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.

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.

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.

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.

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.

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.

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.

A one-step reverse-transcription recombinase aided PCR assay for the rapid and sensitive detection of human enteroviruses

Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR),the gold-standard method,still has shortfalls in diagnostic sensitivity and timeliness.Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay(RT-RAP)to detect HEV fragment within an hour.The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains.Among 15 types of HEV(species A-C),the sensitivity of RT-RAP was approximately 2-8 folds lower than that of the qRT-PCR in 9 types,and no-cross reaction with other viruses was observed.RT-RAP was further applied to analyze CSF and fecal specimens;the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results.These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.

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。

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。

人在环路模拟驾驶仿真实验系统研发

汽车是人机协同控制的复杂系统,基于NI PXIe8108实时控制器、PXI7842R数据采集卡、模拟驾驶输入设备及相关软硬件搭建了驾驶模拟仿真系统;用LabVIEW编程语言建立了车辆动力学模型;用虚拟引擎Unity3D软件开发了视景系统;并通过数据库技术使仿真过程与视景系统相结合,实现仿真过程与三维动画实时视景系统的数据交互。结果表明:通过人在环路模拟驾驶仿真实验,使仿真过程中引入了驾驶模拟操纵,实现了人在环路的人机交互式仿真,得到汽车相关性能仿真结果,为驾驶过程中人和汽车协同控制仿真和极端工况下的仿真,提供了一种安全、可靠的人车协同控制仿真环境。

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。

缺氧诱导因子-1在病毒感染中的作用

缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)是一种由β亚单位和α亚单位组成的异二聚体转录因子,其表达产物参与细胞的许多生理过程。越来越多的研究提示HIF-1在病毒感染中发挥着重要作用。通过检索相关文献,对人病毒感染中HIF-1活性改变及其在病毒感染中的作用进行了综述,为研究HIF-1在病毒感染中的作用提供参考。

戊型肝炎病毒IgG型抗体水平的检测

应用ＥＬＩＳＡ抗体滴度测定法对２０例含戊型肝炎病毒ＩｇＧ型抗体（抗－ＨＥＶ－ＩｇＧ）阳性的健康献血员及５０例急性戊型病毒性肝炎（急性戊肝）病人血清进行了抗－ＨＥＶ－ＩｇＧ水平的检测。结果显示：健康献血员与急性戊肝病人血清抗－ＨＥＶ－ＩｇＧ几何平均滴度分别为１２２和１１２１（Ｐ＜０．０１）。当抗－ＨＥＶＩｇＧ抗体滴度＞１４０时，对诊断急性戊肝有重要的价值。

慢性戊型肝炎的研究动态

器官移植接受者戊肝病毒的感染是一个新发并引起关注的问题。在免疫力正常的人群中,戊肝病毒的感染通常表现为急性自限性肝炎,但是,目前有研究报道在器官移植接受者及免疫缺陷人群,包括人类免疫缺陷病毒感染者中,HEV感染可转为慢性。在这些患者中,戊肝病毒感染可能更为严重,甚至发展为肝硬化,最终导致移植失败。在免疫抑制人群中,HEV感染的诊断主要依赖于HEV RNA的检测。

人肠道病毒复制子的研究进展

人肠道病毒(human enteroviruses,HEVs)感染人类并造成严重的健康问题。目前缺乏针对HEVs的特异性抗病毒药物,且预防HEVs感染的疫苗的种类有限。HEVs的基因组结构简单且保守,故基于病毒基因组结构的病毒复制子已成为研究HEVs的重要工具,可用于病毒的分子生物学特性、病毒与宿主的关系及抗病毒药物的筛选及疫苗的研发等方面。现就HEVs复制子的研究进展作一综述。

Zoonotic transmission of hepatitis E:Implications for public health worldwide

Since the year 1982,the Hepatitis E virus(HEV),enteric transmission,has been widely spread in many tropical and subtropical countries.HEV has multiple routes of transmission,such as food or water contaminated with faecal matter,person-to-person transmission or by the ingestion of raw or undercooked shellfish.It is important to consider the significance of zoonotic infections propagation,both in endemic areas as well as in developed countries,where there have been some sporadic cases.This article summarizes the research conducted to date,which supports the zoonotic transmission of HEV,its risks and how they are often underestimated.

Dynamic change of mother-source neutralizing antibodies against enterovirus 71 and coxsackievirus A16 in infants

Background Enterovirus 71 （EV71） and coxsackievirus A16 （Cox A16） are major causative agents for hand, foot and mouth disease （HFMD）. Studies indicate that the frequent HFMD outbreaks result in a few hundreds children＇s death in China in recent years. The vaccine and other research for HFMD need to be developed urgently. The aims of our study were： to explore dynamic development of mother-source neutralizing antibodies against EV71 and Cox A16 in infants from Jiangsu Province, China, and to provide the fundamental data for further establishing of corresponding immunization course. Methods Peripheral blood samples were collected from 133 of parturient women once immediately before delivery and their infants at two and seven months of age. Method of micro-dose cytopathogenic effect was used to measure neutralizing antibodies against EV71 and Cox A16, respectively. Results Seropositive rates of anti-EV71 and anti-Cox A16 in prenatal women were 79.7% （106/133） and 92.5% （123/133）, respectively; geometric mean titers （GMTs） were 29.0 and 61.9; 75.9% （101/133） prenatal women were both positive in anti-EV71 and anti-Cox A16; seropositive rates of anti-EV71 and anti-Cox A16 were 25.6% （34/133） and 38.3% （51/133） in infants at two months of age; GMTs were 12.3 and 18.0, respectively. GMTs of anti-EV71 were significantly higher for infants at seven months （82.6） compared with that at two months （P 〈0.05）, showing infants had inapparently infected by EV71 during two to seven months. Although only one offspring （0.75%） at seven months was found having anti-Cox A16 transfered from maternal, this observation suggested no maternal antibody may remain in infants at seven months. Conclusions The prevalence of EV71 and Cox A16 were relatively high in Jiangsu Province. Bivalent vaccine against both EV71 and Cox A16 should be developed, and the ideal time point for prime immunization for infants is around 2-5 months of age.

Pattern Classification of Enterovirus 71-Associated Hand, Foot, and Mouth Disease in Chinese Medicine: A Retrospective Study in 433 Cases

Objective： To determine whether patterns of enterovirus 71（EV71）-associated hand, foot, and mouth disease（HFMD） were classified based on symptoms and signs, and explore whether individual characteristics were correlated with membership in particular pattern. Methods： Symptom-based latent class analysis（LCA） was used to determine whether patterns of EV71-HFMD existed in a sample of 433 cases from a clinical data warehouse system. Logistic regression was then performed to explore whether demographic, and laboratory data were associated with pattern membership. Results： LCA demonstrated a two-subgroup solution with an optimal fit, deduced according to the Bayesian Information Criterion minima. Hot pattern（59.1% of all patients） was characterized by a very high fever and high endorsement rates for classical HFMD symptoms（i.e., rash on the extremities, blisters, and oral mucosa lesions）. Non-hot pattern（40.9% of all patients） was characterized by classical HFMD symptoms. The multiple logistic regression results suggest that white blood cell counts and aspartate transaminase were positively correlated with the hot pattern（adjust odds ratio=1.07, 95% confidence interval： 1.006–1.115; adjust odds ratio=1.051, 95% confidence interval： 1.019–1.084; respectively）. Conclusions： LCA on reported symptoms and signs in a retrospective study allowed different subgroups with meaningful clinical correlates to be defined. These findings provide evidence for targeted prevention and treatment interventions.