6-Thioguanine inhibits severe fever with thrombocytopenia syndrome virus through suppression of EGR1.

The severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus, recently being officially renamed as Dabie bandavirus, and a causative agent for an emerging infectious disease associated with high fatality. Effective therapeutics and vaccines are lacking and disease pathogenesis is yet to be fully elucidated. In our effort to identify new SFTSV inhibitory molecules, 6-Thioguanine (6-TG) was found to potently inhibit SFTSV infection. 6-TG has been widely used as therapeutic agent since the approval of the Food and Drug Administration in the 1960s. In the current study, we showed that 6-TG was a potent inhibitor of SFTSV infection with 50% effective concentrations (EC50) of 3.465 µM in VeroE6 cells, and 1.848 µM in HUVEC cells. The selectivity index (SI) was >57 in VeroE6 cells and >108 in HUVEC cells, respectively. The SFTSV RNA transcription, protein synthesis, and progeny virions were reduced in a dose dependent manner by the presence of 6-TG in the in vitro infection assay. Further study on the mechanism of the anti-SFTSV activity showed that 6-TG downregulated the production of early growth response gene-1 (EGR1). Using gene silencing and overexpression, we further confirmed that EGR1 was a host restriction factor against SFTSV. Meanwhile, treatment of infected experimental animals with 6-TG inhibited SFTSV infection and alleviated multi-organ dysfunction. In conclusion, we have identified 6-TG as an effective inhibitor of SFTSV replication via the inhibition of EGR1 expression. Further studies are needed to evaluate of 6-TG as a potential therapeutic for treating SFTS.

Calcium Influx Regulates the Replication of Several Negative-Strand RNA Viruses Including Severe Fever with Thrombocytopenia Syndrome Virus.

Negative-strand RNA viruses (NSVs) represent one of the most threatening groups of emerging viruses globally. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic emerging virus that was initially reported in 2011 from China. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV. Here, L-type calcium channel blockers obtained from a U.S. Food and Drug Administration (FDA)-approved compound library were identified as effective anti-SFTSV compounds. Manidipine, a representative L-type calcium channel blocker, restricted SFTSV genome replication and exhibited inhibitory effects against other NSVs. The result from the immunofluorescent assay suggested that manidipine inhibited SFTSV N-induced inclusion body formation, which is believed to be important for the virus genome replication. We have shown that calcium possesses at least two different roles in regulating SFTSV genome replication. Inhibition of calcineurin, the activation of which is triggered by calcium influx, using FK506 or cyclosporine was shown to reduce SFTSV production, suggesting the important role of calcium signaling on SFTSV genome replication. In addition, we showed that globular actin, the conversion of which is facilitated by calcium from filamentous actin (actin depolymerization), supports SFTSV genome replication. We also observed an increased survival rate and a reduction of viral load in the spleen in a lethal mouse model of SFTSV infections after manidipine treatment. Overall, these results provide information regarding the importance of calcium for NSV replication and may thereby contribute to the development of broad-scale protective therapies against pathogenic NSVs. IMPORTANCE SFTS is an emerging infectious disease and has a high mortality rate of up to 30%. There are no licensed vaccines or antivirals against SFTS. In this article, L-type calcium channel blockers were identified as anti-SFTSV compounds through an FDA-approved compound library screen. Our results showed the involvement of L-type calcium channel as a common host factor for several different families of NSVs. The formation of an inclusion body, which is induced by SFTSV N, was inhibited by manidipine. Further experiments showed that SFTSV replication required the activation of calcineurin, a downstream effecter of the calcium channel. In addition, we identified that globular actin, the conversion of which is facilitated by calcium from filamentous actin, supports SFTSV genome replication. We also observed an increased survival rate in a lethal mouse model of SFTSV infection after manidipine treatment. These results facilitate both our understanding of the NSV replication mechanism and the development of novel anti-NSV treatment.

Potential efficacy of existing drug molecules against severe fever with thrombocytopenia syndrome virus: an in silico study.

Severe fever with thrombocytopenia syndrome (SFTS) is a zoonotic disease caused by the SFTS virus (SFTSV). SFTS can be considered a life-threatening notifiable infectious disease. The unavailability of specific therapeutics encourages the investigation of potential efficacy of existing drugs against this infection. Drug repurposing was done by performing  virtual screening of already established drug molecules followed by 100 ns molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area-based binding-energy calculation by targeting the SFTS L protein. On the basis of binding energy and protein-ligand interactions, top 10 promising hits were identified, showing stable binding with SFTS L protein. Further 100 ns atomistic MD simulation refined the hits from top 10 to top 4 with docking-based binding energy lesser than -8.0 kcal/mol toward the SFTS L protein and engaged in π-π interactions with pivotal amino acid residues. Various parameters and binding affinity of top 4 ligands towards L protein was computed. Ligand zaltoprofen exhibited best binding energy -220.095 kJ/mol. The present work is the first in silico study to assess bromfenac, cinchophen, elliptinium, and zaltoprofen; four promising hits against SFTS. Nonetheless, further proper biological evaluation is necessary to determine their efficacy against SFTS.

In Silico Structure-Based Design of Antiviral Peptides Targeting the Severe Fever with Thrombocytopenia Syndrome Virus Glycoprotein Gn.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus in Asia that causes severe disease. Despite its clinical importance, treatment options for SFTSV infection remains limited. The SFTSV glycoprotein Gn plays a major role in mediating virus entry into host cells and is therefore a potential antiviral target. In this study, we employed an in silico structure-based strategy to design novel cyclic antiviral peptides that target the SFTSV glycoprotein Gn. Among the cyclic peptides, HKU-P1 potently neutralizes the SFTSV virion. Combinatorial treatment with HKU-P1 and the broad-spectrum viral RNA-dependent RNA polymerase inhibitor favipiravir exhibited synergistic antiviral effects in vitro. The in silico peptide design platform in this study may facilitate the generation of novel antiviral peptides for other emerging viruses.

A longitudinal sampling study of transcriptomic and epigenetic profiles in patients with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is a novel tick-borne infectious disease caused by a new type of SFTS virus (SFTSV). Here, a longitudinal sampling study is conducted to explore the differences in transcript levels after SFTSV infection, and to characterize the transcriptomic and epigenetic profiles of hospitalized patients. The results reveal significant changes in the mRNA expression of certain genes from onset to recovery. Moreover, m6A-seq reveals that certain genes related with immune regulation may be regulated by m6A. Besides the routine tests such as platelet counts, serum ALT and AST levels testing, distinct changes in myocardial enzymes, coagulation function, and inflammation are well correlated with the clinical data and sequencing data, suggesting that clinical practitioners should monitor the above indicators to track disease progression and guide personalized treatment. In this study, the transcript changes and RNA modification may lend a fresh perspective to our understanding of the SFTSV and play a significant role in the discovery of drugs for effective treatment of this disease.

Peptides derived from viral glycoprotein Gc Inhibit infection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by a novel phlebovirus (SFTSV), characterized by fever, thrombocytopenia and leukocytopenia which lead to multiple organ failure with high mortality in severe cases. The SFTSV has spread rapidly in recent years and posed a serious threat to public health in endemic areas. However, specific antiviral therapeutics for SFTSV infection are rare. In this study, we demonstrated that two peptides, SGc1 and SGc8, derived from a hydrophobic region of the SFTSV glycoprotein Gc, could potently inhibit SFTSV replication in a dose-dependent manner without apparent cytotoxicity in various cell lines and with low immunogenicity and good stability. The IC50 (50% inhibition concentration) values for both peptides to inhibit 2 MOI of SFTSV infection were below 10 µM in L02, Vero and BHK21 cells. Mechanistically, SGc1 and SGc8 mainly inhibited viral entry at the early stage of the viral infection. Inhibition of SFTSV replication was specific by both peptides because no inhibitory effect was shown against other viruses including Zika virus and Enterovirus A71. Taken together, our results suggested that viral glycoprotein-derived SGc1 and SGc8 peptides have antiviral potential and warrant further assessment as an SFTSV-specific therapeutic.

M Segment-Based Minigenome System of Severe Fever with Thrombocytopenia Syndrome Virus as a Tool for Antiviral Drug Screening.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans with case fatality rates of approximately 30%. There are few treatment options for SFTSV infection. SFTSV RNA synthesis is conducted using a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are, therefore, potential antiviral targets. A library of small molecule compounds was processed using a high-throughput screening (HTS) based on an SFTSV minigenome assay (MGA) in a 96-well microplate format to identify potential lead inhibitors of SFTSV RNA synthesis. The assay confirmed inhibitory activities of previously reported SFTSV inhibitors, favipiravir and ribavirin. A small-scale screening using MGA identified four candidate inhibitors that inhibited SFTSV minigenome activity by more than 80% while exhibiting less than 20% cell cytotoxicity with selectivity index (SI) values of more than 100. These included mycophenolate mofetil, methotrexate, clofarabine, and bleomycin. Overall, these data demonstrate that the SFTSV MGA is useful for anti-SFTSV drug development research.

Loperamide Inhibits Replication of Severe Fever with Thrombocytopenia Syndrome Virus.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by the SFTS virus (SFTSV). SFTS is mainly prevalent in East Asia. It has a mortality rate of up to 30%, and there is no approved treatment against the disease. In this study, we evaluated the effect of loperamide, an antidiarrheal and antihyperalgesic agent, on the propagation of SFTSV in a cell culture system. METHODS: SFTSV-infected human cell lines were exposed to loperamide, and viral titers were evaluated. To clarify the mode of action of loperamide, several chemical compounds having shared targets with loperamide were used. Calcium imaging was also performed to understand whether loperamide treatment affected calcium influx. RESULTS: Loperamide inhibited SFTSV propagation in several cell lines. It inhibited SFTSV in the post-entry step and restricted calcium influx into the cell. Furthermore, nifedipine, a calcium channel inhibitor, also blocked post-entry step of SFTSV infection. CONCLUSIONS: Loperamide inhibits SFTSV propagation mainly by restraining calcium influx into the cytoplasm. This indicates that loperamide, a Food and Drug Administration (FDA)-approved drug, has the potential for being used as a treatment option against SFTS.

Fludarabine Inhibits Infection of Zika Virus, SFTS Phlebovirus, and Enterovirus A71.

Viral infections are one of the leading causes in human mortality and disease. Broad-spectrum antiviral drugs are a powerful weapon against new and re-emerging viruses. However, viral resistance to existing broad-spectrum antivirals remains a challenge, which demands development of new broad-spectrum therapeutics. In this report, we showed that fludarabine, a fluorinated purine analogue, effectively inhibited infection of RNA viruses, including Zika virus, Severe fever with thrombocytopenia syndrome virus, and Enterovirus A71, with all IC50 values below 1 µM in Vero, BHK21, U251 MG, and HMC3 cells. We observed that fludarabine has shown cytotoxicity to these cells only at high doses indicating it could be safe for future clinical use if approved. In conclusion, this study suggests that fludarabine could be developed as a potential broad-spectrum anti-RNA virus therapeutic agent.

The NF-κB inhibitor, SC75741, is a novel antiviral against emerging tick-borne bandaviruses.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) cause viral hemorrhagic fever-like illnesses in humans due to an aberrant host inflammatory response, which contributes to pathogenesis. Here, we established two separate minigenome (MG) systems based on the M-segment of SFTSV and HRTV. Following characterization of both systems for SFTSV and HRTV, we used them as a platform to screen potential compounds that inhibit viral RNA synthesis. We demonstrated that the NF-κB inhibitor, SC75741, reduces viral RNA synthesis of SFTSV and HRTV using our MG platform and validated these results using infectious SFTSV and HRTV. These results may lead to the use of MG systems as potential screening systems for the identification of antiviral compounds and yield novel insights into host-factors that could play role in bandavirus transcription and replication.

Activation of platelet-derived growth factor receptor ß in the severe fever with thrombocytopenia syndrome virus infection.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging viral infectious disease with a high case fatality rate, is caused by the SFTS virus (SFTSV). Although several cellular molecules involved in viral entry have been identified, the entry mechanisms of SFTSV remain unclear. In this study, we screened the protein kinase inhibitors in inhibitory effects on the infection of Vero cells with SFTSV using InhibitorSelect™ Protein Kinase Library Series (Merck & Co., Inc., Kenilworth, NJ, USA). Several types of inhibitors targeted to platelet-derived growth factor receptor ß (PDGFRß) inhibited the infection of Vero, Huh7, and NIH3T3 cells with SFTSV in a dose-dependent manner within the noncytotoxic range. In addition, these protein kinase inhibitors also inhibited the infection of the target cells with SFTSV glycoprotein (SFTSV-GP) pseudotyped virus (SFTSVpv). Activation of PDGFRß phosphorylation was detected in SFTSV-treated cells. The infectivities of SFTSVpv were specifically decreased not only in NIH3T3 cells treated with siRNA for PDGFRß but also in NIH3T3 cells treated with anti-PDGFRß neutralizing antibody in a dose-dependent manner. SFTSV growth and entry of SFTSVpv were also inhibited by Akt inhibitors. Activation of Akt phosphorylation was also detected in SFTSV-treated cells. These data indicate that PDGFRß is one of the important host factors in the entry steps of SFTSV.

The Cap-Snatching SFTSV Endonuclease Domain Is an Antiviral Target.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%-30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (IC50) of ∼100 nM in enzyme inhibition and an EC50 value of ∼250 nM against SFTSV and HRTV in plaque assays. Together, our data support sNSV endonucleases as an antiviral target.

Proteasome Inhibitor PS-341 Effectively Blocks Infection by the Severe Fever with Thrombocytopenia Syndrome Virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever disease caused by SFTSV, a newly discovered phlebovirus that is named after the disease. Currently, no effective vaccines or drugs are available for use against SFTSV infection, as our understanding of the viral pathogenesis is limited. Bortezomib (PS-341), a dipeptide-boronic acid analog, is the first clinically approved proteasome inhibitor for use in humans. In this study, the antiviral efficacy of PS-341 against SFTSV infection was tested in human embryonic kidney HEK293T (293T) cells. We employed four different assays to analyze the antiviral ability of PS-341 and determined that PS-341 inhibited the proliferation of SFTSV in 293T cells under various treatment conditions. Although PS-341 did not affect the virus absorption, PS-341 treatment within a non-toxic concentration range resulted in a significant reduction of progeny viral titers in infected cells. Dual-luciferase reporter assays and Western blot analysis revealed that PS-341 could reverse the SFTSV-encoded non-structural protein (NS) mediated degradation of retinoic acid-inducible gene-1 (RIG-I), thereby antagonizing the inhibitory effect of NSs on interferons and blocking virus replication. In addition, we observed that inhibition of apoptosis promotes virus replication. These results indicate that targeting of cellular interferon pathways and apoptosis during acute infection might serve as the bases of future therapeutics for the treatment of SFTSV infections.

Interferon-γ-Directed Inhibition of a Novel High-Pathogenic Phlebovirus and Viral Antagonism of the Antiviral Signaling by Targeting STAT1.

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening infectious disease caused by a novel phlebovirus, SFTS virus (SFTSV). Currently, there is no vaccine or antiviral available and the viral pathogenesis remains largely unknown. In this study, we demonstrated that SFTSV infection results in substantial production of serum interferon-γ (IFN-γ) in patients and then that IFN-γ in turn exhibits a robust anti-SFTSV activity in cultured cells, indicating the potential role of IFN-γ in anti-SFTSV immune responses. However, the IFN-γ anti-SFTSV efficacy was compromised once viral infection had been established. Consistently, we found that viral nonstructural protein (NSs) expression counteracts IFN-γ signaling. By protein interaction analyses combined with mass spectrometry, we identified the transcription factor of IFN-γ signaling pathway, STAT1, as the cellular target of SFTSV for IFN-γ antagonism. Mechanistically, SFTSV blocks IFN-γ-triggered STAT1 action through (1) NSs-STAT1 interaction-mediated sequestration of STAT1 into viral inclusion bodies and (2) viral infection-induced downregulation of STAT1 protein level. Finally, the efficacy of IFN-γ as an anti-SFTSV drug in vivo was evaluated in a mouse infection model: IFN-γ pretreatment but not posttreatment conferred significant protection to mice against lethal SFTSV infection, confirming IFN-γ's anti-SFTSV effect and viral antagonism against IFN-γ after the infection establishment. These findings present a picture of virus-host arm race and may promote not only the understanding of virus-host interactions and viral pathogenesis but also the development of antiviral therapeutics.

Heartland virus antagonizes type I and III interferon antiviral signaling by inhibiting phosphorylation and nuclear translocation of STAT2 and STAT1.

Heartland virus (HRTV) is a pathogenic phlebovirus recently identified in the United States and related to severe fever with thrombocytopenia syndrome virus (SFTSV) emerging in Asia. We previously reported that SFTSV disrupts host antiviral responses directed by interferons (IFNs) and their downstream regulators, signal transducer and activator of transcription (STAT) proteins. However, whether HRTV infection antagonizes the IFN-STAT signaling axis remains unclear. Here, we show that, similar to SFTSV, HRTV also inhibits IFN-α- and IFN-λ-mediated antiviral responses. As expected, the nonstructural protein (NSs) of HRTV (HNSs) robustly antagonized both type I and III IFN signaling. Protein interaction analyses revealed that a common component downstream of type I and III IFN signaling, STAT2, is the target of HNSs. Of note, the DNA-binding and linker domains of STAT2 were required for an efficient HNSs-STAT2 interaction. Unlike the NSs of SFTSV (SNSs), which blocks both STAT2 and STAT1 nuclear accumulation, HNSs specifically blocked IFN-triggered nuclear translocation only of STAT2. However, upon HRTV infection, IFN-induced nuclear translocation of both STAT2 and STAT1 was suppressed, suggesting that STAT1 is an additional HRTV target for IFN antagonism. Consistently, despite HNSs inhibiting phosphorylation only of STAT2 and not STAT1, HRTV infection diminished both STAT2 and STAT1 phosphorylation. These results suggest that HRTV antagonizes IFN antiviral signaling by dampening both STAT2 and STAT1 activities. We propose that HNSs-specific targeting of STAT2 likely plays an important role but is not all of the "tactics" of HRTV in its immune evasion.

Screening of an FDA-Approved Drug Library with a Two-Tier System Identifies an Entry Inhibitor of Severe Fever with Thrombocytopenia Syndrome Virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans with case-fatality rates of up to 30%. There are currently very limited treatment options for SFTSV infection. We conducted a drug repurposing program by establishing a two-tier test system to rapidly screen a Food and Drug Administration- (FDA)-approved drug library for drug compounds with anti-SFTSV activity in vitro. We identified five drug compounds that inhibited SFTSV replication at low micromolar concentrations, including hexachlorophene, triclosan, regorafenib, eltrombopag, and broxyquinoline. Among them, hexachlorophene was the most potent with an IC50 of 1.3 ± 0.3 µM and a selectivity index of 18.7. Mechanistic studies suggested that hexachlorophene was a virus entry inhibitor, which impaired SFTSV entry into host cells by interfering with cell membrane fusion. Molecular docking analysis predicted that the binding of hexachlorophene with the hydrophobic pocket between domain I and domain III of the SFTSV Gc glycoprotein was highly stable. The novel antiviral activity and mechanism of hexachlorophene in this study would facilitate the use of hexachlorophene as a lead compound to develop more entry inhibitors with higher anti-SFTSV potency and lower toxicity.

Severe fever with thrombocytopenia syndrome phlebovirus non-structural protein activates TPL2 signalling pathway for viral immunopathogenesis.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV), listed in the World Health Organization Prioritized Pathogens, is an emerging phlebovirus with a high fatality1-4. Owing to the lack of therapies and vaccines5,6, there is a pressing need to understand SFTSV pathogenesis. SFSTV non-structural protein (NSs) has been shown to block type I interferon induction7-11 and facilitate disease progression12,13. Here, we report that SFTSV-NSs targets the tumour progression locus 2 (TPL2)-A20-binding inhibitor of NF-κB activation 2 (ABIN2)-p105 complex to induce the expression of interleukin-10 (IL-10) for viral pathogenesis. Using a combination of reverse genetics, a TPL2 kinase inhibitor and Tpl2-/- mice showed that NSs interacted with ABIN2 and promoted TPL2 complex formation and signalling activity, resulting in the marked upregulation of Il10 expression. Whereas SFTSV infection of wild-type mice led to rapid weight loss and death, Tpl2-/- mice or Il10-/- mice survived an infection. Furthermore, SFTSV-NSs P102A and SFTSV-NSs K211R that lost the ability to induce TPL2 signalling and IL-10 production showed drastically reduced pathogenesis. Remarkably, the exogenous administration of recombinant IL-10 effectively rescued the attenuated pathogenic activity of SFTSV-NSs P102A, resulting in a lethal infection. Our study demonstrates that SFTSV-NSs targets the TPL2 signalling pathway to induce immune-suppressive IL-10 cytokine production as a means to dampen the host defence and promote viral pathogenesis.

Therapeutic effects of favipiravir against severe fever with thrombocytopenia syndrome virus infection in a lethal mouse model: Dose-efficacy studies upon oral administration.

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV), is a viral hemorrhagic fever with a high case fatality rate. Favipiravir was reported to be effective in the treatment of SFTSV infection in vivo in type I interferon receptor knockout (IFNAR-/-) mice at treatment dosages of both 60 mg/kg/day and 300 mg/kg/day for a duration of 5 days. In this study, the efficacy of favipiravir at dosages of 120 mg/kg/day and 200 mg/kg/day against SFTSV infection in an IFNAR-/- mouse infection model was investigated. IFNAR-/- mice were subcutaneously infected with SFTSV at a 1.0 × 10(6) 50% tissue culture infectious dose followed by twice daily administration of favipiravir, comprising a total dose of either 120 mg/kg/day or 200 mg/kg/day. The treatment was initiated either immediately post infection or at predesignated time points post infection. Neutralizing antibodies in the convalescent-phase mouse sera was examined by the pseudotyped VSV system. All mice treated with favipiravir at dosages of 120 mg/kg/day or 200 mg/kg/day survived when the treatment was initiated at no later than 4 days post infection. A decrease in body weight of mice was observed when the treatment was initiated at 3-4 days post infection. Furthermore, all control mice died. The body weight of mice did not decrease when treatment with favipiravir was initiated immediately post infection at dosages of 120 mg/kg/day and 200 mg/kg/day. Neutralizing antibodies were detected in the convalescent-phase mouse sera. Similar to the literature-reported peritoneal administration of favipiravir at 300 mg/kg/day, the oral administration of favipiravir at dosages of 120 mg/kg/day and 200 mg/kg/day to IFNAR-/- mice infected with SFTSV was effective.

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.

Caffeic acid, a coffee-related organic acid, inhibits infection by severe fever with thrombocytopenia syndrome virus in vitro.

Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) causes tick-borne hemorrhagic fever in East Asia. The disease is characterized by high morbidity and mortality. Here, we evaluated the effects of caffeic acid (CA), a coffee-related organic acid with antiviral effects, against SFTSV infection. CA dose-dependently inhibited SFTSV infection in permissive human hepatoma Huh7.5.1-8 cells when SFTSV was added into the culture medium with CA. However, quinic acid (QA), another coffee-related organic acid, did not inhibit SFTSV infection. The 50% inhibitory concentration (IC50) of CA against SFTSV was 0.048 mM, whereas its 50% cytotoxic concentration was 7.6 mM. The selectivity index (SI) was 158. Pre-incubation of SFTSV with CA for 4 h resulted in a greater inhibition of SFTSV infection (IC50 = 0.019 mM; SI = 400). The pre-incubation substantially decreased viral attachment to the cells. CA treatment of the SFTSV-infected cells also inhibited the infection, albeit less effectively. CA activity after cell infection with SFTSV was more pronounced at a low multiplicity of infection (MOI) of 0.01 per cell (IC50 = 0.18 mM) than at a high MOI of 1 per cell (IC50 > 1 mM). Thus, CA inhibited virus spread by acting directly on the virus rather than on the infected cells. In conclusion, CA acted on SFTSV and inhibited viral infection and spread, mainly by inhibiting the binding of SFTSV to the cells. We therefore demonstrated CA to be a potential anti-SFTSV drug for preventing and treating SFTS.