Four FDA-approved drugs exhibited inhibition effect on severe fever with thrombocytopenia syndrome virus in vitro

Objective:To screen the anti-SFTSV drugs from 1430 FDA-approved drugs via mini-genome system,and to investigate which stage of the infection process could be suppressed by the identified drugs.Methods:The SFTSV mini-genome system was used to screen drugs with inhibitory effect on SFTSV replication and transcription,and the 50%inhibitory concentration(IC_(50))of each drug was calculated by drug concentration gradient inhibition experiment.Drugs were used to pre-incubate with virus and then incubate with cells,to incubate with virus and cells simultaneously,to incubate with cells after virus invading into cells,or to incubate during the whole infection process,and then qRT-PCR was used to measure the viral RNA copies in the culture supernatant.These experiments were performed to quantitatively determine the inhibition effects of drugs on SFTSV indifferent stages of the whole process including virion stability,entry and post-entry stages,so as to clarify the inhibition mechanism of these drugs.Results:Four drugs including Mycophenolate mofetil,Mycophenolic acid,Nitazoxanide,and Vidofludimus were identified having efficient inhibitory effects on SFTSV RNA replication via minigenome system,with the IC_(50) of 0.014μmol/L,0.627μmol/L,1.283μmol/L,and 0.059μmol/L,respectively.All four drugs showed effective inhibition when adding during the whole SFTSV infection process as well as the post-entry stage.Conclusion:Mycophenolate mofetil,Mycophenolic acid,Nitazoxanide and Vidofludimus show efficient anti-viral effects on SFTSV infection.

Hsp90βis 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 Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.

Antiviral activity of vitamin D derivatives against severe fever with thrombocytopenia syndrome virus in vitro and in vivo

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 dosedependent 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.

Close Correlation between Development of MODS during the Initial 72h of Hospitalization and Hospital Mortality in Severe Fever with Thrombocytopenia Syndrome

An emerging infectious disease was identified as severe fever with thrombocytopenia syn- drome （SFTS） in central China since late March 2009. We found the patients with SFTS had severe clinical symptoms, and progressed rapidly to multiple organ dysfunction syndrome （MODS） with high fatality rate of 25%-30%. The aim of this study was to assess the significance of risk factors predicting the development of MODS and death in SFTS patients. Consecutive SFTS admissions between May 2009 and September 2011 were analyzed for parameters of organ function during hospitalization using Marshall scoring system for MODS, and platelet counts were recorded on admission and at 24, 48, 72 h and one week after admission. We investigated the kinetics of organ failures and analyzed the associa- tion between age, platelet count and development of MODS or death. A total of 92 SFTS patients were enrolled in this study. Among them, 32 patients with dysfunction of over 4 organs were identified, 45% of them died within 72 h, 72% died within 5 days, and 76% died within 7 days after admission. We also found cumulative Marshall score was significantly higher in death patients （11.76＋2.05） than in survival patients （4.22~1.98） （P〈0.001）. In addition, SFTS patients had older age and lower platelet counts in MODS and death groups. Furthermore, we also observed that there was a close correlation between platelet count on admission and Marshall score （P〈0.001）. High Marshall score, advanced age and lower platelet counts were the main risk factors for the development of MODS, and those factors could predict mortality in SFTS patients, suggesting prompt treatment and close monitoring of severe complications, especially MODS, are of great importance in saving patients＇ lives.

View Point on Severe Fever with Thrombocytopenia Syndrome

Severe fever with thrombocytopenia syndrome (SFTS), a new disease of zoonotic natural foci has been increasing in recent years. In this review, the disease of human granulocytic anaplasmosis infection due to tick-borne and new Bunia virus infection, have similar clinical symptoms. Total of 11 articles were retrieved. The emergence, etiology, nomenclature and pathogenic mechanisms, clinical diagnosis and genotyping of SFTS were summarized, and the current situation of treatment was also evaluated. In conclusion, early detection, identiifcation and treatment are the key points to SFTS, but how to reduce the mortality of critically ill patients is still the focus in the future.

Laboratory indicators of death in severe fever with thrombocytopenia syndrome:a metaanalysis and systematic review

Background:Severe fever with thrombocytopenia syndrome(SFTS)is a tick-borne disease characterized by a fever-induced by severe fever with thrombocytopenia syndrome virus(SFTSV)infection.Methods:We systematically searched the literature on Web of Science,PubMed,Cochrane Library,Embase,Chinese National Knowledge Infrastructure databases,Sinomed Database,Wan Fang Data,and VIP Database,which describing the death characteristics SFTS(published between January 1,2009 and May 5,2021)and used STATA version 15.0 software to combine data from the selected study.Results:In the 14150 identified studies,23 were eligible and were included in our analysis.The death characteristics of SFTS are associated with old age,high viral load,high neutrophil ratio,low lymphocyte ratio,low platelet count,elevated liver enzymes,high serum potassium,and high blood urea nitrogen.Conclusions:The results of this meta-analysis can improve our knowledge of the death features of SFTS and necessitate increased attention to the above laboratory indicators to improve the understanding and therapeutic strategies of SFTS.

Time series models in prediction of severe fever with thrombocytopenia syndrome cases in Shandong province,China

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV). Predicting the incidence of this disease in advance is crucial for policymakers to develop prevention and control strategies. In this study, we utilized historical incidence data of SFTS (2013–2020) in Shandong Province, China to establish three univariate prediction models based on two time-series forecasting algorithms Autoregressive Integrated Moving Average (ARIMA) and Prophet, as well as a special type of recurrent neural network Long Short-Term Memory (LSTM) algorithm. We then evaluated and compared the performance of these models. All three models demonstrated good predictive capabilities for SFTS cases, with the predicted results closely aligning with the actual cases. Among the models, the LSTM model exhibited the best fitting and prediction performance. It achieved the lowest values for mean absolute error (MAE), mean square error (MSE), and root mean square error (RMSE). The number of SFTS cases in the subsequent 5 years in this area were also generated using this model. The LSTM model, being simple and practical, provides valuable information and data for assessing the potential risk of SFTS in advance. This information is crucial for the development of early warning systems and the formulation of effective prevention and control measures for SFTS.

Entry of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus （SFTSV） is a globe-shaped virus covered by a dense icosahedral array of glycoproteins Gn/Gc that mediate the attachment of the virus to host cells and the fusion of viral and cellular membranes. Several membrane factors are involved in virus entry, including C-type lectins and nonmuscle myosin heavy chain IIA. The post-fusion crystal structure of the Gc protein suggests that it is a class II membrane fusion protein, similar to the E/E1 protein of flaviviruses and alphaviruses. The virus particles are internalized into host cell endosomes through the clathrin-dependent pathway, where the low pH activates the fusion of the virus with the cell membrane. With information from studies on other bunyaviruses, herein we will review our knowledge of the entry process of SFTSV.

Isolation,characterization,and phylogenic analysis of three new severe fever with thrombocytopenia syndrome bunyavirus strains derived from Hubei Province,China

Hubei Province is a major epidemic area of severe fever with thrombocytopenia syndrome bunyavirus(SFTSV) in China. However, to date, a few SFTSV strains have been isolated from Hubei Province, preventing effective studies of epidemic outbreaks. Here, we report three confirmed patients(2015–2016) with typical symptoms of severe fever with thrombocytopenia syndrome disease(SFTS) who were farmers resident in different regions in Hubei Province. Three new SFTSV strains were isolated from the serum samples of each patient. Characterization of viral growth properties showed that there were no significant differences in virus production. All strains were completely sequenced, and phylogenetic analysis showed that unlike the other strains from Hubei province, which belonged to the SFTSV C3 genotype, one of the three strains belonged to the SFTSV C2 genotype. These results suggested that multiple SFTSV genotypes have been circulating in Hubei Province, providing insights into SFTSV evolution and improving our understanding of SFTSV prevalence in Hubei Province.

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

Severe fever with thrombocytopenia syndrome(SFTS)is an emerging hemorrhagic fever disease caused by SFTSV,a newly discovered phlebovirus that is named after the disease.Currently,no effective vaccines or drugs are available for use against SFTSV infection,as our understanding of the viral pathogenesis is limited.Bortezomib(PS-341),a dipeptideboronic 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 nonstructural 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.

Establishment of a Reverse Genetic System of Severe Fever with Thrombocytopenia Syndrome Virus Based on a C4 Strain

Severe fever with thrombocytopenia syndrome virus(SFTSV)is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease(SFTS)in humans with a case fatality rate up to 30%.To date,the molecular biology involved in SFTSV infection remains obscure.There are seven major genotypes of SFTSV(C1-C4 and J1-J3)and previously a reverse genetic system was established on a C3 strain of SFTSV.Here,we reported successfully establishment of a reverse genetics system based on a SFTSV C4 strain.First,we obtained the 5’-and 3’-terminal untranslated region(UTR)sequences of the Large(L),Medium(M)and Small(S)segments of a laboratory-adapted SFTSV C4 strain through rapid amplification of cDNA ends analysis,and developed functional T7 polymerase-based L-,M-and S-segment minigenome assays.Then,fulllength cDNA clones were constructed and infectious SFTSV were recovered from co-transfected cells.Viral infectivity,growth kinetics,and viral protein expression profile of the rescued virus were compared with the laboratory-adapted virus.Focus formation assay showed that the size and morphology of the foci formed by the rescued SFTSV were indistinguishable with the laboratory-adapted virus.However,one-step growth curve and nucleoprotein expression analyses revealed the rescued virus replicated less efficiently than the laboratory-adapted virus.Sequence analysis indicated that the difference may be due to the mutations in the laboratory-adapted strain which are more prone to cell culture.The results help us to understand the molecular biology of SFTSV,and provide a useful tool for developing vaccines and antivirals against SFTS.

Prevalence of severe fever with thrombocytopenia syndrome virus in animals in Henan Province, China

Severe fever with thrombocytopenia syndrome(SFTS)is an emerging infectious disease caused by severe fever with thrombocytopenia syndrome virus(SFTSV).SFTSV has been found in humans,ticks and animals,and SFTS has high mortality and increasing prevalence in East Asia.In the study,the samples(heart,liver,lung,kidney,spleen,brain tissue and serum)were collected from 374 domestic animals and 241 wild animals in Pingqiao District and Xinxian County of Xinyang in Henan Province,China.275(44.72%,275/615)animals were positive for anti-SFTSV antibodies,the anti-SFTSV antibodies positive ratios of domestic and wild animals were 43.58%(163/374)and 46.47%(112/241),respectively.There was no significant difference in domestic and wild animals,but significant differences were detected among different species of animals(x^2=112.59,P<0.0001).Among 615 animals,105(17.07%,105/615)animals were positive for SFTSV RNA,and only one SFTSV strain was isolated from heart tissue of a yellow weasel.The phylogenetic analysis shows that the sequence from animals belonged to the same group with viral sequences obtained from humans.The animals maybe play a reservoir host in maintaining the life cycle of SFTSV in nature.

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Phlebovirus in Domesticated Deer in South Korea

Severe fever with thrombocytopenia syndrome phlebovirus(SFTSV)has a wide host range.Not only has it been found in humans,but also in many wild and domesticated animals.The infection of breeding deer on farms is a particularly worrisome public health concern due to the large amount of human contact and the diverse use of deer products,including raw blood.To investigate the prevalence of breeding domesticated deer,we examined the SFTSV infection rate on deer farms in South Korea from 2015 to 2017.Of the 215 collected blood samples,0.9%(2/215)were found to be positive for viral RNA by PCR,and sequence analysis showed the highest homology with the KADGH human isolate.Both SFTSVspecific recombinant N and Gn protein-based ELIS As revealed that 14.0%(30/215)and 7.9%(17/215)of collected blood specimens were positive for SFTSV antibody.These results demonstrate that the breeding farm deer are exposed to SFTSV and could be a potential infection source for humans through direct contact or consumption of byproducts.

Depletion but Activation of CD56dimCD16+ NK Cells in Acute Infection with Severe Fever with Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome(SFTS)is an emerging infectious disease with high mortality(12%–30%).The mechanism by which the SFTS bunyavirus(SFTSV)causes severe illness remains unclear.To evaluate the phenotypic and functional characteristics of the NK cell subsets in SFTS patients,twenty-nine SFTS patients were sequentially sampled from admission until recovery.Phenotypic and functional characteristics of NK cell subsets in circulating blood were analysed via flow cytometry.Then,correlations between NK cell subset frequencies and the SFTS index(SFTSI)were evaluated in all SFTS patients(15 mild,14 severe)upon admission.The frequencies of CD56dimCD16+NK cells were greatly decreased in early SFTSV infection and were negatively correlated with disease severity.Additionally,higher Ki-67 and granzyme B expression and relatively lower NKG2 A expression in CD56dimCD16+NK cells were observed in acute infection.Moreover,the effector function of CD56dimNK cells was increased in the acute phase compared with the recovery phase in nine severe SFTS patients.Additionally,interleukin(IL)-15,interferon(IFN)-a,IL-18 and IFN-c secretion was markedly increased during early infection.Collectively,despite depletion of CD56dimCD16+NK cells,activation and functional enhancement of CD56dimCD16+NK cells were still observed,suggesting their involvement in defence against early SFTSV infection.

Experimental infections of mosquitoes with severe fever with thrombocytopenia syndrome virus

Background:Severe fever with thrombocytopenia syndrome(SFTS)is a newly identified emerging infectious disease,which is caused by a novel bunyavirus(termed SFTSV)in Asia.Although mosquitoes have not been identified as the primary vectors,as revealed by epidemiological surveys,their role in transmitting this SFTSV as a suspicious vector has not been validated.Findings:In this study,we conducted experimental infections of mosquitoes with SFTSV to examine the role of mosquitoes in the transmission of the virus.We did not detect viral replication in Culex pipiens pallens,Aedes aegyptis and Anopheles sinensis as revealed by qRT-PCR assay.In addition,we failed to isolate SFTSV from the Vero cells cultured with suspensions of SFTSV-infected mosquitoes.Conclusion:The results of the present study demonstrate little possibility that mosquitoes act as vectors for the emerging pathogen SFTSV.

Antigenicity of severe fever with thrombocytopenia syndrome virus nucleocapsid protein and its potential application in the virus serodiagnosis

Dear Editor,Severe fever with thrombocytopenia syndrome virus(SFTSV)is a newly identified viral pathogen of the genus Phlebovirus in the family Bunyaviridae(Sun et al.,2012).SFTSV was first identified from patient serum samples in China(Li et al.,2013;Ning et al.,2015).SFTSV can cause a severe hemorrhagic fever-like disease with a reported case fatality rate ranging from 2.5%

Quantitative assessment on the epidemic characteristics of severe fever with thrombocytopenia syndrome virus infection in China

Severe fever with thrombocytopenia syndrome(SFTS)was first detected in China in 2009.The incidence of SFTS increases year by year,and there is no effective treatment.Considering that the reported prevalence of SFTSV infection varies from region to region,we aimed to quantitatively evaluate the epidemic characteristics of SFTSV infection in China from 2010 to 2020,including the distribution differences in infectious season,sex,age,occupation,and region.A meta-analysis framework was used to search for the related published data with keywords in electronic databases(CNKI,WanFang,CBM,and PubMed).According to the PRISMA statement,the studies that included SFTS diagnosed in China were analyzed.Furthermore,we used Revman and Stata to merge statistical effects,and used I2 and P-values for heterogeneity test and quality assessment.Eleven studies containing 4,932 cases confirmed by SFTSV infection were included in this meta-analysis.The ratio of male-to-female is 1.04 to 1.Cases were concentrated between 40 and 80 years of age(MD=92%,95%CI:91%–93%).Farmers are at the highest risk of SFTSV infection(MD=84%,95%CI:77%–90%).The risk of infection for consecutive period of April-August was significantly higher than the sum of the remaining months(MD=82%,95%CI:78%–85%).In addition,the patient has an extensive history of exposure,including living in the mountains,exposure to ticks,livestock,mouse and the patient.We came to the conclusion that SFTSV is transmitted primarily through tick bites in China,so middle-aged and older adults living in mountains regions are at the highest risk for SFTSV infection in April through August each year.

Inactivation of severe fever with thrombocytopenia syndrome virus for improved laboratory safety

Severe fever with thrombocytopenia syndrome(SFTS),a tick-borne infectious disease with high mortality,is diagnosed by the serological testing of serum samples from patients in the acute and convalescent phases and/or by direct detection of the SFTS virus(SFTSV).Conventionally,heat and UV treatments have been used to inactivate the viruses present in serum samples before performing the serological assays.Here,we examined the inactivation conditions optimal for SFTSV-containing serum samples to ensure the safety of laboratory workers while maintaining the accuracy of serological assay results simultaneously.Heating human serum samples spiked with SFTSV to 60
C for 30 min or exposing them to UV irradiation for 30 min,reduced the infectious virus titer below the limit of detection.SFTSV in sera from patients in the acute phase of SFTS was completely inactivated by heating to 60
C for 30 min,followed by UV irradiation for 10 min.This inactivation procedure had minimal impact on the performance of SFTSV antibody detection tests.The data provided herein can serve as a guide for laboratory workers and researchers working with SFTS serological tests.

Current status of severe fever with thrombocytopenia syndrome in China

Severe fever with thrombocytopenia syndrome （SFTS） is an emerging infectious disease caused by SFTS virus （SFTSV）. SFTSV is associated with a high mortality rate and has been reported in China, South Korea and Japan. SFTSV undergoes rapid changes owing to evolution, gene mutations, and reassortment between different strains of SFTSV. In this review, we summarize the recent cases and general properties of SFTS, focusing on the epidemiology, genetic diversity, clinical features, and diagnostics of SFTSV in China. From 2010 to October 2016, SFTS cases were reported in 23 provinces of China, with increased numbers yearly. Infection and death cases are mainly found in central China, where the Haemaphysalis Iongicornis ticks are spread. The national average mortality rate of SFTS infection was 5.3%, with higher risk to elder people. The main epidemic period was from May to July, with a peak in May. Thus, SFTS reminds a significant public health problem, and development of prophylactic vaccines and effective antiviral drugs will be highly needed.

Transmission and mortality risk assessment of severe fever with thrombocytopenia syndrome in China:results from 11-years’study

Background: The transmission and fatal risk of severe fever with thrombocytopenia syndrome (SFTS), an emerging infectious disease first discovered in China in 2009, still needed further quantification. This research aimed to analyze the SFTS clusters and assess the transmission and mortality risk for SFTS.Methods: Both epidemiological investigation and case reports regarding SFTS clusters in China during 2011-2021 were obtained from the Public Health Emergency Information Management System of the Chinese Center for Disease Control and Prevention Information System. The transmission risk was evaluated by using the secondary attack rate (SAR) and relative risk (RR). Mortality risk factors were analyzed using a logistic regression model.Results: There were 35 SFTS clusters during 2011-2021 involving 118 patients with a fatality rate of 22.0%. The number of clusters annually increased seasonally from April to September. The clusters mainly occurred in Anhui (16 clusters) and Shandong provinces (8 clusters). The SAR through contact with blood or bloody fluids was much higher than that through contact with non-bloody fluids (50.6% vs 3.0%;χ^(2) = 210.97,P < 0.05), with anRR of 16.61 [95% confidence interval (CI): 10.23-26.97]. There was a statistically significant difference in the SAR between exposure to the blood of a deceased person during burial preparation and exposure to the living patients’ blood (66.7% vs 34.5%;χ^(2)= 6.40,P < 0.05), with anRR of 1.93 (95%CI: 1.11-3.37). The mortality risk factors were a long interval from onset to diagnosis [odds ratio (OR)= 1.385), 95%CI: 1.083-1.772,P= 0.009) and advanced age (OR: 1.095, 95%CI: 1.031-1.163,P= 0.01).Conclusions: The SFTS clusters showed a high mortality rate and resulted in a high SAR. Contact with a bleeding corpse was associated with a higher infection risk, compared with contacting the blood from living patients. It is important to promote early detection and appropriate case management of patients with SFTS, as well as improved handling of their corpses, to prevent further transmission and mortality.