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.

Genetic Analysis of the VP1 Region of Human Enterovirus 71 Strains Isolated in Fuyang,China,During 2008

Enterovirus 71 （EV71） is a common cause of Hand, foot, and mouth disease （HFMD） and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences （891 nueleotides） from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous tO members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine（H） at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.

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.

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.

Comparative Genomic Analysis of Enterovirus 71 Revealed Six New Potential Neurovirulence-associated Sites

In the present study,the complete genomes of four common（4/EV71/Wenzhou/CHN/2014,15/EV71/Wenzhou/CHN/2014,116/EV71/Wenzhou/CHN/2014,and 120/EV71/Wenzhou/CHN/2014）and two virulent（11/EV71/Wenzhou/CHN/2014and 109/EV71/Wenzhou/CHN/2014）enterovirus 71（EV71）isolates were sequenced and described.They are 7405 bp in length and belong to EV71 sub-genotype C4 （C4a cluster）.

Clinical characteristics and treatment of severe encephalitis associated with neurogenic pulmonary edema caused by enterovirus 71 in China

BACKGROUND： Hand-foot-mouth disease has become a major public health issue in children in China. In the present prospective study we investigated the clinical characteristics and emergency management of children with severe encephalitis associated with NPE caused by enterovirus 71.METHODS： The study was conducted in 2 pediatric intensive care units （PICUs） over a 2-month period. Clinical records were reviewed of critically ill children with severe encephalitis associated with NPE caused by EV71 who were admitted to PICUs during the period of May to June 2008 in Fuyang.RESULTS： We reviewed the complete records of 36 children, of whom 23 （63.9%） were male and 13 （36.1%） female. Their age ranged from 4 to 48 months, with an average of 15.8 months. All children except one were under 3 years of age. The overall mortality in these children was 19.4%. The average duration of critical life threatening signs and symptoms was 2.1 days （12 hours-5 days）. Nervous system diseases included brainstem encephalitis in 27 children （75%）, brainstem encephalitis associated with myelitis in 6 children （16.7%）, and general encephalitis in 3 chidren （8.3%）, respectively. In 12 patients of NPE （33.3%） pink or bloody bubble sputum and asymmetric pulmonary edema or hemorrhage was the primary manifestation but no typical exanthema was observed. Five children died of acute onset of NPE and / or pulmonary hemorrhage with rapid progression of cardiopulmonary failure within hours after admission. Therapeutic management consisted of mechanical ventilation and administration of mannitol, methylprednisolone, intravenous immunoglobulin （IVIG） and vasoactive drugs, associated with the need of fluid volume resuscitation in 9 （25%） of the 36 children.CONCLUSION： In children less than 3 years of age found to be affected by severe EV71 encephalitis associated with NPE, one fifth may die. The major organ systems infected by severe EV71 include the central nervous system, the respiratory system, and the cardiovascular system. Early diagnosis and evaluation, respiratory support, treatment of intracranial hypertension, and mainttenance of function of the cardiovascular system are the most important therapeutic measures.

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.

Antiviral Activity of GuiQi Polysaccharides against Enterovirus 71 in vitro

In this study,we have investigated the antiviral activity of GuiQi polysaccharides (GQP) upon enterovirus 71 (EV71) in vitro.An assay using methyl thiazolyl tetrazolium (MTT),and analyses of cytopathic effects (CPE)were used to examine the antiviral activity of GQP upon Vero cells infected with EV71.The results revealed that GQP at concentrations below 31.2μg/mL exhibited significant antiviral effects upon EV71 when applied under three different experimental protocols.GQP was most strongly active in preventing the adsorption of EV71 to target cells and in this respect it was significantly more effective than ribavirin.In addition,it was clear that GQP could inhibit viral replication when added to cells 2 h after infection,but if added at the point of infection its effect was weak.GQP is considered to be less toxic than ribavirin,and may warrant further evaluation as a possible agent in the treatment of hand,foot and mouth disease (HFMD).

Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.

Contribution of 3CD Region to the Virulence of Enterovirus 71

Enterovirus 71 （EV71） and Coxsackievirus A16 （CoxA16） are the major pathogens causing hand, foot, and mouth disease （HFMD） that occurs primarily in children and infants with mild clinical manifestations. HFMD caused by EV71 could develop to a fatal neurological complication.

Rational design of a DNA-launched live attenuated vaccine against human enterovirus 71

Human Enterovirus 71(EV71)has emerged as one of the predominant causative agents of hand,foot and mouth disease(HFMD)with global impact.Despite the inactivated vaccine being licensed,other vaccine candidates based on advanced technology platforms are under development.In this report,we rationally designed and constructed two DNA-launched live attenuated vaccine candidates(pDL-EV71)under the control of specific promoters.In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71.More importantly,the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR/mice,demonstrating its safety profile.Moreover,a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice.A single-dose immunization with 10μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice.Overall,our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development.

重症EV71型手足口病炎症因子的临床意义

目的了解5种炎症因子MCP-1、IL-1β、IL-18、HMGB1、IL-10在重症EV71型手足口病(HFMD)中的临床意义。方法选取某院2014年3—8月确诊为重症EV71型HFMD的住院患儿为HFMD组,同期本院门诊体检的健康儿童为对照组,动态观察两组外周血MCP-1、IL-1β、IL-18、HMGB1、IL-10表达水平的变化,并收集HFMD组临床资料。结果 HFMD组共102例患儿,平均年龄为(2.18±0.91)岁,其中主要以≤3岁为主,占80.39%;HFMD组患儿入院时为第2期77例,第3期16例,第4期9例。经过第2期共77例,第3期共52例,第4期共21例,第5期共88例。HFMD组第2、3、4期分别与对照组的5种炎症因子表达水平比较,差异均有统计学意义(均P<0.05);HFMD组第3期与第4期的IL-10表达水平比较,差异无统计学意义(P>0.05)。HFMD组第2、3期进展组HMGB1表达水平均高于痊愈组,差异均有统计学意义(均P<0.05)。死亡组与存活组在入院时5种炎症因子表达水平的比较,差异均有统计学意义(均P<0.05)。结论 MCP-1、HMGB1、IL-1β、IL-10、IL-18的表达水平与HFMD病情轻重相关,对患儿预后的预测有一定临床意义。HMBG1在HFMD中对病情转归有一定预测价值。

检测EV71和EV-U的TaqMan-LNA探针多重荧光RT-PCR的建立

目的建立一种针对手足口病病原体的快速准确检测方法。方法利用TaqMan-LNA探针建立多重荧光定量RT-PCR方法,同时检测肠道病毒71型(EV71)和肠道病毒通用型(EV-U),用含有目的序列RNA的标准品制备标准曲线,对病毒进行准确定量,同时对该方法的特异性、灵敏度、重复性进行评估。结果用TaqMan-LNA探针多重荧光RT-PCR法检测CoxA16、CoxB2、EV71病毒和其他人类病毒RNA,证实其特异性为100%;对EV71和EV-U的检测灵敏度均达到102拷贝/μl;将EV71和EV-U的RNA标准品进行重复性实验,其批内、批间变异系数分别为0.97%～2.02%和0.89%～2.13%。结论本方法灵敏度较高,特异性强,重复性好,适用于手足口病的临床检测。

EV71-VP1抗原在双歧杆菌中表达的免疫效果检测

目的检测表达肠道病毒71型(enterovirus 71,EV71)VP1基因的重组双歧杆菌免疫后小鼠Ig G、Ig A表达变化,并对免疫的孕鼠产下的新生小鼠注射EV71病毒,以检测免疫保护作用可否从母代传给子代。方法 ELISA法检测免疫小鼠血清Ig G、Ig A抗体效价;用EV71毒株对免疫孕鼠所产下的新生小鼠攻毒。结果两种抗体效价都有明显升高(P<0.01);实验组受病毒攻击新生小鼠存活率明显高于对照组。结论口服表达VP1蛋白的双歧杆菌能够激活体内免疫系统产生特异性抗体,重组双歧杆菌可通过免疫孕鼠而使新生幼鼠获得保护性抗体。为研制EV71亚单位口服活疫苗奠定了研究基础。

以主要流行EV71 VP1基因高度保守区为靶点的TaqMan荧光定量PCR检测方法的建立

在我国,肠道病毒71型(enterovirus 71,EV71)C4型是引起手足口病的主要流行基因型。为建立EV71C4型TaqMan荧光定量PCR检测方法,在C4型EV71VP1基因的高保守区,设计合成引物和TaqMan探针,将包含此目的区段的基因片段克隆到pcDNA3.1载体中,通过体外转录获得标准品,并以梯度稀释的标准品为模板建立工作曲线,进而在优化反应条件的基础上建立TaqMan荧光实时定量PCR检测方法。实验中,所设计引物、探针的高度保守性保证了C4型EV71的高效扩增。经反应条件优化,引物和探针的最佳工作浓度分别为300 nmol/L和200 nmol/L,在1×10~301×10~3拷贝数检测范围内具有良好的线性关系(R^2=1),灵敏度可达到10~2 copies/μL。通过对该方法进行检验发现,批间和组间重复实验的变异系数均小于0.5%,且该方法对柯萨奇A16(coxsackievirus A16,CA16)柯萨奇B1(coxsackievirus B1,CB1)人轮状病毒(human rotavirus,HRV)单纯疱疹病毒2型(herpes Simplex virus type 2,HSV-2)均无交叉反应,对6份EV71阳性样本检出率为100%。以上数据表明,文中建立的TaqMan荧光定量PCR方法可为我国主要流行C4型EV71感染的快速诊断及疾病监控提供有效途径。

红景天苷体外抗EV71病毒的作用

应用MTT法研究红景天苷对Vero细胞的毒性以及对肠道病毒71型(Enterovirus 71,EV71)的抑制作用,通过吸附试验和穿入试验观察红景天苷是否对EV71的吸附和穿入具有抑制作用。结果表明,红景天苷的细胞毒性低,半数细胞毒性浓度(CC50)为4.25 mg/mL。药物在病毒感染前加入(药物预处理)、药物和病毒同时加入及药物在病毒感染后加入都能够抑制病毒,以药物预处理细胞抗病毒作用最好;红景天苷能够抑制病毒的吸附和穿入作用,对吸附作用抑制效果更强。表明红景天苷能够在体外抑制病毒对细胞的感染。

EV71入侵人神经母细胞瘤SK-N-SH细胞机制的初步研究

目的:初步研究肠道病毒71型(enterovirus type 71,EV71)入侵人神经母细胞瘤SK-N-SH细胞的机制。方法:将临床EV71分离株接种于人横纹肌肉瘤(rhabdomyosarcoma,RD)细胞,扩增和纯化病毒;MTT法检测不同病毒胞吞途径阻断剂对SKN-SH细胞生长抑制作用;用特异性的化学阻断剂预处理靶细胞后,Taq Man real-time PCR验证其对EV71 m RNA表达的影响。结果:RD细胞能够成功扩增出EV71病毒,病毒滴度为1×105 TCID50。随着药物浓度梯度的增加,SK-N-SH细胞的生长增殖受到抑制。Taq Man荧光定量RT-PCR结果显示氯丙嗪(chlorpromazine,CPZ)能够抑制EV71 m RNA的表达(Ρ<0.05),制霉菌素(nystatin,NT)对其影响不大(Ρ>0.05)。结论:初步推测EV71入侵SK-N-SH细胞是通过网格蛋白依赖性的内吞作用入胞。

肠道病毒71型(EV71)通过网格蛋白介导的内吞途径入侵人神经母细胞瘤SK-N-SH细胞

目的研究肠道病毒71型(EV71)入侵人神经母细胞瘤SK-N-SH细胞的内吞机制。方法利用氯丙嗪(CPZ)、制霉菌素(NT)分别预处理SK-N-SH细胞,实时定量PCR(qRT-PCR)检测靶细胞中EV71 mRNA水平,免疫荧光细胞化学染色检测靶细胞病毒蛋白1(VP1)蛋白表达水平;EV71感染SK-N-SH细胞后,激光共聚焦显微镜检测EV71与入侵途径标志分子网格蛋白的共定位。结果 CPZ能抑制靶细胞内EV71 mRNA和VP1蛋白的表达,而NT对其无影响;激光共聚焦显微镜发现EV71与网格蛋白共定位。结论 EV71是通过网格蛋白介导的内吞途径入侵人神经母细胞瘤SK-N-SH细胞。

抗EV71病毒人源抗体Fab段表达基因的构建及序列分析

目的从成人外周血淋巴细胞中扩增抗体可变区并构建人源抗体Fab段表达基因,为构建抗EV71病毒人源化单克隆抗体库奠定基础。方法从EV71病毒中和抗体阳性且免疫功能健全的成人全血中,分离淋巴细胞并提取总RNA,经反转录获得cDNA。用一组人IgG Fab基因特异性引物,从合成的cDNA中分别扩增抗体轻、重链可变区,应用重叠PCR技术构建Fab段表达基因,测序分析抗体基因的多样性。结果成功扩增人抗体可变区基因并组装Fab段表达盒,表达盒的长度约为1 500bp,测序分析结果表明,Fab段的多样性可达94.12%。结论成功构建人源抗体Fab段基因,为抗EV71病毒人源性单克隆抗体库的制备奠定了基础。