重庆市2010年手足口病流行病学特征

目的分析重庆市2010年手足口病流行特征,为手足口病的防治提供科学依据。方法下载＂疾病监测信息报告管理系统＂疫情数据,描述病例流行病学特征,收集重庆市手足口病监测资料,分析死亡病例流行特征及实验室检测结果。结果 2010年报告病例19970例,重症42例,死亡26例,发病率为69.85/10万,死亡率为0.091/10万。4-5月为发病高峰,主城区比远郊区县高发,1～3岁组的散居儿童为高发人群。0～3岁男性散居儿童为手足口病死亡的高危人群,临床表现以高热、手足部出疹为主,伴有不同程度的神经、呼吸、循环、消化系统症状和体征。聚集性病例多发生在学校和幼托机构。荧光PCR检测阳性率为63.26%,轻症病例以柯萨奇病毒A16型为主（39.09%）,但引起死亡的均为肠道病毒71型,病毒分离率为24.30%。结论手足口病疫情的防控重点应放在及时发现重症病例,加强重症病例的临床就治,降低死亡率等方面。     

Infection of primary human tonsillar lymphoid cells by KSHV reveals frequent but abortive infection of T cells

Insertion of nucleotide sequences encoding “tags” that can be expressed in specific viral proteins during an infection is a useful strategy for purifying viral proteins and their functional complexes from infected cells and/or for visualizing the dynamics of their subcellular location over time. To identify regions in the poliovirus polyprotein that could potentially accommodate insertion of tags, transposon-mediated insertion mutagenesis was applied to the entire nonstructural protein-coding region of the poliovirus genome, followed by selection of genomes capable of generating infectious, viable viruses. This procedure allowed us to identify at least one site in each viral nonstructural protein, except protein 2C, in which a minimum of five amino acids could be inserted. The distribution of these sites is analyzed from the perspective of their protein structural context and from the perspective of virus evolution.     

Neonatal rhesus monkey is a potential animal model for studying pathogenesis of EV71 infection

Data from limited autopsies of human patients demonstrate that pathological changes in EV71-infected fatal cases are principally characterized by clear inflammatory lesions in different parts of the CNS; nearly identical changes were found in murine, cynomolgus and rhesus monkey studies which provide evidence of using animal models to investigate the mechanisms of EV71 pathogenesis. Our work uses neonatal rhesus monkeys to investigate a possible model of EV71 pathogenesis and concludes that this model could be applied to provide objective indicators which include clinical manifestations, virus dynamic distribution and pathological changes for observation and evaluation in interpreting the complete process of EV71 infection. This induced systemic infection and other collected indicators in neonatal monkeys could be repeated; the transmission appears to involve infecting new monkeys by contact with feces of infected animals. All data presented suggest that the neonatal rhesus monkey model could shed light on EV71 infection process and pathogenesis.     

Nuclear body formation and PML body remodeling by the human cytomegalovirus protein UL35

The human cytomegalovirus (HCMV) UL35 gene encodes two proteins, UL35 and UL35a. Expression of UL35 in transfected cells results in the formation of UL35 nuclear bodies that associate with promyelocytic leukemia (PML) protein. PML forms the basis for PML nuclear bodies that are important for suppressing viral lytic gene expression. Given the important relationship between PML and viral infection, we have further investigated the association of UL35 with PML bodies. We demonstrate that UL35 bodies form independently of PML and subsequently recruit PML, Sp100 and Daxx. In contrast, UL35a did not form bodies; however, it could bind UL35 and inhibit the formation of UL35 bodies. The HCMV tegument protein pp71 promoted the formation of UL35 bodies and the cytoplasmic localization of UL35a. Similarly, UL35a shifted pp71 to the cytoplasm. These results indicate that the interplay between UL35, UL35a and pp71 affects their subcellular localization and likely their functions throughout infection.     

Introduction of a novel parechovirus RT-PCR clinical test in a regional medical center

BackgroundHuman enterovirus 71 (EV-71) emerged as a significant pathogen able to cause large outbreaks involving severe neurological cases and children fatalities in Asia.ObjectivesTo describe epidemiology of EV-71 infections in France.Study designFifty-nine patients admitted in 12 different hospitals from 1994 to 2009 were included. The entire VP1 coding gene of 58 EV-71 strains was sequenced and phylogenetic analyses were performed to assign strains to genogroups/subgenogroups and to compare French isolates to European and worldwide isolates.ResultsThe median age of the patients was 1.04 years (9 days to 7 years). Among 46 documented EV-71 infections, 39 were self-limited. Seven children developed severe sepsis-like, respiratory or neurological complications. Among them, 2 children died from acute respiratory distress syndrome. All the EV-71 strains belonged to genogroup C: 31 isolates belonged to subgenogroup C1, 26 to subgenogroup C2 and 1 to subgenogroup C4. All the strains were genetically related to other European strains isolated at the same period of time. Although C1 isolates were predominant between 1994 and 2005, C2 strains have been predominant since 2007. No association was found between any genotype and the age or the clinical symptoms.ConclusionsThe C4 subgenogroup, which was associated with large outbreaks in China, did not spread in France. It is important to monitor more carefully the EV-71 strains circulating in France to detect the introduction of new genetic variants that could be associated with major outbreaks.     

中国大陆EV71病毒分离株的分子流行病学研究

目的了解1987-2010年中国大陆肠道病毒71型（EV71）分离株的分子流行病学特点及其种系进化、基因分型和遗传变异性。方法从GenBank/NCBI上获得中国大陆来源的具有完整VP1或近似完整VP1基因的核苷酸序列信息的413株EV71毒株进行分析,采用MEGA5.0软件,构建系统进化树,计算相同或不同基因型及基因亚型毒株的核苷酸与氨基酸的相似性。结果 1987-2010年,中国大陆20个省、市或地区均分离到具有完整VP1序列的毒株,且2008年以来数量陡增;中国大陆流行的主要是C型,只有2008年安徽和2009年湖北发现了A型;各基因型在43、58、142、164、167、184、240、249、292等氨基酸位点发生了特异性变异;从健康人体内分离的HQ129932毒株与其他序列比较,氨基酸无特异性变异。结论 C型株可能具有更强的传染力;氨基酸位点变异对于EV71病毒进化有重要意义;VP1基因与疾病的严重程度无明显关联;应加强C4a亚型疫苗候选疫苗株对其他基因型毒株的交叉保护作用研究。     

广州市2008-2011年手足口病流行特征动态研究

目的分析2008年5月至2011年7月广州市手足口病的流行病学特征,以了解流行现状,探讨流行趋势,为制定有效的防控策略奠定基础。方法采用描述性流行病学方法分析2008-2011年广州市手足口病疫情资料的流行特征。手足口病的肛拭子标本从哨点医院获得,采用RT-PCR方法进行病毒核酸检测。结果疫情暴发月份提前,流行强度增大,高位持续时间延长;市区病例构成从2008年下半年的48.32%逐步降至2011年上半年的35.09%,农村地区则从2008年的11.95%逐步升至24.67%,城乡结合部相对稳定在40.30%左右;病例主要在5岁以下婴幼儿,但呈现出低龄化、成人化的两极化发展趋势,男性高于女性;散居儿童病例数构成比从2008年下半年的60.04%增至2011年的68.92%,托幼儿童则从2008年的38.09%降至2011年的28.27%,学生病例2009年以来相对稳定在2.55%左右;肠道病毒71型（EV71）和柯萨奇病毒A组16型（CoxA16）呈现交替流行的现象。结论广州市手足口病疫情不断向低龄儿童、散居儿童和农村地区扩散,同时不同型别的毒株交替出现。手足口病疫情形势严峻,防控难度加大。     

Human enterovirus D68 in clinical and sewage samples in Israel

BackgroundSince mid-August 2014, North America experienced a wide outbreak of Enterovirus D68 (EV-D68) associated with severe respiratory illness in children. Several other countries also reported cases of EV-D68 in 2014.ObjectivesThe aim of this study was to determine whether EV-D68 circulated in Israel in 2014, caused severe respiratory illness in children and was the causative agent of Acute Flaccid Paralysis.Study designArchived clinical respiratory samples from a cohort of 710 hospitalized pediatric patient’s (<10 years old) with respiratory illness were screened for clade B specific EV-D68 by real-time PCR. The patients were seen at four medical centers covering the entire country between August and November 2014. We also evaluated 49 patient stool samples from 26 AFP cases during 2014 for presence of EV-D68. In addition, RNA from sewage samples collected throughout Israel during the same study period was also tested for EV-D68. Partial VP1 sequencing was performed on all positive samples.ResultsOf the 710 clinical samples evaluated, 7 (1%) were positive for EV-D68. Two patients were from the central part of Israel, while the rest was from the southern part. The majority of the patients did not have any underlying disease. Not only that, but, none of the 26 suspected AFP cases had EV-D68 nucleic acid in their stool samples. EV-D68 RNA was detected in 9 out of 93 sewage samples, mainly from Southern Israel. Sequence analysis of EV-D68 VP1 gene from both sewage and clinical samples indicated that the Israeli EV-D68 RNA belonged to Clade B which was genetically similar to 2014 circulating European and North American EV-D68 virus.ConclusionsEV-D68 circulated in Israel during the 2014 summer-fall season and caused hospitalization of a small percent of the patients with respiratory illness.     

肠道病毒71型感染人神经母细胞瘤细胞(SH-SY5Y)的miRNA表达谱

目的 研究肠道病毒71型(EV71)感染神经细胞的miRNA表达谱,探讨miRNA在病毒感染神经细胞中的可能作用.方法 建立EV71感染人神经母细胞瘤细胞(SH-SY5Y)模型,收集感染后48 h细胞.以Taqman低密度芯片检测miRNA表达谱,使用实时RT-PCR对芯片结果进行验证并在TargetScan和miRanda网站预测靶基因,采用GO和KEGG分析靶基因功能.结果 成功建立EV71感染SH-SY5Y细胞模型,通过低密度芯片筛选出215种显著升高的miRNA和25种显著下调的miRNA.经过RT-PCR验证,3种miRNA(MiR-10a*、miR-15b*和miR-195)显著下调,7种miRNA(miR-10a、miR-342-5p、miR-483-5p、Let-7b、miR-99a、miR-140-5p和miR-21)显著上调,与芯片结果相符.GO分析显示发展进程和信号调节条目最富集靶基因.KEGG路径分析显示靶基因在肿瘤路径、蛋白水解、Wnt信号传导、黑素形成、粘附连接、MAPK信号通道最富集.结论 EV71感染神经细胞48 h后miRNA表达谱发生改变,10种变化的miRNA靶基因预测在发展进程、信号传导及凋亡中起着重要的作用,可为后期机制研究提供参考.     

肠道病毒-D68 VP1蛋白原核表达、纯化及抗原性的评价

目的 原核表达EV-D68结构蛋白VP1,并对其抗原性进行评价.方法 以肠道病毒68型P1基因为模板,设计引物扩增出目的片段VP1,将其连接至大肠杆菌表达载体pGEX-5X-1上.将重组表达载体pGEX-5X-1-VP1转化大肠杆菌表达菌株Rosetta(DE3)pLysS,对该工程菌进行诱导表达.菌体裂解后用SDS-PAGE电泳、Western-blot鉴定融合蛋白;对表达菌体进行超声破碎和亲和层析纯化;使用酶联免疫吸附试验方法评估目的蛋白与不同种属EV-D68阳性血清的结合能力及亲和力.结果 成功构建融合表达载体pGEX-5X-1-VP1,经过双酶切及测序鉴定均正确;成功表达和纯化出EV-D68(En-terovirus D68,EV-D68)VP1融合蛋白,经过聚丙烯酰胺凝胶电泳测定,得到与预期蛋白相对分子质量为61000大小一致的目的条带;纯化蛋白与不同种属的阳性血清可以特异性结合,与兔血清、恒河猴血清、小鼠血清的亲和力常数分别为1.6×106、3.9×105、3.1×106.结论 成功表达了EV-D68 VP1融合蛋白,为研究EV-D68 VP1蛋白的结构、功能及中和抗体筛选奠定基础.