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Induction of type I IFNs during viral infection is crucial for host defense. IRF 3 and IRF7 play a critical role as key transcription factors in the activation of the IFN induction. Viruses have evolved a variety of strategies to evade innate immunity. Our previous studies have shown that the nonstructural protein (NSs) of the severe fever with thrombocytopenia syndrome virus (SFTSV) can suppress the IFN-β induction through its interaction with tank-binding kinase-1 and sequestering the inhibitor of nuclear factor kappa B kinase(IKK) complex into the inclusion bodies formed by NSs. In this study, we characterized the unique function of IRF7 in innate immunity and its role in inducing IFN-α in particular, regulated by NSs during the SFTSV infection in several cell types of human origin. Whereas IRF3 is constitutively expressed, IRF7 was significantly induced differentially in various cell types in response to SFTSV infection, promoted the induction of IFN-α2 and -α4, and further induced IFN-β, thus contributing to suppressing the viral replication. Our data indicate that NSs directly interacted with and sequestered IRF7 into the inclusion bodies, which is different from IRF3 indirectly interacting with NSs. Although interaction of NSs with IRF7 did not inhibit IRF7 phosphorylation, p-IRF7 was trapped in the inclusion bodies, resulting in a significant reduction of the IFN-α2 and -α4 induction and therefore enhanced viral replication. Interaction of the viral NSs with both IRF7 and IRF3 and subsequent sequestration of these transcription factors into viral inclusion bodies, a unique strategy used by this phlebovirus, may ensure effective evasion and suppression of host innate immunity.

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little has been known about the function of synaptogyrin-2. Severe Fever with Thrombocytopenia Syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with a high fatality and caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that viral nonstructural protein NSs formed inclusion bodies (IBs) which are involved in viral immune evasion as well as in viral RNA replication. In this report, we sought to elucidate the mechanism how NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and ADRP. We identified synaptogyrin-2 to be highly upregulated in response to SFTS bunyavirus (SFTSV) infection through a high throughput screening and be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which was in parallel to the reduced number of the large IBs restructured from regular lipid droplets and increased antiviral interferon induction. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs, to be virus factories for viral RNA replication and immune evasion through its interaction with NSs. The findings unveiled the function of synaptogyrin-2 as an enhancer in viral infection.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease, which is caused by SFTS virus (SFTSV), a novel bunyavirus transmitted by ticks from animals. The disease is characterized by high fever and drastic reduction of leukocytes and platelets with high mortality rates in severe cases. We have reported that SFTSV suppresses host antiviral immunity and the viral nonstructural protein NSs plays a role in the immune evasion by interacting with downstream interferon signaling effectors, which is probably significant in virus pathogenesis. We furthered our study and demonstrated in this report that NSs interacted with mitochondrial antiviral signaling protein MAVS, the essential linker for host innate responses. NSs formed unique inclusion bodies (IBs) in the cytoplasm of infected cells and, through NSs and MAVS interaction, MAVS was translocated into the IBs in both transfected and infected cells. The interaction was specific and NSs did not interact with RIG-I or MDA5. Co-localization of NSs and MAVS trapped a significant portion of MAVS in the cytoplasmic IBs and weakened the functions of MAVS in activating downstream NFκB and IRF3 signaling. We examined host responses in NSs-overexpressing or infected cells and observed significant reduction of pro-inflammatory cytokine (IL-6, IL-8, Fas) and IFNs (IFN-α, IFN-β). Our data suggest that MAVS is essential to anti-SFTSV response since the knockdown of MAVS promoted viral replication via decreased IFN-β production, which could be reversed by overexpression of MAVS in the cells. This report demonstrates another player key to host responses, targeted by NSs of SFTSV through sequestration into viral IBs, resulting in inhibited IFN and NF-κB responses.

Most low pathogenicity avian influenza (LPAI) viruses cause no or mild diseases in avian species. The H9N2 subtype are commonly detected LPAI viruses that have crossed the species barrier to infect humans, unlike most other LPAI viruses. We report in this study that chicken macrophages are susceptible to infection with H9N2 and H6N2 viruses and infection led to apoptosis. Additionally, in H9N2 virus-infected macrophages, major histocompatibility complex (MHC) antigens, class II in particular, were downregulated, which also occurred in the lungs of H9N2 infected chickens. Pro-inflammatory cytokines and chemokines were differentially regulated in both infected macrophages and lungs. Among the regulated genes, IL-4, IL-4R, and CD74 were downregulated, all of which are pivotal in the activation of CD4+ helper T cells and humoral immunity. Remarkably, in H9N2 virus-infected chickens the antibody response was largely suppressed. This is in contrast to what was observed in those infected with H6N2 virus. This finding suggests that the pathogenesis of H9N2 infection in chickens may involve humoral immune suppression. Modulation of MHC antigens and the subsequent impact on the immune responses in mammals infected with avian influenza viruses is largely unclear. These findings raise questions about how some LPAI viruses regulate avian immune responses and if they have similar effects on mammalian immune function.

Avian influenza virus (AIV) H9N2 subtype has circulated in wild birds and domestic poultry, has successfully crossed the species boundary to transmit to and infect humans and have contributed to the generation of highly pathogenic H5N1 viruses. Little is known about host responses to H9N2 viruses in human airway respiratory epithelium. We used apically differentiated primary human tracheobronchial epithelial (hTBE) cultures to study host antiviral and apoptotic responses to an H9N2 virus strain. Compared to the immortalized bronchial epithelial cells HBE1, differentiated hTBE were less susceptible to the H9N2 virus. Among the antiviral cytokines, IFN-β was the prominent antiviral component in primary cultures. We found that IL-6, TNF-α, IP10 and CCL5 seemed to play important roles in proinflammatory responses while IL-1β and CCL5 were downregulated in primary hTBE cells. The AIV H9N2 virus caused apoptosis in both HBE1 and primary hTBE cultures. In addition to the extrinsic apoptotic pathway, we found that the mitochondria-mediated intrinsic cascade participated in H9N2-induced apoptosis, demonstrated by the cytosolic presence of active caspase 9 and cytochrome c. Further, our data indicated that FLIP and Bcl-2 family members Bax and Bcl-Xs appeared to be involved in the apoptotic regulation. The findings will further our understanding on host defense mechanisms and pathogenesis in human respiratory epithelium infected with AIV H9N2 virus.