The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.

In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.

Hsp90 ß is critical for the infection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV) is an emerging disease in East Asia with a fatality rate of up to 30%. However, the viral-host interaction of SFTSV remains largely unknown. The heat-shock protein 90 (Hsp90) family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses. Here, we showed that Hsp90 is an important host factor involved in SFTSV infection. Hsp90 inhibitors significantly reduced SFTSV replication, viral protein expression, and the formation of inclusion bodies consisting of nonstructural proteins (NSs). Among viral proteins, NSs appeared to be the most reduced when Hsp90 inhibitors were used, and further analysis showed that their translation was affected. Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90 ß specifically interacted with them. Knockdown of Hsp90 ß expression also inhibited replication of SFTSV. These results suggest that Hsp90 ß plays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.

Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early and late stages of elongation with the RdRp core accommodating a 10-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA 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.

Comparison of RT-PCR, RT-nested PCRs, and real-time PCR for diagnosis of severe fever with thrombocytopenia syndrome: a prospective study.

We designed a highly sensitive reverse transcription nested polymerase chain reaction targeting the M-segment (NPCR-M) of severe fever with thrombocytopenia syndrome (SFTS) virus. NPCR-M was performed in parallel with three other referenced PCR assays QPCR-S, PCR-M, and NPCR-S to assess their clinical usefulness as routine diagnostic techniques for SFTS. In this multi-centered prospective study, 122 blood samples from 38 laboratory-confirmed SFTS patients and 85 control samples were used. The results demonstrated that QPCR-S and NPCR-S had better sensitivity rate up to 21 days after symptom onset however, the PCR-M showed poor sensitivity after 7 days of symptom onset. Our designed NPCR-M had a higher detection rate up to 40 days from symptom onset and revealed the persistence of SFTSV RNA in the early convalescent phase. No false-positive results were seen for the control samples. Additionally, NPCR-M showed positive results for a sample that initially showed negative results from other PCRs and for many other samples collected in the convalescent phase of SFTS. Our designed nested PCR is suitable for SFTSV detection in patients' blood collected in the acute and early convalescent phase of SFTS, and shows better sensitivity and high specificity even up to 40 days after symptom onset.

Clinical effect and antiviral mechanism of T-705 in treating severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) is an emerging tick-borne virus with high fatality and an expanding endemic. Currently, effective anti-SFTSV intervention remains unavailable. Favipiravir (T-705) was recently reported to show in vitro and in animal model antiviral efficacy against SFTSV. Here, we conducted a single-blind, randomized controlled trial to assess the efficacy and safety of T-705 in treating SFTS (Chinese Clinical Trial Registry website, number ChiCTR1900023350). From May to August 2018, laboratory-confirmed SFTS patients were recruited from a designated hospital and randomly assigned to receive oral T-705 in combination with supportive care or supportive care only. Fatal outcome occurred in 9.5% (7/74) of T-705 treated patients and 18.3% (13/71) of controls (odds ratio, 0.466, 95% CI, 0.174-1.247). Cox regression showed a significant reduction in case fatality rate (CFR) with an adjusted hazard ratio of 0.366 (95% CI, 0.142-0.944). Among the low-viral load subgroup (RT-PCR cycle threshold ≥26), T-705 treatment significantly reduced CFR from 11.5 to 1.6% (P = 0.029), while no between-arm difference was observed in the high-viral load subgroup (RT-PCR cycle threshold <26). The T-705-treated group showed shorter viral clearance, lower incidence of hemorrhagic signs, and faster recovery of laboratory abnormities compared with the controls. The in vitro and animal experiments demonstrated that the antiviral efficacies of T-705 were proportionally induced by SFTSV mutation rates, particularly from two transition mutation types. The mutation analyses on T-705-treated serum samples disclosed a partially consistent mutagenesis pattern as those of the in vitro or animal experiments in reducing the SFTSV viral loads, further supporting the anti-SFTSV effect of T-705, especially for the low-viral loads.

Molecular Signatures of Inflammatory Profile and B-Cell Function in Patients with Severe Fever with Thrombocytopenia Syndrome.

Dabie bandavirus (severe fever with thrombocytopenia syndrome virus [SFTSV]) induces an immunopathogenic disease with a high fatality rate; however, the mechanisms underlying its clinical manifestations are largely unknown. In this study, we applied targeted proteomics and single-cell transcriptomics to examine the differential immune landscape in SFTS patient blood. Serum immunoprofiling identified low-risk and high-risk clusters of SFTS patients based on inflammatory cytokine levels, which corresponded to disease severity. Single-cell transcriptomic analysis of SFTS patient peripheral blood mononuclear cells (PBMCs) at different infection stages showed pronounced expansion of B cells with alterations in B-cell subsets in fatal cases. Furthermore, plasma cells in which the interferon (IFN) pathway is downregulated were identified as the primary reservoir of SFTSV replication. This study identified not only the molecular signatures of serum inflammatory cytokines and B-cell lineage populations in SFTSV-induced fatalities but also plasma cells as the viral reservoir. Thus, this suggests that altered B-cell function is linked to lethality in SFTSV infections.IMPORTANCE SFTSV is an emerging virus discovered in China in 2009; it has since spread to other countries in East Asia. Although the fatality rates of SFTSV infection range from 5.3% to as high as 27%, the mechanisms underlying clinical manifestations are largely unknown. In this study, we demonstrated that SFTSV infection in fatal cases caused an excessive inflammatory response through high induction of proinflammatory cytokines and chemokines and the aberrant inactivation of adaptive immune responses. Furthermore, single-cell transcriptome sequencing (RNA-seq) analysis of SFTS patient PBMCs revealed that SFTSV targets the B-cell lineage population, especially plasma cells, as the potential viral reservoir in patients for whom the infection is fatal. Thus, SFTSV infection may inhibit high-affinity antibody maturation and secretion of plasma B cells, suppressing neutralizing antibody production and thereby allowing significant virus replication and subsequent fatality.

Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Phlebovirus Inhibits TBK1 to Evade Interferon-Mediated Response.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging phlebovirus of the Phenuiviridae family that has been circulating in the following Asian countries: Vietnam, Myanmar, Taiwan, China, Japan, and South Korea. Despite the increasing infection rates and relatively high mortality rate, there is limited information available regarding SFTSV pathogenesis. In addition, there are currently no vaccines or effective antiviral treatments available. Previous reports have shown that SFTSV suppresses the host immune response and its nonstructural proteins (NSs) function as an antagonist of type I interferon (IFN), whose induction is an essential part of the host defense system against viral infections. Given that SFTSV NSs suppress the innate immune response by inhibiting type I IFN, we investigated the mechanism utilized by SFTSV NSs to evade IFN-mediated response. Our co-immunoprecipitation data suggest the interactions between NSs and retinoic acid inducible gene-I (RIG-I) or TANK binding kinase 1 (TBK1). Furthermore, confocal analysis indicates the ability of NSs to sequester RIG-I and related downstream molecules in the cytoplasmic structures called inclusion bodies (IBs). NSs are also capable of inhibiting TBK1-interferon regulatory factor 3 (IRF3) interaction, and therefore prevent the phosphorylation and nuclear translocation of IRF3 for the induction of type I IFN. The ability of SFTSV NSs to interact with and sequester TBK1 and IRF3 in IBs demonstrate an effective yet unique method utilized by SFTSV to evade and suppress host immunity.

A RIG-I-like receptor directs antiviral responses to a bunyavirus and is antagonized by virus-induced blockade of TRIM25-mediated ubiquitination.

The RIG-I-like receptors (RLRs) retinoic acid-inducible gene I protein (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are cytosolic pattern recognition receptors that recognize specific viral RNA products and initiate antiviral innate immunity. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic member of the Bunyavirales RIG-I, but not MDA5, has been suggested to sense some bunyavirus infections; however, the roles of RLRs in anti-SFTSV immune responses remain unclear. Here, we show that SFTSV infection induces an antiviral response accompanied by significant induction of antiviral and inflammatory cytokines and that RIG-I plays a main role in this induction by recognizing viral 5'-triphosphorylated RNAs and by signaling via the adaptor mitochondrial antiviral signaling protein. Moreover, MDA5 may also sense SFTSV infection and contribute to IFN induction, but to a lesser extent. We further demonstrate that the RLR-mediated anti-SFTSV signaling can be antagonized by SFTSV nonstructural protein (NSs) at the level of RIG-I activation. Protein interaction and MS-based analyses revealed that NSs interacts with the host protein tripartite motif-containing 25 (TRIM25), a critical RIG-I-activating ubiquitin E3 ligase, but not with RIG-I or Riplet, another E3 ligase required for RIG-I ubiquitination. NSs specifically trapped TRIM25 into viral inclusion bodies and inhibited TRIM25-mediated RIG-I-Lys-63-linked ubiquitination/activation, contributing to suppression of RLR-mediated antiviral signaling at its initial stage. These results provide insights into immune responses to SFTSV infection and clarify a mechanism of the viral immune evasion, which may help inform the development of antiviral therapeutics.