Search

Lipids and lipid metabolism play an important role in RNA virus replication, which typically occurs on host cell endomembrane structures in the cytoplasm through mechanisms that are not yet fully identified. We conducted genome-scale CRISPR screening and identified sphingomyelin synthase 1 (SMS1; encoded by SGMS1) as a critical host factor for infection by severe fever with thrombocytopenia syndrome virus (SFTSV). SGMS1 knockout reduced sphingomyelin (SM) (d18:1/16:1) levels, inhibiting SFTSV replication. A helix-turn-helix motif in SFTSV RNA-dependent RNA polymerase (RdRp) directly binds to SM(d18:1/16:1) in Golgi apparatus, which was also observed in SARS-CoV-2 and lymphocytic choriomeningitis virus (LCMV), both showing inhibited replication in SGMS1-KO cells. SM metabolic disturbance is associated with disease severity of viral infections. We designed a novel SMS1 inhibitor that protects mice against lethal SFTSV infection and reduce SARS-CoV-2 replication and pathogenesis. These findings highlight the critical role of SMS1 and SM(d18:1/16:1) in RNA virus replication, suggesting a broad-spectrum antiviral strategy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Severe Fever with Thrombocytopenia Syndrome (SFTS) is an emerging infectious disease with high mortality and severity rates. However, there is no clear treatment plan, specific effective antiviral drugs, or effective vaccine for SFTS. Recent studies have shown that the therapeutic effect of Ribavirin and other commonly used antiviral drugs such as Favipiravir (T-705), on SFTS is still controversial. This meta-analysis was conducted to evaluate the therapeutic efficacy of antiviral drugs on SFTS., Methods: Relevant studies were searched from Cochrane Library, Web of Science, Embase. and PubMed from inception to June 30, 2022, and updated to 2023. The study screening, data extraction, and quality assessment were conducted by re-searchers independently. A counterpart assessment tool was used to assess methodological quality, and the Stata15 software was employed to perform meta-analysis., Results: The meta-analysis included 8 studies consisting of 4692 patients. The results showed no significant difference in the mortality rate of SFTS patients between the antiviral drug group and the control group (HR: 0.85, 95% CI: 0.46-1.59, p = 0.618). Antiviral drugs had no noticeable effect on this disease (RR: 0.93, 95% CI: 0.58-1.48, p = 0.747)., Conclusions: At present, antiviral drugs (Ribavirin and Favipiravir) used routinely in clinics show no positive effect on the treatment of SFTS yet and fail to reduce the mortality rate. On the other hand, clinical studies specifically evaluating viral load found that antiviral drugs were effective in the treatment of SFTS patients with low viral load, but the efficacy was not statistically significant., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Lumeng Shen, Mingfang Han, Jingjing Lou, Wenya Shen, Shibo Li.)

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by the SFTS virus (SFTSV). CP-COV03 is a novel antiviral candidate that significantly enhanced the bioavailability of niclosamide through inorganic-based drug delivery technology. The active pharmaceutical ingredient of CP-COV03, niclosamide, has been previously shown to possess broad-spectrum antiviral activity against over 30 different viruses in the in vitro tests. The aim of this study is to confirm the antiviral activity of CP-COV03 against the SFTSV in an in vitro model. Vero cells and SFTS viral stock NCCP43270, a 2015 Gangwon Province isolate, were used to obtain the 50% tissue culture infective dose of the virus. Vero cells seeded in 96-well plates were infected with SFTSV for 1 h. SFTSV-infected cells were treated with CP-COV03 at various concentrations of 0.1-100 μM and incubated for 7 days. On the seventh day of the culture, the cytopathic effect (CPE) of SFTSV was checked by microscopy and the cell viability was checked by using Cell Counting Kit-8 assay. The CPE reduced as the CP-COV03 concentration increased. The 50% inhibitory concentration (IC 50 ) range of CP-COV03 was below 0.125 µM, as determined from the viral titers of culture supernatants collected on the third day posttreatment of CP-COV03. The plaque reduction assay showed that the IC 50 of CP-COV03 was 1.893 µM, as determined from the percentage reduction of plaque counts for each drug concentration on the second day posttreatment with CP-COV03. This study suggests that CP-COV03 could be used as a potential antiviral agent for SFTS.IMPORTANCEWe demonstrated a concentration-dependent response and identified low a IC 50 of CP-COV03. This result is comparable to other antiviral drugs used against viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We believe that our study makes a significant contribution to the literature as our findings suggest that CP-COV03 may serve as a potential treatment for SFTS, highlighting its importance in the field of antiviral research., Competing Interests: The authors declare no conflict of interest.

Severe fever with thrombocytopenia syndrome virus (SFTSV), recently named as Dabie bandavirus, belongs to the family Phenuiviridae of the order Bunyavirales, is a newly-identified bunyavirus with a case fatality rate of up to 30%, posing a serious threat to public health. Lipid rafts on plasm membranes are important for the entry of enveloped viruses; however, the role of lipid rafts in bunyavirus entry remains unclear. In this study, we found that methyl-beta-cyclodextrin (MβCD), a drug that disrupts cholesterol in lipid rafts of cell membranes, inhibits SFTSV infection. Additionally, there is a back-complementary effect of SFTSV infection upon the addition of cholesterol. Moreover, the concentration of SFTSV particles in lipid rafts during entry directly indicated the role of lipid rafts as a gateway, whereas MβCD could inhibit SFTSV entry by affecting the structure of lipid rafts. In an in vivo study, MβCD also reduced the susceptibility of mice to SFTSV infection. Our results suggest that SFTSV can interact with Talin1 proteins on lipid rafts to enter host cells by endocytosis of lipid rafts and reveal the potential therapeutic value of MβCD for SFTSV infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus that causes the severe fever thrombocytopenia syndrome, which manifests as fever and haemorrhage, accompanied by severe neurological complications. To date, no specific antiviral drugs have been approved for this indication. Herein, we investigated whether vitamin D derivatives inhibit SFTSV both in vitro and in vivo. An in vitro study demonstrated that vitamin D derivatives significantly suppressed viral RNA replication, plaque formation, and protein expression in a dose-dependent manner. Subsequently, in vivo studies revealed that doxercalciferol and alfacalcidol were associated with increased survival and reduced viral RNA load in the blood. Time-of-addition assay suggested that vitamin D derivatives primarily acted during the post-entry phase of SFTSV infection. However, cytopathic effect protective activity was not observed in RIG-I immunodeficient cell line Huh7.5, and the administration of vitamin D derivatives did not improve the survival rates or reduce the blood viral loads in adult A129 mice. Further transcriptome exploration into the antiviral mechanism revealed that alfacalcidol stimulates host innate immunity to exert antiviral effects. To expand the application of vitamin D derivatives, in vitro and in vivo drug combination assays were performed, which highlighted the synergistic effects of vitamin D derivatives and T-705 on SFTSV. The combination of alfacalcidol and T-705 significantly enhanced the therapeutic effects in mice. This study highlights the potential of vitamin D derivatives against SFTSV and suggests that they may have synergistic effects with other compounds used in the treatment of SFTSV infection., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Severe fever with thrombocytopenia syndrome virus (SFTSV), also known as the Dabie Banda virus, is an emerging tick-borne Bunyavirus that causes severe fever with thrombocytopenia syndrome (SFTS). Currently, symptomatic treatment and antiviral therapy with ribavirin and favipiravir are used in clinical management. However, their therapeutical efficacy is hardly satisfactory in patients with high viral load. In this study, we explored the antiviral effects of selective estrogen receptor modulators (SERMs) on SFTSV infection and the antiviral mechanisms of a representative SERM, bazedoxifene acetate (BZA). Our data show that SERMs potently inhibited SFTSV-induced cytopathic effect (CPE), the proliferation of infectious viral particles, and viral RNA replication and that BZA effectively protected mice from lethal viral challenge. The mode of action analysis reveals that BZA exerts antiviral effects during the post-entry stage of SFTSV infection. The transcriptome analysis reveals that GRASLND and CYP1A1 were upregulated, while TMEM45B and TXNIP were downregulated. Our findings suggest that SERMs have the potential to be used in the treatment of SFTSV infection.

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is prevalent in East Asia. However, the use of glucocorticoids (GCs) in the treatment of SFTS remains controversial., Methods: In this retrospective cohort study, we collected the data from patients with SFTS at Wuhan Union Hospital to evaluate the effect of GC therapy. Mortality and secondary infections were compared as outcomes. After searching public databases, we also included articles that examined GC use in patients with SFTS for meta-analysis., Results: Patients treated with GC had higher fatality rates (21.1% vs. 11.9%, respectively; P =0.006) and a longer length of stay (10.6 ± 5.1 vs. 9.5 ± 4.2, respectively; P =0.033). In cohorts adjusted using propensity score matching and inverse probability of treatment weighting, no significant differences in fatality rates and length of stay were observed. A meta-analysis of 4243 SFTS patient revealed that those treated with GCs had significantly higher mortality (OR=3.46, 95% CI =2.12-5.64, P <0.00001) and secondary infection rate (OR=1.97, 95% CI=1.45-2.67, P <0.0001)., Discussion: GC should be used cautiously when treating SFTS. No significant differences were identified in terms of mortality and secondary infection rates between patients with SFTS treated with or without GC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Wang, Zhou and Guo.)

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 (EC 50 ) 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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.

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., (© 2021. The Author(s).)

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.

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 IC 50 (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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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, m 6 A-seq reveals that certain genes related with immune regulation may be regulated by m 6 A. 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., (© 2021. The Author(s).)

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.

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.

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 IC 50 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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 β (PDGFRβ) 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 PDGFRβ phosphorylation was detected in SFTSV-treated cells. The infectivities of SFTSVpv were specifically decreased not only in NIH3T3 cells treated with siRNA for PDGFRβ but also in NIH3T3 cells treated with anti-PDGFRβ 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 PDGFRβ is one of the important host factors in the entry steps of SFTSV., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 (IC 50 ) of ∼100 nM in enzyme inhibition and an EC 50 value of ∼250 nM against SFTSV and HRTV in plaque assays. Together, our data support sNSV endonucleases as an antiviral target., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., (© 2019 Feng et al.)

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.

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 IC 50 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 (SFTSV), listed in the World Health Organization Prioritized Pathogens, is an emerging phlebovirus with a high fatality 1-4 . Owing to the lack of therapies and vaccines 5,6 , there is a pressing need to understand SFTSV pathogenesis. SFSTV non-structural protein (NSs) has been shown to block type I interferon induction 7-11 and facilitate disease progression 12,13 . 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 P 102 A and SFTSV-NSs K 211 R 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 P 102 A, 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.

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-β 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., (Copyright © 2018 Wuerth et al.)

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., Competing Interests: The authors declare no conflicts of interest in association with the present study.

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 (IC 50 ) 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 (IC 50  = 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 (IC 50  = 0.18 mM) than at a high MOI of 1 per cell (IC 50  > 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., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by the SFTS virus (SFTSV). Clinical symptoms of SFTS often involve encephalopathy and other central neurological symptoms, particularly in seriously ill patients; however, pathogenesis of encephalopathy by SFTSV is largely unknown. Herein, we present case reports of three patients with SFTS, complicated by encephalopathy, admitted to Tokushima University hospital: one patient was a 63-year-old man, while the other two were 83- and 86-year-old women. All of them developed disturbance of consciousness around the 7th day post onset of fever. After methylprednisolone pulse therapy of 500 mg/day, all of them recovered without any neurological sequelae. SFTSV genome was not detected in the cerebrospinal fluid of 2 out of the 3 patients that were available for examination. In these patients, disturbance of consciousness seemed to be an indirect effect of the cytokine storm triggered by SFTSV infection. We propose that short-term glucocorticoid therapy might be beneficial in the treatment of encephalopathy during early phase of SFTSV infection., (Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC 50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC 50  ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).

Background/aims: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV), a novel bunyavirus. As yet, there is no effective antiviral therapy for SFTS. Ribavirin is a broad-spectrum antiviral agent, which has been tried for treatment of SFTS. In this study, antiviral activity of ribavirin against SFTSV has been investigated., Methods: Vero cell-grown SFTSV strain Gangwon/Korea/2012 was treated with ribavirin at various concentrations. Antiviral activity of ribavirin was evaluated by inhibition of the SFTSV cytopathic effect in Vero cells and quantification of viral RNA load in culture supernatant using one-step real-time reverse transcription polymerase chain reaction. Cytotoxicity of ribavirin was determined by a tetrazolium-based colorimetric method., Results: Ribavirin reduced SFTSV titers in a dose-dependent manner, with a half-maximal inhibitory concentration ranged from 3.69 to 8.72 μg/mL. Cytopathic effects were reduced as ribavirin concentration increased. No significant cytotoxicity was detected at ribavirin concentrations of ≤ 31.3 μg/mL., Conclusions: Ribavirin exhibited inhibitory activity against SFTSV replication in vitro , which suggests that ribavirin can be used as a potential antiviral agent for SFTS.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by SFTS virus and characterized by a high case fatality rate. Currently, there is no effective therapy for the disease. While the administration of ribavirin does not improve the case fatality rate or viral load in patient blood, it can inhibit viral infection in vitro., Methods: Vero cells were pre-treated with interferons (IFNs) α, β, and γ alone and in combination with ribavirin drugs and inoculated with SFTS virus. Three days later, supernatants were harvested and subjected to virus titration. An unpaired t-test was used for statistical analysis of the drugs' effects., Results: While the effects of IFNγ at high concentrations were slightly weaker than those of the other IFNs, all IFNs showed dose-dependent inhibitory effects. The combined usage of IFNs with ribavirin at 90 % effective concentrations showed large inhibitory effects, with over a 3 log10 reduction in viral titers., Conclusions: The combined usage of one of type-I/II IFNs with ribavirin drastically reduced SFTS virus infection and therefore may be useful in the treatment of SFTS.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tickborne virus in the Bunyaviridae family. This virus has recently been found in China, Japan and Korea. The risk of transfusion-transmitted SFTSV infection (TTI-SFTSV) is a concern because person-to-person transmission resulting from contact with SFTSV-contaminated blood has been reported. Therefore, we investigated the efficacy of the Mirasol pathogen reduction technology (PRT) system for inactivating SFTSV in vitro. The Mirasol PRT system achieved a > 4.11 log10 reduction value (LRV) for SFTSV. In conclusion, we showed that the Mirasol PRT system could potentially be used to reduce the risk of TTI-SFTSV., (© 2015 International Society of Blood Transfusion.)

Severe Fever with Thrombocytopenia Syndrome (SFTS) is associated with high mortality rate, for which antiviral therapy with ribavirin was recommended. Based on our previous study, no visible effect of ribavirin therapy in improving clinical outcome was observed. Here we have accumulated the sample size to 634, and by performing prospective observation on the clinical progress and laboratory parameters, we found a significantly higher incidence of anemia and hyperamylasemia in patients who received ribavirin therapy in comparison with those who received no therapy. Generalized estimating equation model disclosed a significant effect on hemoglobin reduction and blood amylase augmentation from ribavirin administration. The occurrence of anemia and hyperamylasemia was associated with SFTS patients receiving ribavirin therapy, which might be adverse event of this drug administration. The recommendation of ribavirin for treating SFTS should be applied with caution., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A novel series of 5-arylcarbamoyl- and 5-arylmethyl-2-methylisoxazolidin-3-yl-3-phosphonates have been synthesized via cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone with N-substituted naphthalimide acrylamides and N-allylnaphthalimides. All cis- and trans-isoxazolidine phosphonates obtained herein were assessed for antiviral activity against a broad range of DNA and RNA viruses. Isoxazolidines trans-9d and trans-9f exhibited the highest activity (EC50=8.9μM) toward cytomegalovirus. Compounds cis- and trans-9d as well as cis- and trans-9f were found potent against HSV and Vaccinia viruses (EC50 in the 45-58μM range), whereas isoxazolidines 10a and 10d suppressed replication of Coxsackie B4 and Punta Toro viruses (EC50 in the 45-73μM range). Antiproliferative evaluation of all obtained isoxazolidines revealed the promising activity of cis-9b, cis-9d, trans-9d, cis-9e, trans-9e, cis-9f and trans-9f toward tested cancer cell lines with IC50 in the 1.1-19μM range., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Severe fever with thrombocytopenia syndrome (SFTS) is a disease with a high case fatality rate that is caused by infection with the recently identified tick-borne SFTS virus (SFTSV), for which there are no specific countermeasures. We examined the effects of ribavirin and mizoribine, which are nucleoside analogue drugs with broad antiviral activities, on SFTSV proliferation in vitro. When 3 cell lines were treated with these drugs before and during infection with a Chinese SFTSV strain, the 99% effective concentrations (EC99) of ribavirin were 19-64 μg/ml (78-262 μM); in contrast, the EC99 of mizoribine was >500 μg/ml (1,929 μM). Similar levels of inhibitory effects of ribavirin were observed with 4 Japanese SFTSV strains. However, when Vero cells were treated with ribavirin 3 days after inoculation, the inhibitory effect was dramatically decreased, indicating that ribavirin did not effectively reduce virus production in pre-infected cells. These results suggest that ribavirin could be used as post-exposure prophylaxis for the prevention of SFTS.

Severe fever with thrombocytopenia syndrome is an emerging infectious disease caused by a novel bunyavirus (SFTSV). Lack of vaccines and inadequate therapeutic treatments have made the spread of the virus a global concern. Viral nucleocapsid protein (N) is essential for its transcription and replication. Here, we present the crystal structures of N from SFTSV and its homologs from Buenaventura (BUE) and Granada (GRA) viruses. The structures reveal that phleboviral N folds into a compact core domain and an extended N-terminal arm that mediates oligomerization, such as tetramer, pentamer, and hexamer of N assemblies. Structural superimposition indicates that phleboviral N adopts a conserved architecture and uses a similar RNA encapsidation strategy as that of RVFV-N. The RNA binding cavity runs along the inner edge of the ring-like assembly. A triple mutant of SFTSV-N, R64D/K67D/K74D, almost lost its ability to bind RNA in vitro, is deficient in its ability to transcribe and replicate. Structural studies of the mutant reveal that both alterations in quaternary assembly and the charge distribution contribute to the loss of RNA binding. In the screening of inhibitors Suramin was identified to bind phleboviral N specifically. The complex crystal structure of SFTSV-N with Suramin was refined to a 2.30-Å resolution. Suramin was found sitting in the putative RNA binding cavity of SFTSV-N. The inhibitory effect of Suramin on SFTSV replication was confirmed in Vero cells. Therefore, a common Suramin-based therapeutic approach targeting SFTSV-N and its homologs could be developed for containing phleboviral outbreaks.

Intracellular pathogens with widespread drug-resistance contribute substantially to the increasing rates in morbidity and mortality due to emerging and reemerging diseases. Thus, the development of new drugs, including those that can enhance the immune response, is urgently needed. The immunomodulator, P-MAPA, a proteinaceous aggregate of ammonium and magnesium phospholinoleate-palmitoleate anhydride derived from Aspergillus oryzae, have been shown to induce antitumor activities. The ability of this compound to elicit protective immunity against viral infections has not been fully explored. Here, we report findings on the use of P-MAPA as an antiviral agent in a mouse model of acute phleboviral (Punta Toro virus) disease. A dose administered i.p. 24h post-infectious challenge (100mg/kg dose of P-MAPA) was remarkably effective at preventing death due to Punta Toro virus infection. This dose also reduced systemic viral burden and liver discoloration assayed on day 3 of infection. Taken together, our findings indicate that non-specific immunotherapy with P-MAPA appears to be an effective treatment for blocking Punta Toro virus-induced disease and suggest that further exploration with other viral disease models is warranted.

Punta Toro virus (PTV; genus Phlebovirus, family Bunyaviridae) causes apoptosis of hepatocytes in vivo in experimentally infected hamsters and in vitro in cultured HepG2 cells. Screening for expression of apoptosis-related genes has shown alterations in the genes for tumour necrosis factor-alpha (TNF-alpha) and the TNF receptor family. This study examined the roles of the TNF receptor-related extrinsic pathway and the Bcl-2 family-associated mitochondrial pathway in PTV-induced cell death. The effects of caspase inhibitors (caspIs) and TNF on cellular viability, virus replication, and morphological and biochemical changes in apoptosis were examined in HepG2 cells at different time points after infection with PTV (Adames strain). The results showed that caspIs dampened the virus-induced reduction in cellular viability, partially suppressed and delayed viral titres and antigen expression, and partially decreased the expression of apoptotic genes, caspase activities and DNA fragmentation. TNF treatment further decreased cellular viability after PTV infection and increased the level of apoptosis, whilst caspIs partially inhibited these effects. These findings indicate that TNF, caspase-8 and caspase-9 contribute to PTV-induced hepatocytic apoptosis and that additional mediators are probably also involved in this process. These mediators from different pathways correlated with one another and may be interlinked.

Punta Toro virus (PTV) is a member of the Bunyaviridae family, genus Phlebovirus, related to the highly pathogenic Rift Valley fever virus (RVFV). It produces a disease in hamsters that models severe Rift Valley fever (RVF) in humans. The recent outbreak of RVF in Kenya stresses the need to identify prophylactic and therapeutic measures for preventing and treating severe forms of disease. To this end, interferon (IFN) alfacon-1 (consensus IFN-alpha) was evaluated in cell culture against RVFV and PTV, and in the hamster PTV infection model. Survival outcome following treatment initiated pre- and post-virus challenge and the suppression of viral burden and liver disease in infected hamsters was determined. Pre-treatment of cell cultures with IFN alfacon-1 induced marked antiviral activity against both viruses. Intraperitoneal treatment of hamsters initiated 4 h prior to infection with PTV was highly protective and greatly limited liver disease and systemic and liver viral burden. Complete protection from a highly lethal challenge dose was afforded by treatment initiated 36 h following viral inoculation. Although efficacy was much reduced, IFN alfacon-1 therapy was still beneficial when started as late as 3-5 days post-virus exposure. These studies suggest that IFN alfacon-1 may be an effective treatment for early intervention following infection with RVFV.

The possible effect of virus adaptation to different transmission routes on virus stability in the environment is not well known. In this study we have compared the stabilities of three viruses within the Bunyaviridae family: the rodent-borne Hantavirus Hantaan virus (HTNV), the sand fly-borne Phlebovirus sandfly fever Sicilian virus (SFSV), and the tick-borne Nairovirus Crimean-Congo hemorrhagic fever virus (CCHFV). These viruses differ in their transmission routes: SFSV and CCHFV are vector borne, whereas HTNV is spread directly between its hosts, and to humans, via the environment. We studied whether these viruses differed regarding stability when kept outside of the host. Viral survival was analyzed at different time points upon exposure to different temperatures (4 degrees C, 20 degrees C, and 37 degrees C) and drying at 20 degrees C. We observed clearly different stabilities under wet conditions, particularly at 4 degrees C, where infectious SFSV, HTNV, and CCHFV were detectable after 528, 96, and 15 days, respectively. All three viruses were equally sensitive to drying, as shown by drying on aluminum discs. Furthermore, HTNV and SFSV partially survived for 2 min in 30% ethanol, whereas CCHFV did not. Electron microscopy images of HTNV, SSFSV, and CCHFV stored at 37 degrees C until infectivity was lost still showed the occurrence of virions, but with abnormal shapes and densities compared to those of the nonincubated samples. In conclusion, our study points out important differences in ex vivo stability among viruses within the Bunyaviridae family.

Sandfly fever Sicilian virus (SFSV) was used in our laboratory to screen antiviral substances active toward viruses of the Bunyaviridae family. Antiviral activity was estimated by the reduction of the cytopathic effect of SFSV on infected Vero cells. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion. The specificity of action of each tested compound was estimated by the selectivity index (CD50/ED50). Selectivity indices of human recombinant interferon-alpha (IFN alpha) (Roferon and Introna), iota-, kappa- and lambda- carrageenans, fucoidan and 6-azauridine were much higher than that of ribavirin, the only antiviral substance which has been previously investigated for its inhibitory effects on Phlebovirus infections. Other compounds showed significant antiviral activity: glycyrrhizin, suramin sodium, dextran sulphate and pentosan polysulphate. All these compounds caused a concentration-dependent reduction in the virus yield. Ribavirin, 6-azauridine and IFN alpha have been shown to inhibit a late step of the virus replicative cycle, whereas glycyrrhizin and suramin sodium were active at an early step and the sulphated polysaccharides inhibited adsorption of SFSV on the cells. The antiviral compounds selected in this study as specific inhibitors of in vitro replication of SFSV are promising candidates for the chemotherapy of haemorrhagic fevers caused by viruses of the Bunyaviridae family. The combination of IFN alpha and ribavirin, which showed a synergistic antiviral effect, should be evaluated for the treatment of these infections.

A major component of a US Army Medical Research and Development Command-supported program to discover and develop new drugs for the treatment of Rift Valley fever, sandfly fever, and Crimean-Congo hemorrhagic fever has been to study candidate test materials against hepatotropic infections of C57BL/6 mice induced by the related but less biohazardous Punta Toro virus (PTV). The effects of 75 compounds, some of which were considered immunomodulators in their primary mechanism of activity, were studied in the PTV infection model. Of these, ribavirin, ribamidine, ribavirin 2',3',5'-triacetate, tiazofurin, tiazofurin-5'-monophosphate, tiazofurin-2',3',5'-triacetate, selenazofurin, pyrazofurin, 3-deazaguanine, and 3-deazaguanosine were considered significantly inhibitory, acting against the infection by a direct antiviral (non-immunomodulatory) fashion. These compounds had therapeutic indices (TI) ranging from > or = 5 to 65, using increased survivors as the evaluation parameter. Immunomodulators considered significantly inhibitory to this infection were poly (ICLC), ampligen, human recombinant interferon-alpha-A/D, MVE-1, MVE-2, AM-3, AM-5, mannozym, bropirimine, CL246,738, phenyleneamine, and 7-thia-8-oxoguanosine. Utilizing increased survivor numbers as measure of activity, these inhibitors had TI ranging from > or = 16 to 1000. Other antiviral effects exerted by the active compounds included reduction of hepatic icterus, lowered serum glutamic oxaloacetic and pyruvic acid transaminases, and inhibition of recoverable serum and liver virus titers. The active immunomodulators were significantly effective when therapy was initiated as late as 48 h after virus inoculation, at a time when clinical signs of the PTV disease were being manifested in the animal.

A novel nucleoside analog, 3'-fluoro-3'-deoxyadenosine (3'F3'dAdo), was evaluated for antiviral activity against several arthropod-borne and arenaviruses in Vero cell culture. The following 50% inhibitory concentrations (EC50) of virus plaque formation were obtained against the test viruses: Semliki Forest (10.3 microM) and Venezuelan equine encephalitis (5.3 microM) alphaviruses, lymphocytic choriomeningitis (7.7 microM) and Pichinde (greater than 32 microM) arenaviruses, Punta Toro (greater than 32 microM) and San Angelo (1.6 microM) bunyaviruses, banzi flavivirus (4.0 microM), and Colorado tick fever orbivirus (0.6 microM). By comparison, the broad-spectrum antiviral agent ribavirin was active against lymphocytic choriomeningitis (18 microM), Pichinde (24 microM), Punta Toro (114 microM), and San Angelo (99 microM) viruses, but was less active against the other 4 viruses (greater than 200 microM). Vero cell proliferation and thymidine and uridine incorporation into replicating Vero cells were inhibited by 50% with 3'F3'dAdo concentrations of 36, 45, and 32 microM, respectively. In virus yield reduction assays, increasing the multiplicity of infections of Semliki Forest and Venezuelan equine encephalitis viruses reduced the inhibitory activity of 3'F3'dAdo. Using the same assay, 3'F3'dAdo was found to enhance Punta Toro virus replication up to 5-fold relative to the untreated control. By adding the nucleoside transport inhibitor nitrobenzylthioinosine (100 microM) to the culture medium, antiviral activity against the two alphaviruses was eliminated, indicating that 3'F3'dAdo uses the nucleoside transport system for cell entry. When actinomycin D (5 microM) was used to greatly suppress cellular RNA synthesis in Semliki Forest virus-infected and uninfected cells, 3'F3'dAdo preferentially inhibited viral RNA synthesis. The results of these studies indicate 3'F3'dAdo is a selective inhibitor of most of the viruses tested and should be a promising candidate for in vivo evaluations.

We studied the effects of various oils used to trap sand flies on the recovery of virus from infected adult Phlebotomus papatasi. Both Rift Valley fever and sandfly fever Sicilian viruses were readily recovered from virus-inoculated specimens held at 26 degrees C on mineral, olive or castor oil-soaked sheets for up to 12 h. However, after 50 h on oil-soaked paper, significantly greater titers were recovered from sand flies trapped with mineral oil than from sand flies trapped with either of the other oils. This indicates that sand flies trapped on oil-soaked paper would be suitable for virus isolation attempts and that mineral oil had the least effect on virus recovery.

Punta Toro virus (PTV), a member of the sandfly fever group of bunyaviruses, is assembled by budding at intracellular membranes of the Golgi complex. We have examined PTV glycoprotein transport, assembly, and release and the effects of brefeldin A (BFA) on these processes. Both the G1 and G2 proteins were transported out of the endoplasmic reticulum (ER) and retained in the Golgi complex in a stable structure, either during PTV infection or when expressed from a vaccinia virus recombinant. BFA treatment causes a rapid and dramatic change in the distribution of the G1 and G2 proteins, from a Golgi pattern to an ER pattern. The G1 and G2 proteins were found to be modified by medial but not trans Golgi network enzymes, in the presence or absence of BFA. We found that BFA blocks PTV release from cells but does not interfere with the intracellular assembly of infectious virions. Further, the BFA block of virus release is fully reversible, with high levels of virus release occurring upon removal of the inhibitor. It was also found that the release of PTV virions is polarized, occurring exclusively from the basolateral surfaces of the polarized Vero C1008 epithelial cell line.

The antibiotic pyrazofurin, 3-(beta-D-ribofuranosyl)-4-hydroxypyrazole-5-carboxamide, markedly inhibited the in vitro replication of a number of RNA viruses including Rift Valley fever (RVF), Venezuelan equine encephalomyelitis (VEE), Sandfly, Pichinde, Lassa and LCM virus. Plaque formation was reduced by 80% or more with 2-10 micrograms/ml of pyrazofurin while 2 micrograms/ml reduced by 1000-fold the yield of Lassa and LCM virus in a yield reduction assay. In vivo, pyrazofurin failed to protect mice and guinea pigs against a lethal challenge with VEE and Pichinde virus, respectively. On the other hand, pyrazofurin caused a slight increase in the mean time to death of mice infected with RVF virus.

Dimethyl sulfoxide (DMSO)incorporated into an agar overlay containing DEAE-dextran enhanced plaque formation in Vero cells by Naples sandfly fever virus passaged in mouse brain or Vero cell cultures. No plaques were visible when DMSO was used without the DEAE-dextran, some plaques were rarely visible (less than 0.5mm) when DEAE-dextran was used without the DMSO, and up to 10-fold more plaques were clearly visible (0.5-1.5 mm) when both chemicals were used. The combined enhancing effect of DMSO and DEAE-dextran was also observed with mouse brain passaged, but not Vero passaged Sicilian sandfly fever virus. Other Phlebotomus group viruses produced a bit plaques (3-5 mm) and did not require DMSO for plaque formation, although an increase in plaque clarity was obtained with DMSO for some of them. Plaque reduction neutralization tests were assayed successfully under agar containing DMSO. The alphavirus Sindbis produced slightly larger plaques under agar containing DMSO, but there was no effect on clarity or size of plaques produced by the flavivirus dengue-2.