-β),and tumour necrosis factor-α(TNF-α)were also reached the highest level,which were 96.2±21.4,33.7±5.4,and 17.2±3.4 times of those of the control group,respectively(all for P<0.05).Viral copies were reduced by the pretreatment of CpG-ODN in a dose-and time-dependent manner,with the maximal inhibition when Hep-2 cells were pretreated with CpG-ODN(5μM)for 24 hours(P<0.05).Conclusion The results indicate an innate immune response activation in Hep-2 cells and affirm the antiviral role of CpG-ODN on seasonal influenza A virus.