整合EVE-NG、GNS3及HCL搭建多厂商网络仿真实验平台

文章针对EVE-NG、GNS3仿真平台主要仿真思科设备,HCL仿真平台只能仿真H3C设备,而实际工程项目往往涵盖多厂商设备的问题,探讨将EVE-NG、GNS3、HCL等网络模拟器通过与本地物理网卡桥接的方法进行整合,搭建集成多厂商设备,可用于实际网络工程项目的网络仿真实验平台。文章不仅给出了上述三款网络模拟器桥接整合的基本原理,还给出了与本地物理网卡的具体桥接过程。测试后证实,整合后的网络仿真实验平台可兼容模拟多厂商网络设备运行。

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.

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）.

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.

Coxsackievirus A6 was the most common enterovirus serotype causing hand,foot,and mouth disease in Shiyan City,central China

BACKGROUND Hand,foot,and mouth disease(HFMD)has become one of the most common infectious diseases in China.Before 2016,the primary causal serotypes were enterovirus A71(EV-A71)and coxsackievirus A16(CV-A16).Following the introduction of EV-A71 vaccines in China since 2016,the situation could change.CV-A6 has recently replaced EV-A71 and CV-A16 in some areas of China.However,the epidemiological characteristics of central China remain unknown.AIM To investigate the clinical symptoms and pathogen spectrum of HFMD in Shiyan City,central China,in recent years.METHODS The epidemiological,clinical,and laboratory data from HFMD cases reported to the Shiyan Center for Disease Control and Prevention between January 2016 and December 2020 were analyzed.196 throat swab specimens were collected from hospitalized HFMD patients between January 2018 and December 2020.To detect and genotype enteroviruses,real-time reverse transcription-polymerase chain reaction and sequencing of the 5'-untranslated region were used.In Shiyan,168 laboratory-confirmed HFMD cases were studied using a logistic regression model to determine the effect of predominant enterovirus serotypes.Based on the logistic regression model,the least absolute shrinkage and selection operator model was used to analyze the correlation between CV-A6 infection and various clinical characteristics in HFMD patients in Shiyan.RESULTS From 2016 to 2020,35840 HFMD cases were reported in Shiyan.The number of cases decreased by 48.4%from 2016 to 2017.Approximately 1.58-fold increases were found in 2018 and 2019 when compared to the previous year,respectively.In 2020,a decrease of about 85.5%was reported when compared to 2019.The most common serotypes shifted from EV-A71 and CV-A16(about 60%-80%in 2016 and 2018)to others(more than 80.0%in 2017,2019,and 2020).EV-A71 lost its dominance in 2017 in Shiyan.Among 196 confirmed HFMD cases,85.7%tested positive for enterovirus,with CV-A6 being the most common serotype(121/168,72.0%).The positive rates for CV-A16 and CVA10 were 4.8%and 3.0%,respectively.There was no EV-A71 discovered.Infection with CV-A6 was linked to fever,myocardial damage,increased creatine kinase MB isoenzyme,and lactate dehydrogenase levels.CONCLUSION CV-A6 was the most common enterovirus serotype in Shiyan City,replacing EV-A71 and CV-A16 as the HFMD pathogen.Developing vaccines against CV-A6 or multiple pathogens,as well as rising CV-A6 surveillance,will help prevent HFMD in central China.

Evolutionary pattern of 5'-UTR of enteroviruses and primer update for the detection of enteroviral RNA in environmental samples

Objective:To study the recombination events among enterovirus strains and the development of specific primers for the detection of enteroviruses in environmental samples.Methods: Nucleotide sequence analysis of enteroviruses deposited in the international database GenBank （www.ncbi.nlm.nih.gov/Genbank） was conducted to develop specific primers for the detection of these viruses.The specificity and sensitivity of the method were tested using coxackicvirus B3 strain Nancy,environmental isolate of human hepatitis A virus and human rotavirus strain WA.Seventy sewage samples were analyzed.Results:Enterovirus genome was detected in all positive samples.The genome of enterovirus was not detected in negative samples.The level of detection of these viruses was 102 TCID50mI..Conclusions:The development of new primers is an important issue for the detection of enteroviruses in the environment and the assessment of risk factors to human health.

潮州市儿童手足口病EV71疫苗犹豫及影响因素分析

目的调研潮州市6月龄~5岁儿童EV71疫苗接种的现状,围绕儿童家长对EV71疫苗的疫苗犹豫、认知情况,分析其影响因素,为改善EV71疫苗接种现状提供建议。方法于2023年5-7月在潮州市每个县区(湘桥区、潮安区、饶平县)各选取2个卫生院(社区卫生服务中心)进行整群抽样。使用调查问卷的方式,对社区卫生服务中心周边儿童家长资料的收集,包括家长基本信息、疫苗犹豫、疫苗认知情况等。结果发放问卷共487份,有效回收453份。调查数据显示,接受调查的家长中有45.92%已接种EV71疫苗。Logistic回归分析显示,家长存在一定的疫苗犹豫,主要在于疫苗安全性及有效性方面。家庭年收入>20万元的、受教育水平在大专/本科的、居住地在城镇的家长犹豫得分较低。而疫苗犹豫得分低的更倾向为孩子接种EV71疫苗,疫苗认知水平得分高的家长更倾向孩子接种。结论政府相关部门应保证EV71疫苗的质量和安全性;定期进行培训和考核疫苗接种人员;加大手足口病和EV71疫苗的宣传科普力度,特别是疫苗安全性和有效性的相关科普,树立疫苗接种的信心。

Chronic Enterovirus D68 Bronchiolitis Causing Severe Respiratory Insufficiency

Human enteroviruses are less well-known causes of acute bronchiolitis. In recent years, Enterovirus D68 [EV D68] has emerged as significant cause of epidemic viral bronchiolitis and pneumonia in the United States and other countries. Chronic bronchiolitis has not been previously attributed to EV D68. We documented EV D68 in open lung biopsies of a young adult patient who was frequently admitted to the hospital for severe exacerbation of respiratory infections and subsequently developed progressive respiratory insufficiency. The difficulty of diagnosis and potential economic impact of this illness is discussed.

吉利帝豪EV450低压无法上电故障诊断与排除

随着环保意识的不断提高和新能源技术的迅速发展,电动汽车在市场上的份额逐渐增加。吉利帝豪EV450作为一款热门的电动汽车,其低压系统的正常上电是车辆正常运行的关键。本文将详细介绍吉利帝豪EV450低压无法上电的故障诊断与排除方法,通过对故障现象的分析、可能原因的探讨以及诊断步骤的详细阐述,为维修人员提供一定的参考。

Distribution Pattern of the Non Polio Enterovirus (NPEV) Rate in Children with Acute Flaccid Paralysis Reported to the Surveillance System in Nigeria 2010-2015

Objective: This study focuses on the reverse cold chain system of stool sample management from cases reported to the AFP surveillance system between 2010 and 2015. Through the distribution of their NPEV isolation rates, we identify lapses in the reverse cold chain management and provide recommendations that should help improve the reverse cold chain system and the AFP surveillance system in general as Nigeria once again matches towards a polio free certification. Methods: A descriptive retrospective study was conducted using AFP surveillance data routinely collected between January 2010 and December 2015 by the Disease Surveillance and Notification Officers (DSNOs) in Nigeria and the WHO accredited Polio Laboratories. All AFP cases reported to the Disease Surveillance network during this period from all the states were included in the study. EPIINFO—veritable customized software was used to run queries on the access database and obtain the specific data sets required. Results: A total of 52,879 AFP cases were reported from 2010-2015, in which 7288 non-polio enteroviruses were isolated. NPEV isolation rate ranged from 10.8% in the southeast to 20.3% in the northeast with the states in the northern geopolitical zones having higher NPEV rates in comparison to the states in the southern geopolitical zones. The WHO Polio laboratory in Ibadan serves twenty-seven states in the country;the average non-polio AFP rate among states served by this laboratory is 11.7% from a total of 4012 AFP cases recorded. The WHO Polio laboratory in Maiduguri recorded 3276 with an average non-polio AFP rate of 19.2%. Conclusion: Though the country’s NPEV isolation rate remain slightly higher above the 10% recommended by WHO, steps should be taken to enhance the reverse cold-chain system particularly in the southern states. This would increase confidence in the AFP surveillance system in Nigeria as she proceeds once again towards complete polio eradication and meeting certification standards.

灰树花水溶物对EV71病毒的抑制作用

【目的】探讨灰树花水溶物(GFWE)的化学成分及其对EV71病毒的抑制作用,为促进灰树花综合利用、提高其经济附加值,以及开发预防和治疗EV71病毒感染的功能性食品提供依据。【方法】采用色谱法测定了GFWE的主要化学成分组成、多糖组成和分子质量。基于体外细胞培养采用CCK-8法确定了GFWE的安全上样量;以EV71病毒感染Vero细胞为模型,探究GFWE对EV71病毒的抑制率及对Vero细胞形态的影响,并采用荧光定量PCR检测了EV71病毒衣壳蛋白(VP1)编码基因mRNA的表达水平,初步评价了GFWE对EV71病毒的抑制作用。【结果】GFWE主要由低分子质量多糖、氨基酸、肽及其衍生物组成。GFWE富含的13种氨基酸中包含7种必需氨基酸;其多糖由葡萄糖、半乳糖、盐酸氨基葡萄糖和古罗糖醛酸4种单糖组成,物质的量比为0.907∶0.038∶0.029∶0.026,重均分子质量分别为15.67、10.10、6.60 ku和<1.00 ku。GFWE表现出较高的抗EV71病毒活性,100、200μg·mL^(-1) GFWE对EV71病毒的抑制率分别为91.32%、91.83%,25~400μg·mL^(-1) GFWE能显著降低EV71 VP1 mRNA的表达水平。【结论】GFWE主要由低分子质量多糖、氨基酸、肽及其衍生物组成,具有较高的抗EV71病毒活性,能够抑制感染EV71病毒的Vero细胞中VP1的合成。

2020款比亚迪秦EV高压互锁结构原理及故障诊断

介绍2020款比亚迪秦EV高压互锁结构和原理,基于结构和原理,分析和排除实车故障,分享整体检修思路和过程,以便体现掌握高压互锁结构和原理对故障检修的重要性。

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.

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).

Clinical and neuroimaging features of enterovirus71 related acute flaccid paralysis in patients with hand-foot-mouth disease

Objective:To investigate clinical and neuroimaging features of enterovirus71(EV71) related acute flaccid paralysis in patients with hand-fool-mouth disease.Methods:Nine patients with acute flaccid paralysis met the criterion of EV71 induced hand-foot-mouth disease underwent spinal and brain MR imaging from May 2008 to Sep 2012.Results:One extremity flaccid was found in four cases(3 with lower limb,1 with upper limb),two limbs flaccid in three cases(2 with lower limbs,1 with upper limbs),and four limbs flaccid in two cases.Spinal MRI studies showed lesion with high signal in T2-weighted images(T2WI) and low signal T1-weighted images(T1WI) in the spinal cord of all nine cases,and the lesions were mainly in bilateral and unilateral anterior hom of cervical spinal cord and spinal cord below thoracic 9(T9) level.In addition,the midbrain,pons, and medulla,which were involved in 3 cases with brainstem encephalitis,demonstrated abnormal signal.Moreover,spinal cord contrast MRI studies showed mild enhancement in corresponding anterior hom of the involved side,and strong enhancement in its ventral root.Conclusions: EV71 related acute flaccid paralysis in patients with hand-foot-mouth disease mainly affected the anterior hom regions and ventral root of cervical spinal cord and spinal cord below T9 level. MR imaging could efficiendy show the characteristic pattern and extent of the lesions which correlated well with the clinical features.

秦EV轿车无钥匙启动失效故障诊断与排除

随着新能源汽车的渗透率不断升高,售后维修的压力随之增大。文章以比亚迪秦EV为例,阐述了无钥匙进入系统和无钥匙启动的工作原理,引入秦EV无钥匙启动系统组合故障案例,在诊断过程中,从故障现象、故障原因分析、维修资料查阅、诊断测试、故障机理五个方面详细的介绍了组合故障的排除过程。结果表明,该诊断思路能够快速排除故障,提高效率,可为职业院校师生和售后维修从业人员在维修教学过程提供借鉴意义。

血清25-羟基维生素D与EV71感染手足口病患儿Th1/Th2细胞因子及病情进展关系研究

目的血清25-羟基维生素D[25-(OH)D]与肠道病毒71型(EV71)感染手足口病患儿辅助性T(Th)1/Th2细胞因子及病情进展的关系研究。方法选择2020年10月至2023年10月在我院就诊的135例EV71感染手足口病患儿作为研究对象,依据病情严重程度分为普通型组(n=95)、重型组(n=40),对比两组血清25-(OH)D、Th1/Th2细胞因子[血清白细胞介素-2(IL-2)、肿瘤坏死因子-α(TNF-α)、血清IL-6]水平,Pearson相关性分析血清25-(OH)D、Th1/Th2细胞因子的关系,多因素Logistic回归模型探讨血清25-(OH)D与病情进展的关系。结果两组血清25-(OH)D水平比较,重型组更低,血清IL-2、TNF-α、IL-6水平比较,重型组更高(P<0.05);血清25-(OH)D与IL-2/TNF-α/IL-6水平负相关(r=-0.344、-0.344、-0.424,P均<0.05);重型组血清25-(OH)D、体液免疫IgG水平及个人卫生习惯良好比例低于普通型组,手足口病病史与白细胞(中性粒细胞)计数升高比例高于普通型组(P<0.05);血清25-(OH)D为患儿病情进展的保护因素(P<0.05),手足口病史、CRP升高为患儿病情进展独立危险因素(P<0.05)。结论血清25-(OH)D与EV71感染手足口病患儿IL-2、TNF-α、IL-6水平均呈负相关,且血清25-(OH)D是EV71感染手足口病患儿病情进展的保护因素,应重视对患儿血清25-(OH)D水平的动态追踪并合理补充维生素D。

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.

Expression,purification and characterization of enterovirus-71 virus-like particles

AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.