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.     

Nonstructural protein of severe fever with thrombocytopenia syndrome phlebovirus targets STAT2 and not STAT1 to inhibit type I interferon-stimulated JAK-STAT signaling

The nonstructural protein NSs of severe fever with thrombocytopenia syndrome phlebovirus blocks type I interferon (IFN)-stimulated JAK-STAT signaling. However, there is continuing controversy as to whether NSs targets STAT1 or STAT2 or both for this blockade. The present study was designed to gain a further understanding of the blockade mechanism. Immunoprecipitation experiments revealed a stronger interaction of NSs with STAT2 than with any other component constituting the JAK-STAT pathway. Expression of NSs resulted in the formation of cytoplasmic inclusion bodies (IBs), and affected cytoplasmic distribution of STAT2. STAT2 was relocated to NSs-induced IBs. Consequently, NSs inhibited IFN-α-stimulated tyrosine phosphorylation and nuclear translocation of STAT2. These inhibitory effects as well as the signaling blockade activity were not observed in NSs mutant proteins lacking the STAT2-binding ability. In contrast, NSs affected neither subcellular distribution nor phosphorylation of STAT1 in response to IFN-α and IFN-γ, demonstrating that NSs has little physical and functional interactions with STAT1. Taken together, these results suggest that NSs sequesters STAT2 into NSs-induced IBs, thereby blocking type I IFN JAK-STAT signaling.     

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 longicornis 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.

Open image in new window

     

新布尼亚病毒的流行病学及临床特征

发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)是引起人类的一种病死率较高的新发传染病——发热伴血小板减少综合征(Severe fever with thrombocytopenia syndrome,SFTS)的病原体。本文从SFTSV的病原学、流行病学及临床特征等作一综述。     

发热伴血小板减少综合征布尼亚病毒微复制子的建立

发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国新发现的一种布尼亚病毒,可引起人类严重发热伴血小板减少综合征。我们利用RNA聚合酶Ⅰ体系,分别构建SFTSV三个片段L、M、S微复制子,研究其非编码区调控功能。将报告基因绿色荧光蛋白(GFP)或荧光素酶(Luciferase)分别插入SFTSV三个片段5′和3′非编码区之间,所形成的嵌合cDNA反向插入含RNA聚合酶I的表达载体pHH21中,获得SFTSV微复制子重组质粒L-GFP-pHH21、M-GFP-pHH21、S-GFP-pHH21、L-Luc-pHH21、M-Luc-pHH21和S-Luc-pHH21,分别与成功表达SFTSV聚合酶蛋白(L蛋白)和结构蛋白(N蛋白)的质粒VR1012-L和VR-1012-NP共同转染293T细胞,24～48h后观察GFP表达情况或检测萤光素酶表达量。L、M、S片段GFP微复制子均可观察到特异性绿色荧光。荧光素酶定量结果显示其在不同节段非编码区中的表达量不同,提示SFTSV三个节段的非编码区启动微复制子转录和复制的强度不同。     

发热伴血小板减少综合征布尼亚病毒结构和非结构蛋白表达研究

发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国2010年新发现的新布尼亚病毒,可导致人类严重发热伴血小板减少综合征。SFTS新布尼亚病毒全基因组已解析,但病毒分子生物学结构蛋白特征及功能尚需更多研究。本文通过蔗糖密度梯度离心确定发热伴血小板减少综合征布尼亚病毒(HB29株)病毒颗粒的沉降密度及超离纯化条件,得出该病毒颗粒在蔗糖中的沉降密度为1.135g/mL。利用PCR方法扩增SFTSV病毒株HB29株病毒RNA聚合酶(RdRp)、糖蛋白前体蛋白(M)、包膜糖蛋白(Gn)、包膜糖蛋白(Gc)、核蛋白(NP)及非结构蛋白(NSs)的编码区基因片段,分别克隆入真核表达载体pcDNA5/FRT或VR1012,在293T细胞上获得上述基因表达。通过SDS-PAGE分析纯化病毒颗粒和重组蛋白,并通过免疫印迹(Western blotting)和间接免疫荧光(IFA)确定蛋白活性和分子量。本研究结果将有利于对新布尼亚病毒分子生物学特征的认识,为后期研究提供基础。     

无血清细胞培养在发热伴血小板减少综合征布尼亚病毒生长中的应用

目的建立无血清培养基培养Vero细胞制备发热伴血小板减少综合征布尼亚病毒(severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)的工艺。方法分别采用含10%牛血清的MEM(10%MEM培养基)和无血清M2培养基(SF-M2培养基)在方瓶中培养Vero细胞制备SFTSV,比较无血清与含血清培养基培养Vero细胞制备SFTSV在病毒滴度及病毒繁殖曲线之间的差异。在生物反应器里用无血清培养的方式进行工艺放大,收获病毒原液并进行检定。结果无血清培养的Vero细胞能够满足SFTSV培养需求,与含血清细胞培养相比,单位细胞病毒产量没有降低,达到30~60个活病毒/细胞。可以实现在生物反应器的工艺放大,病毒高峰时病毒滴度均在7.0lg PFU/m L以上。结论无血清细胞培养可以应用于SFTSV的培养,有利于降低疫苗生产过程中的纯化难度,提高疫苗安全性。     

The nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate RNA encapsidation

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 ? resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identifi ed through mutational analysis and identifi ed to have a signifi cant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The fi ndings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.     

发热伴血小板减少综合征病毒中和抗体酶联免疫吸附试验方法的建立及应用

本文旨在对发热伴血小板减少综合征(severe fever with thrombocytopenia syndrome,SFTS)患者中和抗体进行定性和效价评估,建立中和抗体酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)。用96孔微量培养板培养非洲绿猴肾细胞(Vero-E6)并接种发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV),以抗核衣壳蛋白(nucleocapsid protein,NP)单克隆抗体为一抗,使用间接ELISA检测SFTSV NP,根据光密度(optical density,OD)判断阳性孔数,采用ReedMuench方法计算病毒半数组织培养感染剂量(50%tissue culture infective dose,TCID_(50)),以反映SFTSV在Vero-E6细胞中的复制水平。ELISA检测中和抗体作用后的病毒残余量,可间接反映中和抗体的作用效果并进行定量。应用以上建立的微量中和-ELISA对10例SFTS患者的双份血清进行中和抗体效价测定,8例患者恢复期血清效价较急性期增高4倍以上,7份患者恢复期血清效价达1∶1 280,急性期血清效价最高为1∶640。结果提示,本研究建立的ELISA操作简便,结果判定客观,所需时间短,可用于临床血清抗体诊断,也可用于血清流行病学调查和疫苗效果临床评价等。     

Severe fever with thrombocytopenia syndrome and its pathogen SFTSV

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever in East Asia with case fatality up to 50%. SFTS is caused by SFTSV, a tick borne bunyavirus. In endemic area in China 1%–3% population was infected with SFTSV, but age is critical risk factor for hospitalization and death of SFTS patients.     

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

<正>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%     

A new luciferase immunoprecipitation system assay provided serological evidence for missed diagnosis of severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) infection, was first reported in 2010 in China with an initial fatality of up to 30%. The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy. In this study, a new luciferase immunoprecipitation system (LIPS) assay based on pREN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase (Rluc), was established and used to investigate the levels of antibody responses to SFTSV. Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019. The results showed that 82 of the 464 patients (17.7%) had antibody response to SFTSV, which were further supported by immunofluorescence assays (IFAs). Further, qRT-PCR and microneutralization tests showed that among the 82 positive cases, 15 patients had viremia, 10 patients had neutralizing antibody, and one had both (totally 26 patient). However, none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations. All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases. The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019, and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.     

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 10³ TCID₅₀ and 10⁵ TCID₅₀. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS.     

Deep sequencing and phylogenetic analysis of severe fever with thrombocytopenia syndrome virus from the tick,Haemaphysalis longicornis,in Korea

The Asian longhorned tick, Haemaphysalis longicornis, is widely distributed in China, Japan, and Korea and may transmit infectious diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an important tick‐borne disease caused by the SFTS virus (SFTSV). Deep sequencing to confirm the presence of SFTSV in ticks has not been reported in Korea. To detect SFTSV, RNA was extracted from tick samples and analyzed using high‐throughput deep sequencing. Based on BLASTN results, numerous SFTSV reads were identified. Moreover, a nearly complete genome of SFTSV (JNU‐1 isolate) was obtained using Sanger sequencing. The genome of the JNU‐1 isolate includes three segments of 6,286, 3,299 and 1,642 nucleotides (nt) termed large (L), medium (M), and small (S), respectively. Also, phylogenetic and recombination analyses for each segment of SFTSV were performed using the JNU‐1 isolate. The three segments of JNU‐1 isolate were closely related to the genotype B known human‐derived Korean SFTSV isolate; the JNU‐1 isolate showed no recombination sites with other isolates. This study is the first report of detection of SFTSV from ticks using deep sequencing in Korea and provides information on the genetic diversity of SFTSV in East Asia.     

Tilorone confers robust in vitro and in vivo antiviral effects against severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging pathogen, is a tick-borne bunyavirus belonging to the genus Bandavirus in the family Phenuiviridae (Kuhn et al., 2020). This pathogen was first identified in China during the heightened surveillance of acute febrile illness in 2009, and has been reported to cause several outbreaks in eastern Asia areas, including China, Japan, and Korea (Yu et al., 2011). Besides, Vietnam has also reported several confirmed SFTS cases (Tran et al., 2019). The mortality rate in hospitalised patients with SFTSV infection is up to 10%–30%. Moreover, SFTSV has been reported to possibly transmitted by the contact of body fluids from person-to-person, and extensive SFTSV contamination was detected in the patient rooms (Kim et al., 2015). These reports suggest that more stringent isolation measures are needed for the prevention of massive SFTSV outbreak.     

Johnson-McMillin syndrome: report of a new case with novel features

BACKGROUND: Johnson-McMillin syndrome (JMS) is a rare neuroectodermal disorder characterized by alopecia, ear malformations, conductive hearing loss, anosmia/hyposmia, and hypogonadotropic hypogonadism. It is inherited in an autosomal dominant manner; however, the causative gene has not yet been identified. CASE: Herein we report a patient with this condition who exhibits many of the features previously described, including alopecia, malformed auricles, conductive hearing loss, facial asymmetry, and developmental delays. Interestingly, she also has features that have not yet been reported, such as preauricular pits and tags, broad depressions at the lateral aspects of the eyes, and an abnormal left lower eyelid. CONCLUSIONS: In addition to demonstrating a pattern of anomalies consistent with JMS, this patient has several unique features. This phenotype supports the involvement of the branchial arches in the embryologic basis of this condition.     

The S segment of rift valley fever phlebovirus (Bunyaviridae) carries determinants for attenuation and virulence in mice

Unlike all the other Rift Valley fever virus strains (Bunyaviridae, Phlebovirus) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the NSs nonstructural protein in the nuclei of infected cells (R. Muller, J. F. Saluzzo, N. Lopez, T. Drier, M. Turell, J. Smith, and M. Bouloy, Am. J. Trop. Med. Hyg. 53:405-411, 1995). This defect is correlated with a large in-frame deletion in the NSs coding region of the S segment of the tripartite genome. Here, we show that the truncated NSs protein of clone 13 is expressed and remains in the cytoplasm, where it is degraded rapidly by the proteasome. Through the analysis of reassortants between clone 13 and a virulent strain, we localized the marker(s) of attenuation in the S segment of this attenuated virus. This result raises questions regarding the role of NSs in pathogenesis and highlights, for the first time in the Bunyaviridae family, a major role of the S segment in virulence and attenuation, possibly associated with a defect in the nonstructural protein.     

Cytokine and chemokine levels in patients with severe Fever with thrombocytopenia syndrome virus

Background

Severe fever with thrombocytopenia syndrome virus (SFTSV), which can cause hemorrhagic fever–like illness, is a newly discovered bunyavirus in China. The pathogenesis of SFTSV infection is poorly understood. However, it has been suggested that immune mechanisms, including cytokines and chemokines, play an important role in disease pathogenesis. In the present study, we investigated host cytokine and chemokine profiles in serum samples of patients with SFTSV infection from Northeast China and explored a possible correlation between cytokine levels and disease severity.

Methods and Principal Findings

Acute phase serum samples from 40 patients, diagnosed with SFTSV infection were included. Patients were divided into two groups – severe or non-severe – based on disease severity. Levels of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, interleukin-6, interferon (IFN)-γ, IFN- γ-induced protein (IP)-10 and RANTES were measured in the serum samples with commercial ELISAs. Statistical analysis showed that increases in TNF-α, IP-10 and IFN-γ were associated with disease severity.

Conclusions

We suggest that a cytokine-mediated inflammatory response, characterized by cytokine and chemokine production imbalance, might be in part responsible for the disease progression of patients with SFTSV infection.