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。     

The Effects of Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs) on Protein–Protein Interactions

Non-synonymous single nucleotide polymorphisms (nsSNPs) are single base changes leading to a change to the amino acid sequence of the encoded protein. Many of these variants are associated with disease, so nsSNPs have been well studied, with studies looking at the effects of nsSNPs on individual proteins, for example, on stability and enzyme active sites. In recent years, the impact of nsSNPs upon protein–protein interactions has also been investigated, giving a greater insight into the mechanisms by which nsSNPs can lead to disease.     

Identification of a novel MYH9 mutation (p. V782Y) in a Chinese patient with thrombocytopenia: a case report

MYH9-related diseases (MYH9-RD) are a group of autosomal dominant diseases caused by mutations in the MYH9 gene, which are featured by thrombocytopenia, giant platelets and granulocyte cytoplasmic inclusion bodies. MYH9-RD patients generally suffer from bleeding syndromes, progressive kidney disease, deafness, or cataracts. Here, we reported on a case of MYH9-RD. A novel heterozygous mutation of MYH9 (c.2344-2345delGTinsTA, p.T782Y) was discovered by targeted sequencing technology. Immunofluorescence analysis of neutrophils confirmed abnormal aggregation of MYH9 protein. The results of this study should expand the MYH9 gene mutation spectrum and provide reference for subsequent researchers and genetic counseling.     

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...     

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.     

Indirubin modulates CD4+ T‐cell homeostasis via PD1/PTEN/AKT signalling pathway in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by an immune mediated decrease in platelet number. Disturbance of CD4⁺ T‐cell homeostasis with simultaneous decrease of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4⁺ effector T cells underpin the pathophysiology of ITP. Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L. which is clinically used for the treatment of ITP patients. Whether indirubin targets the Tregs/effector T cell‐axis to restore platelet number is unknown. In our in vitro studies, Indirubin could significantly enhance the number and function of Tregs and meanwhile dampen the activation of effector T cells in a dose‐dependent manner. Indirubin was observed to restore the expression of programmed cell‐death 1 (PD1) and phosphatase and tensin homolog (PTEN) on the CD4⁺ T cells of ITP patients, leading to the subsequent attenuation of the AKT/mTOR pathway. Furthermore, these observations were recapitulated in an active murine model of ITP with a prominent platelet response. Thus, our results identified a potentially novel mechanism of the therapeutic action of indirubin in the treatment of ITP through regulating the homeostasis of CD4⁺ T cells in a PD1/PTEN/AKT signalling pathway.     

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。     

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。     

A case of neonatal thrombocytopenia due to maternal Helicobacter pylori-associated immune thrombocytopenia

Many studies in adults have suggested an association between Helicobacter pylori (H. pylori) infection and chronic immune thrombocytopenia (ITP). In adults with ITP and H. pylori infection, eradicating H. pylori is recommended as the first-line therapy. However, the association between ITP and H. pylori in children remains controversial. Diagnosing thrombocytopenia in pregnant women is challenging but crucial because maternal ITP causes neonatal ITP through transplacental transfer of immunoglobulin G, also known as passive ITP. Herein, we report a case of neonatal passive ITP due to maternal H. pylori-associated ITP. A boy was born at term with neonatal thrombocytopenia to a mother tentatively diagnosed with gestational thrombocytopenia. However, further examination suggested that maternal thrombocytopenia was associated with H. pylori, and neonatal thrombocytopenia was diagnosed as ITP due to maternal ITP. The newborn received intravenous immunoglobulin treatment, and the thrombocytopenia did not recur. The mother was examined using esophagogastroduodenoscopy, and her rapid urease test using gastric mucosa tissue samples was positive. Subsequently, she was diagnosed with H. pylori infection and received H. pylori eradication therapy, after which her platelet count remained normal. To our knowledge, this is the first reported case of neonatal passive ITP secondary to maternal H. pylori-associated ITP. This case suggests that maternal H. pylori infection can lead to the production of platelet autoantibodies, which can destroy antibody-sensitized platelets in the mother and neonate. To summarize, H. pylori infection can also cause ITP in children. Therefore, pregnant women diagnosed with H. pylori-associated ITP should receive H. pylori eradication therapy to prevent their neonates from developing passive ITP.