Identification of a Novel Genotype of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) in Northern Hebei Province, China

Objective To detect the serum viral load, lymphocyte subsets, serum enzymes and blood cell counts of severe fever with thrombocytopenia syndrome (SFTS) patients, and to use logistic regression analysis and receiver operating characteristic (ROC) curve to establish a model to analyze the severity of SFTS. Methods A case-control study of 24 SFTS cases admitted between May 2011 and July 2012 was conducted at the First Affiliated Hospital of Nanjing Medical University. All SFTS cases were defined according to Fever with Thrombocytopenia Syndrome Prevention and Control Guidelines (2010 edition) issued by the Ministry of Health of the People′s Republic of China. According to their disease severity, the patients were divided into two groups, the non-severe group (16 cases) and the severe group (8 cases). In addition, 32 healthy volunteers were also enrolled in this study. Flow cytometry was adopted to detect the CD3+, CD4+, CD8+ lymphocytes and CD3-CD16+CD56+ natural killer cells (NK cells) in the peripheral blood of SFTS patients, and cytometric beads array (CBA) was used to detect Th1/Th2/Th17 cytokines. The serum viral load in patients with severe fever with thrombocytopenia syndrome virus (SFTSV) infection was detected by fluorescent quantitative PCR technology. Besides, white blood cells, platelets and serum enzymes were measured. The multi-index conjunctive model of logistic regression analysis and receiver operating characteristics (ROC) curve were used to analyze the predictive values of indexes on severity of SFTS. Results The ROC analysis of single index found that SFTSV RNA, CD3, CD4, CD8, CD56, AST, LDH, CK, IL-6 and IL-10 have good predictability on severity of SFTS in the early course of the disease; the area under the curve (AUC) were 0.83, 0.84, 0.90, 0.75, 0.94, 0.73, 0.78, 0.87, 0.74 and 0.77 respectively, and the cut-off values of the SFTSV load, CD3+, CD4+, NK cells and CK were 6.19 log10 copies/ml, 57.51%, 19.47%, 15.71% and 696.45 U/L respectively. Using the step-by-step method logistic regression analysis to build a model and analyze the ROC curve of prediction probability, it was found that the predictability of joint indexes of SFTSV RNA/CD3/CD4/CD8/CD56, SFTSV RNA/AST/lactate dehydrogenase (LDH) /CK, or SFTSV RNA/IL-6/IL-10 increases in a certain degree. The areas under the ROC curve were 0.95 (95%CI: 0.00-1.00), 0.87 (95%CI: 0.75-0.99), and 0.83 (95%CI: 0.70-0.97), the sensitivities were 93.3%, 86.70% and 77.30%, and the specificities were 94.4%, 83.30% and 83.30% respectively. Conclusions The level of serum SFTSV is highly related to the severity of the disease. The high levels of serum SFTSV load, LDH, CK, IL-6, and IL-10, together with significant reduction of CD3+cells and CD4+ cells, may indicate poor prognosis of the SFTS patients. Based on the logistic regression model by multiple indexes, the severity of SFTS can be better predicted. (Chin J Lab Med, 2015, 38: 49-54) Key words: Runyaviridae infections; Killer cells, natural; Cytokines; Viral load; Severity of illness index; ROC curve

Severe fever with thrombocytopenia syndrome (SFTS), caused by Dabie bandavirus, generally called SFTS virus (SFTSV), is an emerging zoonosis in East Asia. In Japan, 50-100 cases of SFTS have been reported each year since the first case was reported in 2013. SFTS is a tick-borne infectious disease, and SFTSV has been isolated from ticks in China and South Korea. Haemaphysalis longicornis and Amblyomma testudinarium are considered the primary vectors in Japan. However, the other tick species seldom feeding on humans might also play an important role in maintaining the virus in nature. In this study, we collected ticks on vegetation around the location where two SFTS patients were estimated to have been infected in Miyazaki Prefecture, Japan, isolated live SFTSV, and performed a phylogenetic analysis. A total of 257 ticks were collected, and SFTSV RNA was detected in 19.5% (9/46) of tick pools. A total of 10 infectious SFTSVs were successfully isolated from A. testudinarium, Haemaphysalis flava, Haemaphysalis formosensis, Haemaphysalis hystricis, and Haemaphysalis megaspinosa. Furthermore, the whole viral sequences isolated from ticks were highly homologous to sequences isolated from SFTS patients in the same sampling area in the past. These results suggest that SFTSVs are maintained in these tick species in the sampling area and sporadically transmitted to humans. Surveillance of SFTSV in ticks provides important information about the risk of incidental transmission to humans.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease, caused by severe fever with thrombocytopenia syndrome virus (SFTSV), which is primarily transmitted via tick bites. Clusters of SFTS caused by human-to-human transmission have been reported both at home and abroad, mainly focused on the transmission or exposure modes. However, the correlation between SFTS clusters and viral genotypes has not been investigated. This study mainly reported two clusters of SFTS in Xinyang City, Henan Province, from 2022 to 2023, discussed the possible route of person-to-person transmission of SFTSV infection and analyzed the association between SFTS clusters and virus genotypes. We found that two groups of SFTSV in two clusters were clustered separately into different genotypes through viral sequence analysis of 4 confirmed patients. We also performed phylogenetic analysis, after including SFTSV sequences obtained from SFTS clusters deposited in the GenBank. Three SFTSV genotypes have been reported among cases of human-to-human transmission, suggesting that the occurrence of SFTS clusters may not be related to SFTSV genotypes. This study provided genetic evidence for revealing the chain of human-to-human transmission of SFTS clusters, indicating that contact with patients' blood is an important transmission route of SFTSV. The findings laid the foundation for preventing and controlling human-to-human transmission of SFTS.

Severe fever with thrombocytopenia syndrome (SFTS) is a fatal emerging tick-borne zoonotic disease caused by the SFTS virus (SFTSV). SFTSV infection in humans and companion animals is a matter of concern in endemic areas. Various wild animals are involved in the transmission cycle of SFTSV with vector ticks. Because the home range of medium-sized wild mammals commonly overlaps with humans’ living spheres, this study aimed to reveal the endemicity of SFTSV in such mammals. This study investigated the prevalence of antibodies against SFTSV and viral RNA in medium-sized wild mammals in Miyazaki Prefecture, Japan where human cases have been most frequently reported in Japan and performed a phylogenetic analysis to compare the detected SFTSV with those previously reported. Forty-three of 63 (68%) Japanese badgers (Meles anakuma) and 12 of 53 (23%) Japanese raccoon dogs (Nyctereutes procyonoides viverrinus) had antibodies against SFTSV. Japanese marten (n = 1), weasels (n = 4), and Japanese red fox (n = 1) were negative. Two of 63 (3%) badgers tested positive for SFTSV RNA, whereas the other species were negative. Phylogenetic analysis of the partial nucleotide sequence of SFTSV revealed that viral RNA detected from badgers exhibited 99.8% to 100% similarity to SFTSV, as previously reported in humans, cat, and ticks in the study area. This study demonstrated high seropositivity of antibodies in medium-sized wild mammals and suggested that SFTSV could be shared among these mammals, humans, and companion animals in endemic areas.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease, which causes high fever, thrombocytopenia, and death in humans and animals in East Asian countries. The pathogenicity of SFTS virus (SFTSV) remains unclear. We intraperitoneally infected three groups of mice: wild-type (WT), mice treated with blocking anti-type I interferon (IFN)-α receptor antibody (IFNAR Ab), and IFNAR knockout (IFNAR−/−) mice, with four doses of SFTSV (KH1, 5 × 105 to 5 × 102 FAID50). The WT mice survived all SFTSV infective doses. The IFNAR Ab mice died within 7 days post-infection (dpi) with all doses of SFTSV except that the mice were infected with 5 × 102 FAID50 SFTSV. The IFNAR−/− mice died after infection with all doses of SFTSV within four dpi. No SFTSV infection caused hyperthermia in any mice, whereas all the dead mice showed hypothermia and weight loss. In the WT mice, SFTSV RNA was detected in the eyes, oral swabs, urine, and feces at 5 dpi. Similar patterns were observed in the IFNAR Ab and IFNAR−/− mice after 3 dpi, but not in feces. The IFNAR Ab mice showed viral shedding until 7 dpi. The SFTSV RNA loads were higher in organs of the IFNAR−/− mice compared to the other groups. Histopathologically, coagulation necrosis and mononuclear inflammatory cell infiltration in the liver and white pulp atrophy in the spleen were seen as the main lesions in the IFN signaling lacking mice. Immunohistochemically, SFTSV antigens were mainly detected in the marginal zone of the white pulp of the spleen in all groups of mice, but more viral antigens were observed in the spleen of the IFNAR−/− mice. Collectively, the IFN signaling-deficient mice were highly susceptible to SFTSV and more viral burden could be demonstrated in various excreta and organs of the mice when IFN signaling was inhibited.

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is a severe emerging disease caused by SFTS virus (SFTSV), and the geographical distribution of SFTS has been increasing throughout China in recent years. To assess SFTSV-specific antibody seroprevalence, a cross-sectional study was conducted for healthy people in high SFTS endemic areas of Henan province in 2016.MethodsThis study used a stratified random sampling method to select 14 natural villages as the investigation sites. From April to May 2016, participants completed a questionnaire survey and serum samples were collected. All serum samples were subjected to ELISA to detect SFTSV-specific IgM and IgG. All IgM-positive samples were further tested by real-time RT-PCR, and isolation of virus from serum was attempted. Any participant who was IgM-positive was followed up with a month later to confirm health status.ResultsIn total, 1463 healthy people participated in this study. The average seropositive rates for SFTSV-specific IgG and IgM were 10.46% (153/1463) and 0.82% (12/1463), respectively. IgM was detected in 12 individuals, and SFTSV RNA was detected in six of them. Virus was isolated from five of the six SFTSV RNA-positive individuals, and phylogenetic analyses revealed that all five isolates belonged to SFTSV group A. No IgM-positive participants exhibited any symptoms or other signs of illness at the one-month follow up.ConclusionsThis study identified a relatively high incidence of SFTSV-specific antibody seropositivity in healthy people in Xinyang city. Moreover, our data provide the first evidence for asymptomatic SFTSV infections, which may have significant implications for SFTS outbreak control.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a newly discovered bunyavirus, SFTS virus (SFTSV), and causes high fatality (12% on average and as high as 30%). The objective of this study was to determine whether SFTSV could be transmitted from person to person. We analyzed sera of 13 patients from two clusters of unknown infectious diseases that occurred between September and November of 2006 in Anhui Province of China for SFTSV antibody by indirect immunofluorescence assay and for SFTSV RNA by RT-PCR. We found that all patients (n=14) had typical clinical symptoms of SFTS including fever, thrombocytopenia, and leukopenia and all secondary patients in both clusters got sick at 6-13 days after contacting or exposing to blood of index patients. We demonstrated that all patients in cluster 1 including the index patient and nine secondary patients and all three secondary patients in cluster 2 had seroconversion or fourfold increases in antibody titer to SFTSV and/or by RT-PCR amplification of SFTSV RNA from the acute serum. The index patient in cluster 2 was not analyzed because of lack of serum. No person who contacted the index patient during the same period, but were not exposed to the index patient blood, had got illness. We concluded that SFTSV can be transmitted from person to person through contacting patient's blood.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a lethal threat. Increasing Severe fever with thrombocytopenia syndrome (SFTS) risk in Asia and the United States stems from the spread of natural host, Haemaphysalis longicornis. Rapid and accurate SFTSV molecular diagnosis is crucial for treatment decisions, reducing fatality risk. Blood samples from 17 suspected SFTS patients at Chuncheon Sacred Heart Hospital (September-December 2022) were collected. SFTSV was diagnosed using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays from Gangwon Institute of Health and Environment (RT-qPCR/GIHE) and Asan Medical Center (RT-qPCR/AMC). To address RT-qPCR disparities, amplicon-based MinION sequencing traced SFTSV genomic sequences in clinical samples. In two samples (N39 and N50), SFTSV was detected in both RT-qPCR/GIHE and RT-qPCR/AMC. Among 11 samples, RT-qPCR/AMC exclusively detected SFTSV. In four samples, both assays yielded negative results. Amplicon-based MinION sequencing enabled nearly whole-genome sequencing of SFTSV in samples N39 and N50. Among 11 discordant samples, five contained significant SFTSV reads, aligning with the RT-qPCR/AMC findings. However, another six samples showed insufficient viral reads in accordance with the negativity observed in RT-qPCR/GIHE. The phylogenetic pattern of SFTSV demonstrated N39 formed a genetic lineage with genotype A in all segments. SFTSV N50 grouped with the B-1 sub-genotype for L segment and B-2 sub-genotype for the M and S segments, indicating genetic reassortment. The study demonstrates the robust sensitivity of amplicon-based MinION sequencing for the direct detection of SFTSV in clinical samples containing ultralow copies of viral genomes. Next-generation sequencing holds potential in resolving SFTSV diagnosis discrepancies, enhancing understanding of diagnostic capacity, and risk assessment for emerging SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel emerging virus that has been identified in China, South Korea, and Japan, and it induces thrombocytopenia and leukocytopenia in humans with a high case fatality rate. SFTSV is pathogenic to humans, while immunocompetent adult mice and golden Syrian hamsters infected with SFTSV never show apparent symptoms. However, mice deficient for the gene encoding the α chain of the alpha- and beta-interferon receptor (Ifnar1-/- mice) and golden Syrian hamsters deficient for the gene encoding signal transducer and activator of transcription 2 (Stat2-/- hamsters) are highly susceptible to SFTSV infection, with infection resulting in death. The nonstructural protein (NSs) of SFTSV has been reported to inhibit the type I IFN response through sequestration of human STAT proteins. Here, we demonstrated that SFTSV induces lethal acute disease in STAT2-deficient mice but not in STAT1-deficient mice. Furthermore, we discovered that NSs cannot inhibit type I IFN signaling in murine cells due to an inability to bind to murine STAT2. Taken together, our results imply that the dysfunction of NSs in antagonizing murine STAT2 can lead to inefficient replication and the loss of pathogenesis of SFTSV in mice.IMPORTANCE Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTSV, which has been reported in China, South Korea, and Japan. Here, we revealed that mice lacking STAT2, which is an important factor for antiviral innate immunity, are highly susceptible to SFTSV infection. We also show that SFTSV NSs cannot exert its anti-innate immunity activity in mice due to the inability of the protein to bind to murine STAT2. Our findings suggest that the dysfunction of SFTSV NSs as an IFN antagonist in murine cells confers a loss of pathogenicity of SFTSV in mice.

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

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic tick-borne disease in East Asia caused by the SFTS virus (SFTSV). It is to investigate the presence of SFTSV RNA and antibodies in horses from a slaughterhouse and equestrian centers in the Republic of Korea (ROK). A prevalence study of SFTSV-specific RNA and antibodies was designed from 889 horses in the ROK. Materials and Methods: Serum samples were collected from horses at a slaughterhouse and equestrian centers from 2018 to 2020. To detect the presence of SFTSV, RNA was extracted from the serum samples, and a nested reverse transcription-polymerase chain reaction (RT-PCR) was conducted. Sequencing data were analyzed, and a phylogenetic tree was constructed using the maximum-likelihood method with Molecular Evolutionary Genetics Analysis Version 7.0 software. The horse sera were also tested for SFTSV-specific immunoglobulin G antibodies using enzyme-linked immunosorbent assay (ELISA). Results: Twelve of 889 (1.3%) horse sera were positive for SFTSV RNA, and 452 of 887 (51.0%) horse sera were seropositive by ELISA. Among the RT-PCR-positive samples, 12 of the SFTSV S-segment sequences were classified as sub-genotypes B-2 (n = 6) and B-3 (n = 6). ELISA analysis was evaluated by comparison with neutralization test. We investigated SFTSV infection in horses over a 3-year period, but sampling was not performed evenly by season; continuous surveillance of SFTSV in horses is needed. Conclusions: We report the detection of SFTSV RNA and provide serological data on SFTSV prevalence in horses in the ROK. The detection of SFTSV-specific RNA and antibodies in horses, which are in close proximity to humans, suggests that SFTS is an emerging and important health issue, indicating that more attention to its relevance for equestrian workers is needed.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by Dabie bandavirus, commonly called SFTS virus (SFTSV). In the Republic of Korea (ROK), 1,504 cases of SFTS have been reported since the first human case was identified in 2013 until 2021. However, no case exists to provide molecular evidence between questing tick and patients with confirmed SFTS in the same living environment. In this study, we investigated the presence of ticks near the area of a patient infected with SFTSV. Ticks were collected by flagging and dry ice-baited traps at three spots in the vegetation around the patients’ residence in Chuncheon City, Gangwon Province (ROK). Among the tick samples collected, the presence of SFTSV was genetically determined using reverse transcription PCR, followed by the phylogenetic analysis of the tick virus sequences and SFTSV found in the patient. In total 1,212 Haemaphysalis longicornis ticks were collected, and SFTSV was detected at a minimum infection rate of 5.3% (33 pools/618 tested ticks). The sequences of SFTSV in ticks were 99.6–100% identical with the patient’s SFTSV in the M segment. To the best of our knowledge, this study is the first case to provide a molecular correlation between SFTSV in questing ticks collected from residence and patient with SFTS in the ROK. The present results provide useful information for the epidemiological investigation of patients with SFTS using ticks as vectors of SFTSV.

Objective To elucidate the thermal stability and inactivation of severe fever with thrombocytopenia syndrome virus (SFTSV).Methods The stability of cell culture-derived SFTSV (SD4 strain) under different environmental temperatures,and the ability of heat,ultraviolet rays,diethyl ether,chloroform,acid,disinfectants treatments to inactivate SFTSV were evaluated.The infectious titers of treated viral samples were determined by titration assay based on a double N protein specific antibodies sandwich ELISA assay for SFTSV in Vero cells.An indirect immunofluorescence assay was performed to determine the replication dynamic of SFTSV in cells.Results SFTSV in culture medium was relatively stable at 4℃ without drastic loss of infectivity for at least 1 week,and the virus was sensitive to heat and could be inactivated in 30 min when incubated at 60℃.UV light irradiation with an intensity of 185 μW/cm2 efficiently inactivated SFTSV within 30 min.Exposures to optimized concentration of diethyl ether,chloroform,β-propionolactone,formaldehyde,Hypochlorous acid all destroyed SFTSV infectivity effectively.Acid conditions brought damage to the virus activity,but virus could not be completely inactivated at pH3.0.Conclusion The results provide quantitative evidence for the potential use of a variety of approaches for samples collection,inactivating SFTSV,and safety protection in scientific research and diseases control and prevention. Key words: Phlebovirus; Thrombocytopenia; Heat stress disorders; Inactivation

Severe fever with thrombocytopenia syndrome (SFTS) caused by SFTS virus (SFTSV), a novel phlebovirus, was reported to be endemic to central and northeastern China, South Korea, and Japan. SFTS is a tick-borne viral infection with high morbidity and mortality. SFTSV is circulating in nature in the SFTS-endemic areas. SFTSV is maintained in nature between animals and some species of ticks. Humans are usually infected with SFTSV by virus-positive ticks, especially Haemaphysalis longicornis and Amblyomma testudinarium. The mechanism of SFTSV maintenance in nature is crucial to control SFTSV infections in humans. Antibody positive rates to SFTSV in some species of animals such as goats, deer, old aged bovine, and wild boar were reported to be relatively high. Virus genomes were detected in some species of ticks such as H. longicornis, H. flava, H. concinna, A. testudinarium, and Ixodes nipponensis. Animal species, which were studied for SFTSV antibody and SFTSV genome prevalence, are still limited. Based on the data reported so far suggest that SFTSV is maintained in nature through intensive transmission between animals such as goats, deer, and wild boars and Haemaphysalis, Ambryomma, and Ixodes ticks. Further studies are needed to clarify the SFTSV maintenance mechanism in nature in detail.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever with a high case fatality rate caused by SFTS virus (SFTSV). Effective vaccines and specific therapies for SFTS are urgently sought, and investigation into virus-host cell interactions is expected to contribute to the development of antiviral strategies. In this study, we have developed a pseudotype vesicular stomatitis virus (VSV) bearing the unmodified Gn/Gc glycoproteins (GPs) of SFTSV (SFTSVpv). We have analyzed the host cell entry of this pseudotype virus and native SFTSV. Both SFTSVpv and SFTSV exhibited high infectivity in various mammalian cell lines. The use of lysosomotropic agents indicated that virus entry occurred via pH-dependent endocytosis. SFTSVpv and SFTSV infectivity was neutralized by serial dilutions of convalescent-phase patient sera. Entry of SFTSVpv and growth of SFTSV were increased in Raji cells expressing not only the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) but also DC-SIGN-related (DC-SIGNR) and liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin). 25-Hydroxycholesterol (25HC), a soluble oxysterol metabolite, inhibited the cell entry of SFTSVpv and the membrane fusion of SFTSV. These results indicate that pH-dependent endocytosis of SFTSVpv and SFTSV is enhanced by attachment to certain C-type lectins. SFTSVpv is an appropriate model for the investigation of SFTSV-GP-mediated cell entry and virus neutralization at lower biosafety levels. Furthermore, 25HC may represent a potential antiviral agent against SFTS. SFTSV is a recently discovered bunyavirus associated with SFTS, a viral hemorrhagic fever with a high case fatality rate endemic to China, South Korea, and Japan. Because little is known about the characteristics of the envelope protein and entry mechanisms of SFTSV, further studies will be required for the development of a vaccine or effective therapies. In this study, we investigated the mechanism of SFTSV cell entry using SFTSVpv and the native virus. SFTSV can grow in nonsusceptible cell lines in the presence of certain C-type lectins. Moreover, 25HC, an oxysterol metabolite, may represent a potential therapeutic inhibitor of SFTSV infection.