Gn protein expressed in plants for diagnosis of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes the highly fatal disease in humans. To facilitate diagnosis, the native form of subunit glycoprotein (Gn), a prime target for potential vaccines and therapies, was produced in Nicotiana benthamiana using a Bamboo mosaic virus-based vector system. By fusion with secretory signal tags, SSExt, derived from the extension protein, and the (SP)10 motif, the yield of the recombinant Gn (rGn) was remarkably increased to approximately 7 mg/kg infiltrated leaves. Ultimately, an rGn-based ELISA was successfully established for the detection of SFTSV-specific antibodies in serum samples from naturally infected monkeys. As validated with the reference method, the specificity and sensitivity of rGn-ELISA were 94% and 96%, respectively. In conclusion, utilizing well-suited fusion tags facilitates rGn production and purification in substantial quantities while preserving its antigenic properties. The rGn-ELISA, characterized by its commendable sensitivity and specificity could serve as a viable alternative diagnostic method for assessing SFTSV seroprevalence. KEY POINTS: • SFTSV Gn, fused with secretory signal tags, was expressed by the BaMV-based vector. • The plant fusion tags increased expression levels and eased the purification of rGn. • The rGn-ELISA was established and validated; its specificity and sensitivity > 94%.

CCR2 is a host entry receptor for severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus causing a high fatality rate of up to 30%. To date, the receptor mediating SFTSV entry remained uncharacterized, hindering the understanding of disease pathogenesis. Here, C-C motif chemokine receptor 2 (CCR2) was identified as a host receptor for SFTSV based on a genome-wide CRISPR-Cas9 screen. Knockout of CCR2 substantially reduced viral binding and infection. CCR2 enhanced SFTSV binding through direct binding to SFTSV glycoprotein N (Gn), which is mediated by its N-terminal extracellular domain. Depletion of CCR2 in C57BL/6J mouse model attenuated SFTSV replication and pathogenesis. The peripheral blood primary monocytes from elderly individuals or subjects with underlying diabetes mellitus showed higher CCR2 surface expression and supported stronger binding and replication of SFTSV. Together, these data indicate that CCR2 is a host entry receptor for SFTSV infection and a novel target for developing anti-SFTSV therapeutics.

The SFTSV Nonstructural Proteins Induce Autophagy to Promote Viral Replication via Interaction with Vimentin.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified phlebovirus associated with severe hemorrhagic fever in humans. Studies have shown that SFTSV nucleoprotein (N) induces BECN1-dependent autophagy to promote viral assembly and release. However, the function of other SFTSV proteins in regulating autophagy has not been reported. In this study, we identify SFTSV NSs, a nonstructural protein that forms viroplasm-like structures in the cytoplasm of infected cells as the virus component mediating SFTSV-induced autophagy. We found that SFTSV NSs-induced autophagy was inclusion body independent, and most phenuivirus NSs had autophagy-inducing effects. Unlike N protein-induced autophagy, SFTSV NSs was key in regulating autophagy by interacting with the host's vimentin in an inclusion body-independent manner. NSs interacted with vimentin and induced vimentin degradation through the K48-linked ubiquitin-proteasome pathway. This negatively regulating Beclin1-vimentin complex formed and promoted autophagy. Furthermore, we identified the NSs-binding domain of vimentin and found that overexpression of wild-type vimentin antagonized the induced effect of NSs on autophagy and inhibited viral replication, suggesting that vimentin is a potential antiviral target. The present study shows a novel mechanism through which SFTSV nonstructural protein activates autophagy, which provides new insights into the role of NSs in SFTSV infection and pathogenesis. IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerging tick-borne pathogen that causes multifunctional organ failure and even death in humans. As a housekeeping mechanism for cells to maintain steady state, autophagy plays a dual role in viral infection and the host's immune response. However, the relationship between SFTSV infection and autophagy has not been described in detail yet. Here, we demonstrated that SFTSV infection induced complete autophagic flux and facilitated viral proliferation. We also identified a key mechanism underlying NSs-induced autophagy, in which NSs interacted with vimentin to inhibit the formation of the Beclin1-vimentin complex and induced vimentin degradation through K48-linked ubiquitination modification. These findings may help us understand the new functions and mechanisms of NSs and may aid in the identification of new antiviral targets.

Development and evaluation of a mouse model susceptible to severe fever with thrombocytopenia syndrome virus by rAAV-based exogenous human DC-SIGN expression.

Experimental animal model is indispensable to evaluate the prophylactic and therapeutic candidates against severe fever with thrombocytopenia syndrome virus (SFTSV). To develop a suitable mouse model for SFTSV infection, we delivered human dendritic cell-specific ICAM-3-grabbing non-integrin (hDC-SIGN) by adeno-associated virus (AAV2) and validated its susceptibility for SFTSV infection. Western blot and RT-PCR assays confirmed the expression of hDC-SIGN in transduced cell lines and a significantly increased viral infectivity was observed in cells expressing hDC-SIGN. The C57BL/6 mice transduced with AAV2 exhibited a stable hDC-SIGN expression in the organs for 7 days. Upon SFTSV challenge with 1 × 105 FAID50, the mice transduced with rAAV-hDC-SIGN showed a 12.5% mortality and reduced platelet and white blood cell count in accordance with higher viral titer than control group. Liver and spleen samples collected from the transduced mice had pathological signs similar to the IFNAR-/- mice with severe SFTSV infection. Collectively, the rAAV-hDC-SIGN transduced mouse model can be used as an accessible and promising tool for studying the SFTSV pathogenesis and pre-clinical evaluation of vaccines and therapeutics against the SFTSV infection.

Development of functional anti-Gn nanobodies specific for SFTSV based on next-generation sequencing and proteomics.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, we demonstrated that SFTSV surface glycoprotein (Glycoprotein N, Gn) was a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti-Gn neutralizing antibodies played a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages, including small molecular weight, high affinity, low immunogenicity, convenient production by gene engineering, etc. In this study, we combined next-generation sequencing (NGS) with proteomics technology based on affinity purification-mass spectrometry (AP-MS) and bioinformatics analysis to high-throughput screen monoclonal anti-Gn nanobodies from camel immunized with Gn protein. We identified 19 anti-Gn monoclonal nanobody sequences, of which six sequences were selected for recombinant protein expression and purification. Among these six anti-Gn nanobodies, nanobody 57,493 was validated to be highly specific for Gn. The innovative high-throughput technical route developed in this study could also be expanded to the production of nanobodies specific for other viruses like SARS-CoV-2.

In silico study on miRNA regulation and NSs protein interactome characterization of the SFTS virus.

Severe fever with thrombocytopenia syndrome causing virus i.e. SFTS virus has increased in the last few years. The underlying cause and mechanism of disease progression and development of symptoms is not well known. Many viruses including Hepatitis B, Hepatitis C, HIV-1, Herpes virus, Dengue virus and many others have been seen to regulate their functions at the miRNA level. This study aimed to find out those cellular miRNAs, which can be mimicked or antagonized by the viral genome and analyze the effect of these miRNAs on various gene functions. Investigations in this study suggest a correlation between miRNA regulation with the disease symptoms and progression. By exhaustive literature survey we have tried to identify the interacting partners of the Non Structural S (NSs) protein and characterized the protein-protein interactions. The binding interface that can serve as target for therapeutic studies involving the interfacial residues was analyzed. This study would serve as an avenue to design therapeutics making use of not only protein-protein interactions but also miRNA based regulation as well.

A Viral NOD to Encephalitis.

In this issue, Gao and colleagues (J Virol 96:e00167-22, https://doi.org/10.1128/JVI.00167-22) dissect innate immune signaling in a microglial cell line infected with severe fever with thrombocytopenia syndrome virus (SFTSV). This virus has been designated a priority pathogen by the World Health Organization due to its capacity to induce a fatal cytokine storm. The study's findings attribute the pathogenesis to induction of the host inflammasome response by the SFTSV nonstructural protein.

Nonstructural Protein NSs Activates Inflammasome and Pyroptosis through Interaction with NLRP3 in Human Microglial Cells Infected with Severe Fever with Thrombocytopenia Syndrome Bandavirus.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne febrile disease caused by SFTS virus (SFTSV), or Dabie bandavirus, in the Phenuiviridae family. Clinically neurological disorders in SFTS have been commonly reported, but their neuropathogenesis has rarely been studied. Microglia are a type of neuroglia accounting for 10 to 12% of all cells in the brain. As resident immune cells, microglial cells are the first line of immune defense present in the central nervous system (CNS). Here, we report that SFTSV was able to infect microglial cells and stimulate interleukin 1ß (IL-1ß) secretion in the brains of infected neonatal BALB/c mice. We characterized the cell death induced in infected human microglial HMC3 cells, also susceptible to SFTSV, and found that the NOD-like receptor protein 3 (NLRP3) inflammasome was activated, leading to secretion of IL-1ß and pyroptosis. Knockdown of NLRP3 or inhibition of the NLRP3 inflammasome activation suppressed the viral replication, suggesting that the activation of the NLRP3 inflammasome may support SFTSV replication in microglial cells. Viral nonstructural protein NSs, a known modulator of immune responses, interacted and colocalized with NLRP3 for the inflammasome activation. It appeared that the N-terminal fragment, amino acids 1 to 66, of NSs was critical to promote the assembly of the inflammasome complex by interacting with NLRP3 for its activation in microglial cells. Our findings provide evidence that SFTSV may cause neurological disorders through infecting microglia and activating the inflammasome through its nonstructural protein NSs for neural cell death and inflammation. This study may have revealed a novel mechanism of SFTSV NSs in dysregulating host response. IMPORTANCE Encephalitis or encephalopathy during severe fever with thrombocytopenia syndrome (SFTS) is considered a critical risk factor leading to high mortality, but there have been no studies to date on the pathogenesis of encephalitis or encephalopathy caused by SFTS virus. Here, we report that SFTSV infection can active the NLRP3 inflammasome and induce IL-1ß secretion in the brains of infected newborn mice. In infected human HMC3 microglia, SFTSV activated the NLRP3 inflammasome via the viral nonstructural protein NSs through interaction with its N-terminal fragment. Notably, our findings suggest that the activation of the NLRP3 inflammasome may promote SFTSV replication in infected microglial cells. This study may reveal a novel mechanism by SFTSV to dysregulate host responses through its nonstructural protein, which could help us understand viral neuropathogenesis in SFTS patients.

A bioactive phlebovirus-like envelope protein in a hookworm endogenous virus.

Endogenous viral elements (EVEs), accounting for 15% of our genome, serve as a genetic reservoir from which new genes can emerge. Nematode EVEs are particularly diverse and informative of virus evolution. We identify Atlas virus-an intact retrovirus-like EVE in the human hookworm Ancylostoma ceylanicum, with an envelope protein genetically related to GN-GC glycoproteins from the family Phenuiviridae. A cryo-EM structure of Atlas GC reveals a class II viral membrane fusion protein fold not previously seen in retroviruses. Atlas GC has the structural hallmarks of an active fusogen. Atlas GC trimers insert into membranes with endosomal lipid compositions and low pH. When expressed on the plasma membrane, Atlas GC has cell-cell fusion activity. With its preserved biological activities, Atlas GC has the potential to acquire a cellular function. Our work reveals structural plasticity in reverse-transcribing RNA viruses.

The AST/ALT Ratio (De Ritis Ratio) Represents an Unfavorable Prognosis in Patients in Early-Stage SFTS: An Observational Cohort Study.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS), a widely prevalent infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV) that carries with it a high mortality rate, has emerged to be a public health concern. This study aimed to investigate the epidemiological and clinical characteristics of patients infected with SFTSV, seeking novel prognostic risk factors for SFTS. METHODS: In this retrospective and cross-sectional study, confirmed SFTS patients from the First Affiliated Hospital of Anhui Medical University were enrolled from September 1, 2019, to December 12, 2020. Cases were analyzed for epidemiological, demographic, clinical, and laboratory data. Logistic regression models were used to assess the association between predictors and outcome variables. A generalized additive mixed model (GAMM) was conducted to analyze the trending shift of aspartate aminotransferase/alanine transaminase-ratio (AST/ALT-ratio) and platelet (PLT) in SFTS patients treated with ribavirin. p values ≤ 0.05 were considered statistically significant. RESULTS: Clinical and laboratory results of 107 hospitalized patients with SFTSV infection were retrospectively described. The mean age at onset of disease was 60.38 ± 11.29 years old and the ratio between male and female was 1:1.2. Fever and thrombocytopenia are hallmark features of SFTS. Furthermore, multiple cases also experienced neurological complications, gastrointestinal/skeletal muscle symptoms together with other non-specific clinical manifestations; laboratory dataset outcomes reported dysregulated levels for routine blood biomarkers, coagulation function, and biochemistry. Overall, 107 patients were segregated into two groups according to patient condition at the clinical endpoint (survivors/non-survivors). SFTS survivors had a higher level of PLT- counts, total protein (TP), and estimated glomerular filtration rate (eGFR), while levels of activated partial thromboplastin time (APTT), thrombin time (TT), D-dimer (D-D), fibrinogen degradation products (FDP), ALT, AST, AST/ALT-ratio, creatinine (Cr), creatine phosphokinase (CK) and procalcitonin (PCT) was higher in non-survivors. Results from univariate Cox regression revealed that elevated levels of FDP, TT, AST/ALT-ratio, PCT, as well as decreased eGFR level and presence of central nervous system symptoms (CNS), were significant predictors for SFTS prognostic, results from multivariate logistic regression analysis in three adjusted models showed AST/ALT-ratio and PCT were independent risk factors for the prognosis of SFTS patients. Kaplan-Meier survival analysis showed that SFTS patients with AST/ALT-ratio >2.683 were associated with a shorter futime (means survival time), therefore indicating an unfavorable prognosis. Treatment with ribavirin could increase PLT count while decreasing AST/ALT-ratio within SFTS patients. CONCLUSION: SFTS is an emerging infectious disease, possibly leading to multiple-organ injury; AST/ALT-ratio was an independent risk factor for the prognosis of SFTS patients. Further investigation should be performed in order to gain more knowledge on this disease and guide clinical management.

Bunyavirus SFTSV exploits autophagic flux for viral assembly and egress.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging negatively stranded enveloped RNA bunyavirus that causes SFTS with a high case fatality rate of up to 30%. Macroautophagy/autophagy is an evolutionarily conserved process involved in the maintenance of host homeostasis, which exhibits anti-viral or pro-viral responses in reaction to different viral challenges. However, the interaction between the bunyavirus SFTSV and the autophagic process is still largely unclear. By establishing various autophagy-deficient cell lines, we found that SFTSV triggered RB1CC1/FIP200-BECN1-ATG5-dependent classical autophagy flux. SFTSV nucleoprotein induced BECN1-dependent autophagy by disrupting the BECN1-BCL2 association. Importantly, SFTSV utilized autophagy for the viral life cycle, which not only assembled in autophagosomes derived from the ERGIC and Golgi complex, but also utilized autophagic vesicles for exocytosis. Taken together, our results suggest a novel virus-autophagy interaction model in which bunyavirus SFTSV induces classical autophagy flux for viral assembly and egress processes, suggesting that autophagy inhibition may be a novel therapy for treating or releasing SFTS.

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.

Nonstructural Protein NSs Hampers Cellular Antiviral Response through LSm14A during Severe Fever with Thrombocytopenia Syndrome Virus Infection.

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) plays multiple functions in the virus life cycle. Proteomic screening for host proteins interacting with NSs identified the cellular protein LSm14A. LSm14A, a member of the LSm family involved in RNA processing in the processing bodies, binds to viral RNA or synthetic homolog and mediates IFN regulatory factor 3 activation and IFN-ß induction. NSs interacted with and colocalized with LSm14A, and this interaction effectively inhibited downstream phosphorylation and dimerization of IFN regulatory factor 3, resulting in the suppression of antiviral signaling and IFN induction in several cell types of human origin. Knockdown of NSs resulted in the suppression of SFTSV replication in host cells. Viral RNA bound to LSm14A-NSs protein complex during the interaction. A newly discovered LRRD motif of NSs functioned to interact with LSm14A. Altogether, our data demonstrated a mechanism used by SFTSV to inhibit host innate immune response.

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.

Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble those of SFTS patients, whereas SFTS-contracted cats have high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals. In this study, a reverse transcription polymerase chain reaction (RT-PCR) was newly developed to diagnose SFTS-suspected animals without non-specific reactions. METHODOLOGY/PRINCIPLE FINDINGS: Four primer sets were newly designed from consensus sequences constructed from 108 strains of SFTSV. A RT-PCR with these four primer sets successfully and specifically detected four clades of SFTSV. Their limits of detection are 1-10 copies/reaction. Using this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients. CONCLUSION/SIGNIFICANCE: This newly developed RT-PCR could detect SFTSV RNA of several clades and from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.

Structure of severe fever with thrombocytopenia syndrome virus L protein elucidates the mechanisms of viral transcription initiation.

Segmented negative-sense RNA viruses (sNSRVs) encode a single-polypeptide polymerase (L protein) or a heterotrimeric polymerase complex to cannibalize host messenger RNA cap structures serving as primers of transcription, and catalyse RNA synthesis. Here, we report the full-length structure of the severe fever with thrombocytopaenia syndrome virus (SFTSV) L protein, as determined by cryogenic electron microscopy at 3.4 Å, leading to an atomic model harbouring three functional parts (an endonuclease, an RNA-dependent RNA polymerase and a cap-binding domain) and two structural domains (an arm domain with a blocker motif and a carboxy-terminal lariat domain). The SFTSV L protein has a compact architecture in which its cap-binding pocket is surprisingly occupied by an Arg finger of the blocker motif, and the endonuclease active centre faces back towards the cap-binding pocket, suggesting that domain rearrangements are necessary to acquire the pre-initiation state of the active site. Our results provide insight into the complete architecture of sNSRV-encoded L protein and further the understanding of sNSRV transcription initiation.

Zwiesel bat banyangvirus, a potentially zoonotic Huaiyangshan banyangvirus (Formerly known as SFTS)-like banyangvirus in Northern bats from Germany.

Bats are reservoir hosts for several emerging and re-emerging viral pathogens causing morbidity and mortality in wildlife, animal stocks and humans. Various viruses within the family Phenuiviridae have been detected in bats, including the highly pathogenic Rift Valley fever virus and Malsoor virus, a novel Banyangvirus with close genetic relation to Huaiyangshan banyangvirus (BHAV)(former known as Severe fever with thrombocytopenia syndrome virus, SFTSV) and Heartland virus (HRTV), both of which have caused severe disease with fatal casualties in humans. In this study we present the whole genome of a novel Banyangvirus, named Zwiesel bat banyangvirus, revealed through deep sequencing of the Eptesicus nilssonii bat virome. The detection of the novel bat banyangvirus, which is in close phylogenetic relationship with the pathogenic HRTV and BHAV, underlines the possible impact of emerging phenuiviruses on public health.

Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway.

Nuclear factor erythroid 2-related factor 2 (Nrf2) dissociates from its inhibitor, Keap1, upon stress signals and subsequently induces an antioxidant response that critically controls the viral life cycle and pathogenesis. Besides intracellular Fc receptor function, tripartite motif 21 (TRIM21) E3 ligase plays an essential role in the p62-Keap1-Nrf2 axis pathway for redox homeostasis. Specifically, TRIM21-mediated p62 ubiquitination abrogates p62 oligomerization and sequestration activity and negatively regulates the Keap1-Nrf2-mediated antioxidant response. A number of viruses target the Nrf2-mediated antioxidant response to generate an optimal environment for their life cycle. Here we report that a nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) interacts with and inhibits TRIM21 to activate the Nrf2 antioxidant signal pathway. Mass spectrometry identified TRIM21 to be a binding protein for NSs. NSs bound to the carboxyl-terminal SPRY subdomain of TRIM21, enhancing p62 stability and oligomerization. This facilitated p62-mediated Keap1 sequestration and ultimately increased Nrf2-mediated transcriptional activation of antioxidant genes, including those for heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, and CD36. Mutational analysis found that the NSs-A46 mutant, which no longer interacted with TRIM21, was unable to increase Nrf2-mediated transcriptional activation. Functionally, the NS wild type (WT), but not the NSs-A46 mutant, increased the surface expression of the CD36 scavenger receptor, resulting in an increase in phagocytosis and lipid uptake. A combination of reverse genetics and assays with Ifnar-/- mouse models revealed that while the SFTSV-A46 mutant replicated similarly to wild-type SFTSV (SFTSV-WT), it showed weaker pathogenic activity than SFTSV-WT. These data suggest that the activation of the p62-Keap1-Nrf2 antioxidant response induced by the NSs-TRIM21 interaction contributes to the development of an optimal environment for the SFTSV life cycle and efficient pathogenesis.IMPORTANCE Tick-borne diseases have become a growing threat to public health. SFTSV, listed by the World Health Organization as a prioritized pathogen, is an emerging phlebovirus, and fatality rates among those infected with this virus are high. Infected Haemaphysalis longicornis ticks are the major source of human SFTSV infection. In particular, the recent spread of this tick to over 12 states in the United States has increased the potential for outbreaks of this disease beyond Far East Asia. Due to the lack of therapies and vaccines against SFTSV infection, there is a pressing need to understand SFTSV pathogenesis. As the Nrf2-mediated antioxidant response affects viral life cycles, a number of viruses deregulate Nrf2 pathways. Here we demonstrate that the SFTSV NSs inhibits the TRIM21 function to upregulate the p62-Keap1-Nrf2 antioxidant pathway for efficient viral pathogenesis. This study not only demonstrates the critical role of SFTSV NSs in viral pathogenesis but also suggests potential future therapeutic approaches to treat SFTSV-infected patients.

Fine epitope mapping of glycoprotein Gn in Guertu virus.

Guertu virus (GTV) is a tick-borne phleboviruses (TBPVs) which belongs to the genus Banyangvirus in the family of Phenuiviridae. In vitro and in vivo studies of GTV demonstrated that it was able to infect animal and human cell lines and could cause pathological lesions in mice. Glycoproteins (GP, including Gn and Gc) on the surface of Guertu virus (GTV) could bind to receptors on host cells and induce protective immunity in the host, but knowledge is now lacking on the information of B cell epitopes (BCEs) present on GTV-GP protein. The aim of this study was to identify all BCEs on Gn of the GTV DXM strain using rabbit pAbs against GTV-Gn. Seven fine BCEs and two antigenic peptides (APs) from nine reactive 16mer-peptides were identified, which are EGn1 (2PIICEGLTHS11), EGn2 (135CSQDSGT141), EGn3 (165IP EDVF170), EGn4 (169VFQEL K174), EGn5 (187IDGILFN193), EGn6 (223QTKWIQ228), EGn7 (237CHKDGIGPC245), AP-8 (299GVRVRPKCYGFSRMMA314) and AP-9 (355CASH FCSSAESGKKNT370), of which six of mapped BCEs were recognized by the IgG-positive sheep serum obtained from sheep GTV-infected naturally. Multiple sequence alignments (MSA) based on each mapped BCE motif identified that the most of identified BCEs and APs are highly conserved among 10 SFTSV strains from different countries and lineages that share relatively close evolutionary relationships with GTV. The fine epitope mapping of the GTV-Gn would provide basic data with which to explore the GTV-Gn antigen structure and pathogenic mechanisms, and it could lay the foundation for the design and development of a GTV multi-epitope peptide vaccine and detection antigen.

Quantitative Proteomic Analysis Reveals Unfolded-Protein Response Involved in Severe Fever with Thrombocytopenia Syndrome Virus Infection.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging, highly pathogenic, infectious disease caused by infection with a newly discovered tick-borne phlebovirus, SFTS virus (SFTSV). Limited information on the molecular mechanism of SFTSV infection and pathogenesis impedes the development of effective vaccines and drugs for SFTS prevention and treatment. In this study, an isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis of SFTSV-infected HEK 293 cells was performed to explore dynamic host cellular protein responses toward SFTSV infection. A total of 433 of 5,606 host proteins involved in different biological processes were differentially regulated by SFTSV infection. The proteomic results highlighted a potential role of endoplasmic reticular stress-triggered unfolded-protein response (UPR) in SFTSV infection. Further functional studies confirmed that all three major branches of the UPR, including the PKR-like endoplasmic reticulum kinase (PERK), the activating transcription factor-6 (ATF6), and the inositol-requiring protein-1 (IRE1)/X-box-binding protein 1 (XBP1) pathways, were activated by SFTSV. However, only the former two pathways play a crucial role in SFTSV infection. Furthermore, expression of SFTSV glycoprotein (GP) alone was sufficient to stimulate the UPR, whereas suppression of PERK and ATF6 notably decreased GP expression. Interestingly, two other newly discovered phleboviruses, Heartland virus and Guertu virus, also stimulated the UPR, suggesting a common mechanism shared by these genetically related phleboviruses. This study provides a global view to our knowledge on how host cells respond to SFTSV infection and highlights that host cell UPR plays an important role in phlebovirus infection.IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe fever with thrombocytopenia syndrome in humans, with a mortality rate reaching up to 30% in some outbreaks. There are currently no U.S. Food and Drug Administration-approved vaccines or specific antivirals available against SFTSV. To comprehensively understand the molecular interactions occurring between SFTSV and the host cell, we exploit quantitative proteomic approach to investigate the dynamic host cellular responses to SFTSV infection. The results highlight multiple biological processes being regulated by SFTSV infection. Among these, we focused on exploration of the mechanism of how SFTSV infection stimulates the host cell's unfolded-protein response (UPR) and identified the UPR as a common feature shared by SFTSV-related new emerging phleboviruses. This study, for the first time to our knowledge, provides a global map for host cellular responses to SFTSV infection and highlighted potential host targets for further research.