The critical role of the IFN system in controlling influenza virus transmission was first demonstrated in a classical experiment wherein mice infected with influenza virus and injected with anti-IFN serum were observed to produce higher titers of the virus and an exacerbated pathology . Subsequent studies in knockout mice with defects in their IFN system have confirmed this basic observation . Recent studies in which the IFN-inducing capacity varied in genetically related A-type influenza virus showed that the strain capable of producing the most IFN was attenuated in its pathogenesis in mice [27, 28]. The above researches indicate that IFN plays an important role in limiting human influenza virus or avian influenza virus pathogenesis or transmission.
Since the first reported human case of avian influenza virus H5N1 infection in Hong Kong in 1997 , the avian to human or human to human transmission of H5N1 poses a potential threat to human health. Aquatic birds are considered the natural host of avian influenza virus H5N1 . But recently, some terrestrial birds, such as tree sparrows, have been found carrying H5N1 with no symptoms . This indicates that the host range of avian influenza virus maybe expanding. In addition, some new H5N1 genotypes are emerging through gene reassortment [13, 31]. How those avian influenza viruses pose a threat to humans deserves further study. In the current assay, we chose a new genotype avian influenza virus A/tree sparrow/Henan/1/04(H5N1) isolated from a free-living tree sparrow in Henan Province of Mainland China and studied its modulation of IFN-β and RIG-I in mammalian cells.
In contrast to human influenza virus H1N1, A/tree sparrow/Henan/1/04(H5N1) infection in mammalian cells resulted in rapid IFN-β production during the early stage of infection, but in the late stage, it significantly inhibited the amplification of IFN-β production and showed the same pattern as the Indian isolate of pandemic (H1N1) 2009 influenza virus . Previous studies have demonstrated that highly pathogenic avian influenza virus H5N1 replicated more efficiently than human influenza viruses due to its PA, PB1, and PB2 [33–35]. Our results also proved that A/tree sparrow/Henan/1/04(H5N1) was consistent with other H5N1 strains and that it rapidly and efficiently amplified in mammalian cells, which resulted in the rapid activation of IFN-β signaling in early infection. However, accompanying rapid replication and viral cytoplasmic RNA accumulating, why the rapid IFN-β production was followed with sharp downregulation during H5N1 infection is unknown. In this study, we observed that although A/tree sparrow/Henan/1/04(H5N1) infection caused rapid IFN-β production and STAT-1 phosphorylation during the early stage of infection, it only induced low level endogenous RIG-I expression in comparison to H1N1 in A549 cells. Furthermore, when preinfecting the 293T cells, H5N1 significantly inhibited the endogenous RIG-I expression induced by exogenous interferon than H1N1. Previous investigation showed that low level RIG-I existed in quiescent epithelial cells and ready to sense invading RNA . When the primary interferon produced, it increased RIG-I expression, which again participates in sensing invading RNA and amplifying interferon production. So inhibiting RIG-I expression unavoidly interrupt the above positive feed-back loop. According to these results we assume that manipulating RIG-I expression might be adopted by influenza virus at least as one of strategies antagonizing host’s innate immune response.
Although a latest literature found the polymerase of H5N1 was involved in antagonzing interferon production in chicken macrophage HD-11 cells , the NS1 has been regarded as the key component inhibiting interferon production through targeting many aspects of interferon signalling pathway . In this study NS1 from H5N1 showed more potent inhibition to exogenous interferon than NS1 from H1N1 in the same level of expression. The same level of NS1 from different subtype or strain of influenza virus exhibiting diverse antagonizing capacity might attribute to its property [38, 39]. So, the accumulating NS1 protein during influenza virus infection might be responsible for the differences in inducing interferon or RIG-I expression. Based on these results, during the early stage of infection, H5N1 replicates more efficiently than H1N1 and induced rapid IFN-β production, but in the late stage of infection, it significantly inhibits IFN-β production with NS1 accumulation as showed in influenza C virus NS1 .