Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.

Therapeutic and prevention strategies against human enterovirus 71 infection

Human enterovirus 71(HEV71) is the cause of hand,foot and mouth disease and associated neurological complications in children under five years of age.There has been an increase in HEV71 epidemic activity throughout the Asia-Pacific region in the past decade,and it is predicted to replace poliovirus as the extant neurotropic enterovirus of highest global public health significance. To date there is no effective antiviral treatment and no vaccine is available to prevent HEV71 infection. The increase in prevalence, virulence and geographic spread of HEV71 infection over the past decade provides increasing incentive for the development of new therapeutic and prevention strategies against this emerging viral infection. The current review focuses on the potential, advantages and disadvantages of these strategies. Since the explosion of outbreaks leading to large epidemics in China, research in natural therapeutic products has identified several groups of compounds with anti-HEV71 activities. Concurrently, the search for effective synthetic antivirals has produced promising results. Other therapeutic strategies including immunotherapy and the use of oligonucleotides have also been explored. A sound prevention strategy is crucial in order to control the spread of HEV71. To this end the ultimate goal is the rapid development, regulatory approval and widespread implementation of a safe and effective vaccine. The various forms of HEV71 vaccine designs are highlighted in this review. Given the rapid progress of research in this area, eradication of the virus is likely to be achieved.

Evaluation of Reverse Transcription Loop-Mediated Isothermal Amplification assays for Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 in Clinical Samples

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.

Positive Selection Analysis of VP1 Genes of Worldwide Human Enterovirus 71 Viruses

Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.

RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A–D from Clinical Specimens

Objective To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses(HEVs)from clinical samples and to contribute to etiological surveillance of HEV-related diseases.Methods A panel of RT-nPCR assays,consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D,was established in this study.The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 perμL and copies perμL,and the newly established methods were tested in clinical specimens collected in recent years.Results The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 perμL and 10 virus copies perμL,and for the complete VP1 gene was 1 CCID50 perμL and 100 virus copies perμL,using serially-diluted virus stocks of five serotypes.As a proof-of-concept,25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons.Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D,providing rapid,sensitive,and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Human Enterovirus 71 DNA Vaccine Constructs Containing 5’UTR with Complete Internal Ribosome Entry Site Sequence Stimulated Improved Anti-Human Enterovirus 71 Neutralizing Immune Responses

Recent improvement in the technologies for efficient delivery of DNA vaccines has renewed interest in the DNA-based vaccines. Several DNA-based vaccines against human enterovirus 71 (EV71), the causative agent for hand, foot and mouth disease (HFMD) have been developed. Here we examined the potential of improving the vaccines by inserting the EV71 5’ untranslated region (5’ UTR) containing the full length internal ribosome entry site (IRES) sequence to the EV71 VP1-based DNA vaccine constructs. Four vaccine constructs designated as 5’ UTR-VP1/EGFP, VP1/EGFP, 5’ UTR-VP1/pVAX and VP1/pVAX, were designed using the pEGFP-N1 and pVAX-1 expression vectors, respectively. Transfection of Vero cells with the vaccine constructs with the 5’-UTR (5’-UTR-VP1/EGFP and 5’ UTR-VP1/pVAX) resulted in higher percentages of cells expressing the recombinant protein in comparison to cells transfected with vectors without the 5’-UTR (67% and 57%, respectively). Higher IgG responses (29%) were obtained from mice immunized with the DNA vaccine construct with the full length 5’ UTR. The same group of mice when challenged with life EV71 produced significantly higher neutralizing antibody (NAb) titers (>5-fold). These results suggest that insertion of the EV71 5’ UTR sequence consisting of the full length IRES to the EV71 DNA vaccine constructs improved the efficacy of the constructs with enhanced elicitation of the neutralizing antibody responses.

蓝芩口服液联合利巴韦林气雾剂对手足口病患儿症状恢复时间及病毒转阴率的影响

目的探讨蓝芩口服液联合利巴韦林气雾剂对手足口病患儿症状恢复时间及病毒转阴率的影响。方法选取2021年7月至2023年6月聊城市第二人民医院收治的96例手足口病患儿作为研究对象,按随机数字表法将其分为对照组(48例)和观察组(48例)。对照组采用利巴韦林气雾剂治疗,观察组在对照组基础上采用蓝芩口服液治疗,两组均治疗2周。比较两组临床疗效、症状恢复时间、血清免疫球蛋白水平与病毒转阴率。结果观察组临床总有效率高于对照组,症状恢复时间及住院时间均短于对照组,观察组治疗后免疫球蛋白M(IgM)、免疫球蛋白G(IgG)、免疫球蛋白A(IgA)水平高于对照组,观察组肠道病毒转阴率与人肠道病毒71型(EV71)病毒转阴率均高于对照组,差异有统计学意义(P<0.05)。结论在利巴韦林气雾剂基础上给予蓝芩口服液治疗能够显著提高临床疗效,快速缓解临床症状,改善免疫球蛋白水平,提升病毒转阴率。

HEV保护性基因工程抗体的制备及功能研究

目的:制备抗戊型肝炎病毒(hepatitis E virus,HEV)的人鼠嵌合基因工程抗体并研究其中和保护作用及特性。方法:将已获得的抗HEV鼠源单克隆抗体基因序列进行优化,设计人鼠嵌合的工程化抗体基因序列,将其克隆入真核表达载体;表达载体共转染293F细胞进行表达优化,高效制备基因工程化抗体;通过酶联免疫反应(enzyme linked immunosorbent assay,ELISA)、免疫荧光及HEV细胞感染模型,研究工程化抗体的结合能力及中和活性。结果:成功构建基因工程化人鼠嵌合HEV保护性抗体表达载体,在293F表达系统中实现高效表达。抗体表达产率达30 mg/L,亲和力为1.202×10^-8mol/L。免疫荧光检测结果显示抗体能够灵敏、特异地与已感染HEV的Kernow细胞结合,实时荧光定量PCR检测结果显示抗体可保护易感的C3A细胞不被HEV感染。结论:基因工程化抗体抗体具有高效的HEV结合能力及中和作用,能有效阻断HEV感染,并实现低成本高效表达。

轻或重症手足口病患儿HEV71分离鉴定及其VP1基因型分析

目的从不同病情手足口病（HFMD）患儿中分离鉴定人肠道病毒71型（HEV71）并分析其VP1基因型及药物结合位点突变情况。方法从2014年浙江省绍兴地区轻、重症各4例HFMD患儿标本中分离HEV71并用RT-PCR进行鉴定。采用RT-PCR扩增病毒分离株全长VP1基因并测序。采用专业生物信息学软件,对HEV71分离株进行VP1基因分型、确定VP1基因中药物结合位点突变情况及构建HEV71分离株系统进化树。结果8例HFMD患儿标本中均分离出HEV71。HEV71分离株与文献报道的VP1-C4a基因亚型的核苷酸和氨基酸序列相似性分别高达97.2%～98.8%和99.3%～99.7%。2例重症患儿HEV71分离株分别在VP1基因序列第179位药物结合位点发生V179L点突变。5株HEV71与安徽和湖南分离株、3株与上海和山东分离株亲缘关系较近。结论本地区不同病情HFMD患儿分离的HEV71均为VP1-C4a基因亚型,重症患儿HEV71分离株VP1基因第179位药物结合位点突变可能与病情严重程度有关。

Different human-simulated characteristics of the driver models in the forward simulation of hybrid electric vehicles

Based on the control theories of PID, fuzzy logic and expert PID, the driver models are built and applied in the forward simulation for hybrid electric vehicles （HEV）. The impact to the vehicle speed tracking and the fuel economy is compared among the different driver models. The different human-simulated characteristics of the driver models are emphatically analyzed. The analysis results indicate that the driver models based on PID, simple fuzzy logic and expert PID are corresponding to the handling characteristics of different drives. The driver models of different human-simulated characteristics bring the handling divergence of drivers with different driving level and habit to the HEV forward simulation, and that is significant to the all-around verification and validation of the control strategy for HEV. System simulation results of different driver models validate the impact of driver models to the dynamic and fuel economy performance of HEV.

肠道病毒感染相关疾病的流行特征研究进展

人肠道病毒(HEV)能感染人类,引起世界范围的暴发流行,同时可诱发多种临床症状,主要包括发热、头痛、呼吸道感染疾病、咽痛,偶尔还有呕吐和腹泻等症状。有些特定血清型HEV能够引发严重疾病,但大部分HEV感染症状轻微甚至无症状。本文综述了已报道的部分与HEV感染相关疾病包括手足口病、急性弛缓性瘫痪、脑炎脑膜炎、糖尿病、腹泻、呼吸系统疾病、急性出血性结膜炎、急性中耳炎的流行特征研究进展;并进一步总结HEV在不同感染性疾病中的分子流行特征、致病机制进展,阐述HEV与人类感染性疾病的关系。

抗人类肠道病毒71药物及疫苗研究进展

手足口病作为一个全球性的公共卫生问题,危害巨大,会引起患儿神经系统疾病,具有高传染性、传播方式多样、病原体复杂多样等特点。引起小儿手足口病的病原体主要为柯萨奇病毒A属和人类肠道病毒71(EV71),作为手足口病主要病原体之一的EV71,感染性极强。目前,已经研制出的抗EV71的有效药物,是预防手足口病的关键途径。该文围绕EV71及抗EV71药物的研究进展进行了综述,进而为今后新药物的研发提供有价值的参考。

肠道病毒ECHO_(13)中国分离株的基因特征

为研究ECHO13病毒中国分离株的分子特征及其与世界其它分离株之间的基因关系,对1998年、2000年从中国福建省分离到2株ECHO13病毒进行基因序列对比分析。2株病毒分别命名为Fujian98-1和Fujian00-1,用逆转录-聚合酶链反应(RT-PCR)扩增出VP1蛋白编码基因全长861个核苷酸片段并进行序列测定,将2株E CHO13病毒的VP1序列与所有已发表的ECHO13病毒VP1基因全长进行同源性比较。结果显示,福建分离株之间核苷酸同源性为79 6%,氨基酸同源性为93 4%;与遗传距离最近的法国CF1089-91(AJ537604)毒株的核苷酸同源性分别为80%和88%,与代表株DelCarmen的核苷酸同源性分别为75 8%和77 9%。通过VP1基因分析,福建2株病毒均属于ECHO13病毒,与血清中和试验鉴定结果一致。下载所有已发表的ECHO13病毒VP1序列并构建进化树,发现福建2株病毒分属不同的分枝,提示这2株病毒来自不同的病毒传播链。进一步分析发现,整个E CHO13病毒可划分为3个不同的基因型:A、B和C基因型。福建Fujian98-1和Fujian00-1分别被划分在基因型B和C中,各基因型之间的核苷酸差异均大于20%。为验证该分型方法,将26株来自不同国家和时间的ECHO13病毒和2株福建分离ECHO13病毒部分VP1基因序列进行对比分析,建立进化树。结果显示,所有ECHO13病毒被分在A。

黄芩茎叶提取物与生脉饮抗柯萨奇病毒B3的体外研究

目的:探讨黄芩茎叶提取物及生脉饮体外抗柯萨奇病毒B3(CVB3)的作用。方法:应用细胞感染模型按照药物对Hela细胞及CVB3不同作用方式设直接抗病毒作用组、增强细胞抗病毒作用组、对感染细胞保护作用组及正交设计组。采用光镜观察并结合MTT测定的方法进行综合判断。结果:与病毒对照组比较黄芩茎叶提取物、生脉饮对细胞病变有抑制作用(P<0.05)。结论:黄芩茎叶提取物、生脉饮均有直接抗病毒作用以及对未感染或已感染Hela细胞的保护作用。生脉饮与黄芩茎叶提取物之间有交互作用。

2009～2011年上海市手足口病流行病学和病原学特征

为分析2009～2011年上海地区手足口病流行病学特征和病原构成，从国家疾病监测信息报告管理系统获取上海市2009～2011年手足口病流行病学资料；采用实时荧光反转录聚合酶链反应（RT-PCR）对来自上海18个区（县）的6676例手足口病病例标本进行肠道病毒核酸检测，对其中257份标本进行病毒分离；对27份人肠道病毒71型（HEV71）毒株进行VPl基因序列全长测定和分析。结果显示，2009～2011年上海市18个区（县）均有手足口病病例报道，地区分布元显著差异；≤5岁的婴幼儿为疾病高发年龄段；4～11月为发病高峰期。HEV71和柯萨奇病毒A组16型（CAl6）为主要病原，不同地区病原构成有所不同。实时荧光RT—PCR对6676例病例标本进行核酸检测，其中肠道病毒通用核酸检测阳性率为69．61％，HEV71和CAl6阳性率分别为38．83％和21．26％。对257份HEV71核酸阳性标本进行病毒分离，获得毒株57株，分离阳性率为22．18％。对其中27株进行VPl基因全长测序，均属C4a基因亚型。

内标多重荧光RT-PCR同时检测肠道病毒71型和柯萨奇病毒A16型

[目的]为对当前爆发的手足口病进行快速准确的检测。[方法]本研究建立了含内标的同时检测EV71和CA16的多重荧光RT-PCR方法，对该方法的特异性、灵敏度等进行评估，并对400多份临床样品进行了检测。[结果]实验结果表明，该检测方法特异性强，对10株EV71病毒、8株CA16病毒和25株其他人类病毒进行了检测，特异性为100%；该检测方法对EV71和CA16的检测灵敏度分别达到0.1 TCID50和1 TCID50；将0.1-104TCID50/mL EV71和CA16样本进行重复性实验，其变异系数分别为0.9%-2.0%和0.9%-2.3%。对400多份临床样品分别进行荧光RT-PCR检测和传统方法检测，结果显示，荧光RT-PCR对EV71和CA16的阳性检出率平均为46.1%和14.2%，比传统方法（34.5%和12.8%）的阳性检测率高。另外，实验数据显示，在粪便、直肠拭子、咽喉拭子样本中，PCR抑制物存在的比例为1.8%-3.4%，表明内标对监控PCR抑制物的存在具有重要作用。[结论]本方法能同时对EV71和CA16进行快速检测，并且灵敏度高，特异性好，由于加入了内标，能有效地监控假阴性的出现，适合于手足口病的临床检测。

石家庄市2009—2012年手足口病病原学特征及变化规律分析

目的分析2009—2012年石家庄市手足口病（HFMD）病原体分布特征及规律。方法选择石家庄市2009—2012年临床诊断为HFMD的部分普通及所有聚集发病、重症和死亡病例,采集病例咽拭子、肛拭子或疱疹液、便等生物学标本进行病原学检测。结合地理信息系统（GIS）对监测数据进行空间分布分析。结果 4年共采集3 842例HFMD病例生物学标本,阳性检出率为69.29%（2 662例）。其中人类肠道病毒71型（EV71）、柯萨奇病毒（Cox）A16和其他肠道病毒比例分别占50.60%（1 347例）、35.05%（933例）和14.35%（382例）。2009—2012年,石家庄市各年份HFMD病原学构成间差异有统计学意义（χ^2=2.367,P〈0.001）;其中2009年以Cox A16流行为主（占56.46%）,2010、2011年以EV71流行为主（占77.34%、57.51%）,2012年再次以Cox A16流行为主（占66.40%）。4年中各型病原在4或5月阳性检出数最高,6月开始迅速下降。Cox A16基本呈单峰变化,而EV71后期会再次增多,呈现＂拖尾＂现象;其他肠道病毒检出例数较少,时间分布特征不明显。同一流行年各县区病原分布存在地区差异;0～岁儿童较易感染EV71;EV71感染者中重症病例所占比例明显高于普通病例,死亡病例均由EV71感染引起。结论石家庄市近4年来HFMD病原学构成有变化,呈现Cox A16与EV71交替成为优势病原的现象。EV71、Cox A16及其他肠道病毒分别呈现多峰、单峰和不显著峰的时间分布特征。在同一流行年,多数县区的优势病原与全市一致,个别县区的优势病原与全市不一致。随着年龄增长,EV71阳性检出构成比逐渐降低,Cox A16阳性检出构成比逐渐增多,其他肠道病毒构成比变化不大。

人肠道病毒71型感染免疫抑制恒河猴的分析

目的:通过建立人肠道病毒71型(EV71)感染在免疫抑制恒河猴的动物模型,进一步分析人EV71在免疫抑制动物体内病毒增殖、病理变化以及病毒抗原在不同组织的表达情况。方法:按照每天15 mg/kg的剂量给8只恒河猴口服环孢菌素A连续7 d后造成其免疫抑制状态,再经呼吸道和消化道感染EV71病毒FY23株,对其进行14 d的临床观察后处死,采集各器官组织进行病毒载量检测,同时对不同的组织进行病理学分析和免疫组化分析。结果:感染EV71病毒后,免疫抑制组的临床症状、病毒载量、病理分析和免疫组化结果都明显比非免疫抑制组严重,从病毒对机体造成的影响来看呼吸系统感染较消化系统感染明显严重。结论:本实验分析了人EV71在免疫抑制动物体内病毒增殖的情况,为建立用于EV71疫苗评价的恒河猴动物模型提供了依据,并对未来的EV71疫苗的使用人群范围做出了新的限定。

人肠道病毒2A蛋白酶生物学作用研究进展

人肠道病毒(HEVs)是近年来危害亚太地区婴幼儿健康的重要病原体。随着HEVs研究的进展,对其2A蛋白在病毒复制、关闭宿主蛋白合成以及逃避机体固有免疫反应等方面的作用有了进一步的认识。本文主要对2A蛋白酶的生物学功能作一综述,对相关病毒致病机制研究以及2A蛋白开发应用有启发作用。

CD8^+细胞毒性T细胞的反应质量在EV71HFMD病程发展中的作用

目的研究中国EV71感染手足口病(HFMD)中CD8+细胞毒性T细胞的活化和第二信号的表达,探讨EV71感染的病程发展机制。方法选取48例EV71HFMD患儿分为轻症组、重症组(其中包括中枢神经病变组、中枢神经病变伴自主神经失调组、神经源性肺水肿组),应用流式细胞术检测CD8+T细胞上CD28和CD38的表达水平,分析CD8+T细胞的质量水平在EV71感染病程发展中的作用。结果 CD8+CD28+细胞、活化分子CD38的表达轻症组与健康同龄对照组、重症各组比较增高明显,差异有统计学意义(P<0.05)。结论 CD8+细胞毒性T细胞在EV71感染中的反应质量可能是病程发展的原因之一。