RIG-I-Like Receptor and Toll-Like Receptor Signaling Pathways Cause Aberrant Production of Inflammatory Cytokines/Chemokines in a Severe Fever with Thrombocytopenia Syndrome Virus Infection Mouse Model.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a tick-borne phlebovirus of the family Bunyaviridae, SFTS virus (SFTSV). Wild-type and type I interferon (IFN-I) receptor 1-deficient (IFNAR1-/-) mice have been established as nonlethal and lethal models of SFTSV infection, respectively. However, the mechanisms of IFN-I production in vivo and the factors causing the lethal disease are not well understood. Using bone marrow-chimeric mice, we found that IFN-I signaling in hematopoietic cells was essential for survival of lethal SFTSV infection. The disruption of IFN-I signaling in hematopoietic cells allowed an increase in viral loads in serum and produced an excess of multiple inflammatory cytokines and chemokines. The production of IFN-I and inflammatory cytokines was abolished by deletion of the signaling molecules IPS-1 and MyD88, essential for retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) and Toll-like receptor (TLR) signaling, respectively. However, IPS-1-/- MyD88-/- mice exhibited resistance to lethal SFTS with a moderate viral load in serum. Taken together, these results indicate that adequate activation of RLR and TLR signaling pathways under low to moderate levels of viremia contributed to survival through the IFN-I-dependent antiviral response during SFTSV infection, whereas overactivation of these signaling pathways under high levels of viremia resulted in abnormal induction of multiple inflammatory cytokines and chemokines, causing the lethal disease.IMPORTANCE SFTSV causes a severe infectious disease in humans, with a high fatality rate of 12 to 30%. To know the pathogenesis of the virus, we need to clarify the innate immune response as a front line of defense against viral infection. Here, we report that a lethal animal model showed abnormal induction of multiple inflammatory cytokines and chemokines by an uncontrolled innate immune response, which triggered the lethal SFTS. Our findings suggest a new strategy to target inflammatory humoral factors to treat patients with severe SFTS. Furthermore, this study may help the investigation of other tick-borne viruses.

NSs Protein of Sandfly Fever Sicilian Phlebovirus Counteracts Interferon (IFN) Induction by Masking the DNA-Binding Domain of IFN Regulatory Factor 3.

Sandfly fever Sicilian virus (SFSV) is one of the most widespread and frequently identified members of the genus Phlebovirus (order Bunyavirales, family Phenuiviridae) infecting humans. Being transmitted by Phlebotomus sandflies, SFSV causes a self-limiting, acute, often incapacitating febrile disease ("sandfly fever," "Pappataci fever," or "dog disease") that has been known since at least the beginning of the 20th century. We show that, similarly to other pathogenic phleboviruses, SFSV suppresses the induction of the antiviral type I interferon (IFN) system in an NSs-dependent manner. SFSV NSs interfered with the TBK1-interferon regulatory factor 3 (IRF3) branch of the RIG-I signaling pathway but not with NF-κB activation. Consistently, we identified IRF3 as a host interactor of SFSV NSs. In contrast to IRF3, neither the IFN master regulator IRF7 nor any of the related transcription factors IRF2, IRF5, and IRF9 were bound by SFSV NSs. In spite of this specificity for IRF3, NSs did not inhibit its phosphorylation, dimerization, or nuclear accumulation, and the interaction was independent of the IRF3 activation or multimerization state. In further studies, we identified the DNA-binding domain of IRF3 (amino acids 1 to 113) as sufficient for NSs binding and found that SFSV NSs prevented the association of activated IRF3 with the IFN-ß promoter. Thus, unlike highly virulent phleboviruses, which either destroy antiviral host factors or sequester whole signaling chains into inactive aggregates, SFSV modulates type I IFN induction by directly masking the DNA-binding domain of IRF3.IMPORTANCE Phleboviruses are receiving increased attention due to the constant discovery of new species and the ongoing spread of long-known members of the genus. Outbreaks of sandfly fever were reported in the 19th century, during World War I, and during World War II. Currently, SFSV is recognized as one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naive troops and travelers. We show how the nonstructural NSs protein of SFSV counteracts the upregulation of the antiviral interferon (IFN) system. SFSV NSs specifically inhibits promoter binding by IFN transcription factor 3 (IRF3), a molecular strategy which is unique among phleboviruses and, to our knowledge, among human pathogenic RNA viruses in general. This IRF3-specific and stoichiometric mechanism, greatly distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of SFSV.

The cholesterol, fatty acid and triglyceride synthesis pathways regulated by site 1 protease (S1P) are required for efficient replication of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a high mortality rate. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV infection. Here, we report that the cholesterol, fatty acid, and triglyceride synthesis pathways regulated by S1P is involved in SFTSV replication, using CHO-K1 cell line (SRD-12B) that is deficient in site 1 protease (S1P) enzymatic activity, PF-429242, a small compound targeting S1P enzymatic activity, and Fenofibrate and Lovastatin, which inhibit triglyceride and cholesterol synthesis, respectively. These results enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection.

Suppression of type I and type III IFN signalling by NSs protein of severe fever with thrombocytopenia syndrome virus through inhibition of STAT1 phosphorylation and activation.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen causing significant morbidity and mortality in Asia. NSs protein of SFTSV is known to perturb type I IFN induction and signalling, but the mechanism remains to be fully understood. Here, we showed the suppression of both type I and type III IFN signalling by SFTSV NSs protein is mediated through inhibition of STAT1 phosphorylation and activation. Infection with live SFTSV or expression of NSs potently suppressed IFN-stimulated genes but not NFkB activation. NSs was capable of counteracting the activity of IFN-α1, IFN-ß, IFN-λ1 and IFN-λ2. Mechanistically, NSs associated with STAT1 and STAT2, mitigated IFN-ß-induced phosphorylation of STAT1 at S727, and reduced the expression and activity of STAT1 protein in IFN-ß-treated cells, resulting in the inhibition of STAT1 and STAT2 recruitment to IFNstimulated promoters. Taken together, SFTSV NSs protein is an IFN antagonist that suppresses phosphorylation and activation of STAT1.

Evasion of antiviral immunity through sequestering of TBK1/IKKε/IRF3 into viral inclusion bodies.

Cells are equipped with pattern recognition receptors (PRRs) such as the Toll-like and RIG-I-like receptors that mount innate defenses against viruses. However, viruses have evolved multiple strategies to evade or thwart host antiviral responses. Viral inclusion bodies (IBs), which are accumulated aggregates of viral proteins, are commonly formed during the replication of some viruses in infected cells, but their role in viral immune evasion has rarely been explored. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness caused by a novel phlebovirus in the Bunyaviridae. The SFTS viral nonstructural protein NSs can suppress host beta interferon (IFN-ß) responses. NSs can form IBs in infected and transfected cells. Through interaction with tank-binding kinase 1 (TBK1), viral NSs was able to sequester the IKK complex, including IKKε and IRF3, into IBs, although NSs did not interact with IKKε or IRF3 directly. When cells were infected with influenza A virus, IRF3 was phosphorylated and active phosphorylated IRF3 (p-IRF3) was translocated into the nucleus. In the presence of NSs, IRF3 could still be phosphorylated, but p-IRF3 was trapped in cytoplasmic IBs, resulting in reduced IFN-ß induction and enhanced viral replication. Sequestration of the IKK complex and active IRF3 into viral IBs through the interaction of NSs and TBK1 is a novel mechanism for viral evasion of innate immunity.

Targeting of severe fever with thrombocytopenia syndrome virus structural proteins to the ERGIC (endoplasmic reticulum Golgi intermediate compartment) and Golgi complex.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) is a newly emerged phlebovirus identified in China, Japan, and South Korea. Phlebovirus glycoproteins (GP) play a key role in targeting viral structural components to the budding compartments in the ER-Golgi intermediate compartment (ERGIC) and Golgi complex. However, the role of SFTSV GP in targeting structural proteins to the ERGIC and Golgi complex remains unresolved. In this study, we show that SFTSV GP plays a significant role in targeting RNA-dependent RNA polymerase (L) and nucleocapsid protein (NP) to the budding sites. Confocal microscopy was used to investigate the subcellular localization of SFTSV structural proteins. In SFTSV-infected cells, GP and L localized to the ER, ERGIC and Golgi complex, whereas NP localized to the ERGIC and Golgi complex. In addition, GP colocalized with L and NP in infected cells. In cells singly transfected with GP, L or NP, GP localized to the same subcellular compartments as in infected cells. However, L or NP alone did not localize to the ER, ERGIC, or Golgi complex. Cotransfection experiments showed that GP altered the localization of L to the ERGIC and Golgi complex but not that of NP. Interestingly, plasmid-expressed NP fused with a hemagglutinin tag localized to the ERGIC and Golgi complex when expressed in SFTSV-infected cells and colocalised with GP, suggesting that GP plays a role in the subcellular localization of L and NP in infected cells. Thus, the SFTSV structural components start to assemble at the ERGIC to Golgi complex. GP is required for transporting L and NP to the ERGIC and Golgi complex. In addition, targeting of NP requires interaction with other factors besides GP.

SFTS phlebovirus promotes LC3-II accumulation and nonstructural protein of SFTS phlebovirus co-localizes with autophagy proteins.

Autophagy is essential for eukaryotic cell homeostasis and can perform both anti-viral and pro-viral roles depending on the kinds of viruses, cell types and cell environment. Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) is a newly discovered tick-borne virus in the Phenuiviridae family that causes a severe hemorrhagic fever disease in East Asia. In this study we determined interactions between SFTSV and autophagy. Our results showed that LC3-II (microtubule associated protein 1 light chain 3-II) protein accumulated from 4 h to 24 h after SFTSV infection compared to mock-infected Vero cells, and the use of E64d and pepstatin A did not affect the expression of LC3-II protein, which indicated that the increased LC3-II may be the result of inhibition of autophagic degradation caused by SFTSV infection. However, knockdown of LC3B promotes SFTSV replication, which indicated a negative role of LC3B protein in SFTSV replication. We also detected co-localization of SFTSV non-structure (NSs) protein with LC3B, p62 and Lamp2b respectively in SFTSV infected Vero cells, which indicated the possibility of selective autophagy or chaperone-mediated autophagy involving in SFTSV infection. Our results indicated that SFTSV infection promotes LC3 accumulation and several proteins of the autophagy pathway co-localize with NSs protein during SFTSV infection.

Relationship between serum chromium concentrations and glucose utilization in normal and infected subjects.

Studies in healthy individuals demonstrate that serum chromium concentrations fall precipitiously following the intravenous administration of a 30-gm. glucose load. Significant decreases from baseline control fasting serum Cr concentrations were also observed when intravenous glucose was given during sandfly fever. Glucose disappearance rates also decreased significantly to approximately one half of pre-illness control values while serum Cr values declined still further. In addition, serum Cr disappearance rates could be calculated. When individual preexposure and postexposure serum glucose and Cr disappearance rates were compared, significant linear correlation was found (P smaller than 0.05). Acute infection appears to reduce the availability of circulating Cr, which may contribute to the altered glucose metabolism characteristic of acute infections even in the presence of elevated insulin levels and other hormonal changes.

Truncation of the C-terminal region of Toscana Virus NSs protein is critical for interferon-ß antagonism and protein stability.

Toscana Virus (TOSV) is a Phlebovirus responsible for central nervous system (CNS) injury in humans. The TOSV non-structural protein (NSs), which interacting with RIG-I leads to its degradation, was analysed in the C terminus fragment in order to identify its functional domains. To this aim, two C-terminal truncated NSs proteins, Δ1C-NSs (aa 1-284) and Δ2C-NSs (aa 1-287) were tested. Only Δ1C-NSs did not present any inhibitory effect on RIG-I and it showed a greater stability than the whole NSs protein. Moreover, the deletion of the TLQ aa sequence interposed between the two ΔC constructs caused a greater accumulation of the protein with a weak inhibitory effect on RIG-I, indicating some involvement of these amino acids in the NSs activity. Nevertheless, all the truncated proteins were still able to interact with RIG-I, suggesting that the domains responsible for RIG-I signaling and RIG-I interaction are mapped on different regions of the protein.

Bovine lactoferrin inhibits Toscana virus infection by binding to heparan sulphate.

Toscana virus is an emerging sandfly-borne bunyavirus in Mediterranean Europe responsible for neurological diseases in humans. It accounts for about 80% of paediatric meningitis cases during the summer. Despite the important impact of Toscana virus infection-associated disease on human health, currently approved vaccines or effective antiviral treatments are not available. In this research, we have analyzed the effect of bovine lactoferrin, a bi-globular iron-binding glycoprotein with potent antimicrobial and immunomodulatory activities, on Toscana virus infection in vitro. Our results showed that lactoferrin was capable of inhibiting Toscana virus replication in a dose-dependent manner. Results obtained when lactoferrin was added to the cells during different phases of viral infection showed that lactoferrin was able to prevent viral replication when added during the viral adsorption step or during the entire cycle of virus infection, demonstrating that its action takes place in an early phase of viral infection. In particular, our results demonstrated that the anti-Toscana virus action of lactoferrin took place on virus attachment to the cell membrane, mainly through a competition for common glycosaminoglycan receptors. These findings provide further insights on the antiviral activity of bovine lactoferrin.

Magnitude of the host nutritional responses to infection.

This review describes the scope, complexity, and magnitude of host nutritional responses throughout the course of an infectious process. These responses include prominent changes in nitrogen and protein metabolism, altered rates of carbohydrate and lipid production and utilization, and changes in mineral, electrolyte, trace element, and vitamin metabolism. It is postulated that these responses develop in a relatively predictable sequence which is influenced by the adequacy of host antimicrobial defense mechanisms, the severity and duration of illness, and specific localization of an infectious process within the body. In addition to hormonal regulatory effects, the metabolic and nutritional responses of the host are also influenced by biologically active substances released when host cells participate in phagocytic activity and local inflammatory responses.

Viral suppression of innate immunity via spatial isolation of TBK1/IKKε from mitochondrial antiviral platform.

For antiviral signaling mediated by retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), the recruitment of cytosolic RLRs and downstream molecules (such as TBK1 and IKKε) to mitochondrial platform is a central event that facilitates the establishment of host antiviral state. Here, we present an example of viral targeting for immune evasion through spatial isolation of TBK1/IKKε from mitochondrial antiviral platform, which was employed by severe fever with thrombocytopenia syndrome virus (SFTSV), a deadly bunyavirus emerging recently. We showed that SFTSV nonstructural protein NSs functions as the interferon (IFN) antagonist, mainly via suppressing TBK1/IKKε-IRF3 signaling. NSs mediates the formation of cytoplasmic inclusion bodies (IBs), and the blockage of IB formation impairs IFN-inhibiting activity of NSs. We next demonstrate that IBs are utilized to compartmentalize TBK1/IKKε. The compartmentalization results in spatial isolation of the kinases from mitochondria, and deprived TBK1/IKKε may participate in antiviral complex assembly, leading to the blockage of IFN induction. This study proposes a new role of viral IBs as virus-built 'jail' for imprisoning cellular factors and presents a novel and likely common mechanism of viral immune evasion through spatial isolation of critical signaling molecules from the mitochondrial antiviral platform.

[Role of T lymphocyte activation in the development of severe fever accompanied by thrombocytopenia syndrome].

OBJECTIVE: To explore the effect of peripheral blood T lymphocyte activation on the blood cells, tissue injury and the development of disease in patients with severe fever with thrombocytopenia syndrome (SFTS). METHODS: The expressions of CD69, HLA-DR, CD28 and CTLA-4 on peripheral blood T lymphocytes were determined dynamically by flow cytometry and the relationships between the above immune molecules and ALT, AST, leukocytes, platelets were analyzed respectively. RESULTS: The expressions of CD69 and HLA-DR on peripheral blood T lymphocytes in patients with SFTS were elevated significantly during the whole course of disease (P<0.05). CD28 expression on CD4(+); lymphocyte subset decreased in the early stage and gradually increased to the normal range. Meanwhile, CTLA-4 expression on T lymphocytes went up in the late stage of viral infection. The levels of serum ALT, AST, LDH and CK were significantly higher than the upper limit of the normal and the counts of WBC and PLT dropped to the lowest at the outset. But all of them returned back to the normal range gradually with the down-regulation of CD69 and HLA-DR and the up-regulation of CTLA-4 on T lymphocyte. CONCLUSION: The overactivation of T lymphocytes may contribute to tissue injury and the high expression of CTLA-4 may be a negative feedback regulation to the overactivation of T lymphocytes, which plays an important role in immunoregulation of SFTS patients.