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.

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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.     

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.     

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。     

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.     

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。     

家养动物体表蜱中发热伴血小板减少综合征布尼亚病毒分离及鉴定

为了解发热伴血小板减少综合征布尼亚病毒(SFTSV)的传播机制,采集了山东疫区家养牛、羊和狗等动物体表蜱,分类鉴定后,通过Real-time PCR筛查、病毒分离培养和基因组序列分析等方法分离鉴定蜱中的病毒。所采集的蜱,以长角血蜱为主,占91.4%。其中3头SFTSV核酸检测阳性,阳性率为2.14%,并在其中一份羊体表蜱标本中分离到SFTSV病毒,命名为SDLZTick12。序列分析显示与我国在不同省份患者标本中分离的病毒全基因序列具有高度同源性,且病毒的抗原性和生长特性与人源病毒相同。本研究首次在山东疫区蜱中分离到新型布尼亚病毒,并与人源病毒进行了系统比较研究,提示蜱可能为该新病原体的传播媒介,对疾病的防控具有重要的指导意义。     

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 HEK293 T(293 T) 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 293 T 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.     

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 ELISAs 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.     

Rapid detection of severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies by up‐converting phosphor technology‐based lateral‐flow assay

In this study, an up‐converting phosphor technology‐based lateral‐flow (UPT‐LF) assay was developed to detect severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies rapidly and specifically. SFTSV recombinant N protein (SFTSV‐rNP) was coated on analytical membrane for sample capture, up‐converting phosphor (UCP) particles were used as the reporter, the luminescence emitted by UCP particles was converted to a measurable signal by a biosensor. The performance of UPT‐LF assay was evaluated by testing 302 field serum samples by ELISA (enzyme‐linked immunosorbent assay), Western blotting and UPT‐LF assay. UPT‐LF assay exhibited a lower detection limit than ELISA, and a satisfied level of agreement was exhibited by Kappa statistics (Kappa coefficient = 0.938). Considering Western blotting as the reference for comparison, the sensitivity and specificity of UPT‐LF assay could reach 98.31% and 100%. UPT‐LF assay showed no specific reaction with hantavirus total serum antibodies, which avoids the misdiagnosis of SFTSV from hantavirus that could cause similar clinical symptoms. UPT‐LF assay was able to achieve acceptable results within 15 min and needed only 10 μL sample for each test. As a whole, UPT‐LF assay is a candidate method for on‐site surveillance of SFTSV total antibodies owing to its excellent sensitivity, specificity, stability, easy operation and for being less time consuming.     

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening

Severe fever with thrombocytopenia syndrome virus(SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome(SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11(SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum(ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1(LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall,these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.     

Novel SFTSV Phylogeny Reveals New Reassortment Events and Migration Routes

Severe fever with thrombocytopenia syndrome virus(SFTSV), the causative agent of a febrile human disease, was first identified from central and eastern provinces in China, and later in Japan and South Korea. Hubei Province is one of the major SFTS epidemic areas in the central part of China. This study reported the isolation of 11 new SFTSV strains from patients in Hubei Province collected in 2017. Extensive phylogenetic analyses were conducted based on the complete coding sequences of SFTSV segments including the new strains. It was suggested that five different SFTSV genotypes were circulating in Hubei, and 15 reassortment patterns and migration pathways correlated with each genotype were identified, which was more than previously recognized. Hubei Province was more involved in the evolutionary events of SFTSV than that previously thought in which the evolutionary events of SFTSV were reported to be independent from those in other epidemic regions. Further divergence of SFTSV strains was suggested by pairwise comparison of SFTSV sequences from each genotype and sequence identity normalized to representative strain in genotype C1. Subsequently,amino acid variations specific for genotype(s), strain(s), or cluster(s) were inspected, which may be related to differential biological activity of SFTSV strains/genotypes. In conclusion, we analyzed the current status of SFTSV phylogeny in Hubei Province and discussed the possible events correlated to SFTSV evolution. It provided an in-depth insight into SFTSV evolution, raising concerns for the use of proper SFTSV strains in future studies.     

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

Virologica Sinica - 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...     

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

Virologica Sinica - Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high mortality (12%–30%). The mechanism by which the SFTS bunyavirus (SFTSV)...     

气候变化和经济发展对肾综合征出血热发生的影响

肾综合征出血热(hemorrhagic fever with renal syndrome, HFRS)是一种啮齿动物传播的自然疫源性疾病, 危害严重, 已成为全球重要的公共卫生问题。本研究采用数理统计模型及小波分析方法, 对陕西省西安市鄠邑区1984-2016年HFRS的发生与鼠类、气候和经济因素的关系进行分析, 探讨气候和经济因素对HFRS发生的影响。小波分析结果表明, 该地区的HFRS暴发史可能分为两个时期, 推测每个时期具有不同的主要宿主, 在2002年褐家鼠(Rattus norvegicus)可能取代黑线姬鼠(Apodemus agrarius)成为HFRS疫源地的主要宿主。广义可加模型模拟结果表明, HFRS的发生与1984-2001年黑线姬鼠密度间存在极显著非线性效应(F_2.06,9.02 = 102.415, P < 0.01), 两者间显现为正相关; 与2002-2016年的褐家鼠密度间呈正相关(F_1.67,9.02 = 73.929, P < 0.01); HFRS主要宿主的这种变化可能与当地气候变化和经济发展有关: HFRS的发生与年平均温度存在极显著的非线性效应(F_2.93,9.02 = 12.164, P < 0.01), 两者间呈负相关; 同样, HFRS的发生与上一年的国内生产总值(GDP)也存在显著非线性效应(F_1.70,9.02 = 2.917, P < 0.05), 两者间也呈负相关。结构方程模型通过直接和间接的影响途径证明了这种转移机制, 发现温度对HFRS发生有显著的直接负向影响以及通过褐家鼠的间接正向影响; GDP对HFRS发生有直接的负向影响。本研究表明HFRS的发生与气候变化和经济发展相关, 两者均能影响HFRS的暴发, 该结论有助于今后更好地对HFRS疾病进行预防和控制。     

Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy

Photodynamic therapy (PDT) is a regulatory-approved modality for treating a variety of malignant tumors. It induces tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. The heat shock protein 70 (HSP70-1) is a stress protein encoded by the HSPA1A gene and is significantly induced by oxidative stress associated with PDT. The aim of this study was to identify the functional region of the HSPA1A promoter that responds to PDT-induced oxidative stress and uses the stress responsiveness of HSPA1A expression to establish a rapid and cost-effective photocytotoxic assessment bioassay to evaluate the photodynamic potential of photosensitizers. By constructing luciferase vectors with a variety of hspa1a promoter fractions and examining their relative luciferase activity, we demonstrated that the DNA sequence from −218 to +87 of the HSPA1A gene could be used as a functional promoter to detect the PDT-induced oxidative stress. The maximal relative luciferase activity level of HSPA1A (HSP70-1) induced by hypericin-PDT was nearly nine times that of the control. Our results suggest that the novel reporter gene assay using a functional region of the HSP70A1A promoter has significant advantages for the detection of photoactivity in terms of both speed and sensitivity, when compared with a cell viability test based on ATP quantification and ROS levels. Furthermore, phthalocyanine zinc and methylene blue both induced significantly elevated levels of relative luciferase activity in a dose-dependent manner.     

A rapid assay for simultaneous detection of Spiroplasma eriocheiris and white spot syndrome virus in Procambarus clarkii by multiplex PCR

Aims: To establish a multiplex PCR method for simultaneous and rapid detection of Spiroplasma eriocheiris and white spot syndrome virus (WSSV) in Procambarus clarkii with recommendations for application to other crustacea. Methods and Results: Three primer sets were mixed at a ratio of 1 : 3 : 1 to amplify specific fragments of the S. eriocheiris, WSSV, P. clarkii crayfish (control organism) genomes, respectively. S. eriocheiris and WSSV were used to challenge the susceptible crustacea in the experimental groups. Total DNA of the samples was purified and detected by multiplex PCR. The PCR‐amplified products produced four groups of results as follows. One fragment of 1195 bp, amplified by the primer set ITS‐crayfish/28S‐crayfish, served as an internal control, showed no pathogen detection, thus confirming the specificity of our positive tests. Two groups represented by: (i) samples challenged by S. eriocheiris alone, or (ii) challenged by WSSV alone, yielded two fragments each; i.e. those from S. eriocheiris (271 bp) plus the internal control and those from WSSV (530 bp) plus the internal control. Finally, for the fourth group, in cases of double challenged treatments, all three amplified products were detected simultaneously. Conclusions: Simultaneous and rapid detection of two pathogens in P. clarkii is important to maintain productive and healthy crayfish in aquaculture. The direct detection of S. eriocheiris and WSSV from P. clarkii is practicable with multiplex PCR. Significance and Impact of the Study: This study shows that the two pathogens are simultaneously and rapidly detected in P. clarkii by multiplex PCR, thus increasing the efficiency of pathogen detection.     

Hyper-IgD and periodic fever syndrome: a new MVK mutation (p.R277G) associated with a severe phenotype

Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS; MIM# 260920) is a rare recessively-inherited autoinflammatory condition caused by mutations in the MVK gene, which encodes for mevalonate kinase, an essential enzyme in the isoprenoid pathway. HIDS is clinically characterized by recurrent episodes of fever and inflammation. Here we report on the case of a 2 year-old Portuguese boy with recurrent episodes of fever, malaise, massive cervical lymphadenopathy and hepatosplenomegaly since the age of 12 months. Rash, arthralgia, abdominal pain and diarrhea were also seen occasionally. During attacks a vigorous acute-phase response was detected, including elevated erythrocyte sedimentation rate, C-reactive protein, serum amyloid A and leukocytosis. Clinical and laboratory improvement was seen between attacks. Despite normal serum IgD level, HIDS was clinically suspected. Mutational MVK analysis revealed the homozygous genotype with the novel p.Arg277Gly (p.R277G) mutation, while the healthy non-consanguineous parents were heterozygous. Short nonsteroidal anti-inflammatory drugs and corticosteroid courses were given during attacks with poor benefits, whereas anakinra showed positive responses only at high doses. The p.R277G mutation here described is a novel missense MVK mutation, and it has been detected in this case with a severe HIDS phenotype. Further studies are needed to evaluate a co-relation genotype, enzyme activity and phenotype, and to define the best therapeutic strategies.