Reassortant DS‐1‐like G1P[4] Rotavirus A strains generated from co‐circulating strains in Vietnam, 2012/2013

One major mechanism by which Rotavirus A (RVA) evolves is genetic reassortment between strains with different genotype constellations. However, the parental strains of the reassortants generated have seldom been identified. Here, the whole genome of two suspected reassortants, RVA/Human‐wt/VNM/SP127/2013/G1P[4] and RVA/Human‐wt/VNM/SP193/2013/G1P[4], with short RNA electropherotypes were examined by Illumina MiSeq sequencing and their ancestral phylogenies reconstructed. Their genotype constellation, G1‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2, indicated that they were G1 VP7 mono‐reassortants possessing DS‐1‐like genetic backbones. The two strains were ≧99.7% identical across the genome. While their VP7 genes were ≧99.7 identical to that of a Wa‐like strain RVA/Human‐wt/VNM/SP110/2012/G1P[8] which co‐circulated during the 2012/2013 season, 10 genes were ≧99.8% identical to that of the DS‐1‐like strains RVA/Human‐wt/VNM/SP015/2012/G2P[4] (and SP108) that co‐circulated during the season. The identities were consistent with the phylogenetic relationships observed between the genes of the reassortants and those of the afore‐mentioned strains. Consequently, the G1P[4] strains appear to have been generated by genetic reassortment between SP110‐like and SP015‐like strains. In conclusion, this study provides robust molecular evidence for the first time that G1P[4] strains detected in Hanoi Vietnam were generated by inter‐genogroup reassortment between co‐circulating G1P[8] and G2P[4] strains within the same place and season.

     

轮状病毒RNA基因图谱变异分析

用聚丙烯酰胺凝胶电泳法对轮状病毒(RV)标准株进行RNA基因图谱分析,发现人轮状病毒(HRV)Wa株在MA104细胞上连续传5代后,其RNA基因图谱由原来的4:2:3:2模式变为4:3:3:2模式。继续分别在CV-1、MA104细胞上传代后,基因图谱进一步演变为4:3:4:2模式。经比较电泳、病毒空斑纯化,初步证实RVWa有变异林存在。目前尚未见RV标准株变异的报告,有待进一步研究。同时对多个RV标准株及1939年南京市区婴幼儿秋季腹泻的RV流行株进行基因图谱分析,发现不同的人RV及牛RV标准株有相同的基因图谱;不同实验室来源的同一标准株有不同的基因图谱。此外,尚证实HRV是1989年南京市区婴幼儿秋季腹泻的主要病源,总检出率45.1％,电泳长型为流行优势株89.2％。本文尚对RV RNA基因图谱变异的原因及意义进行了讨论。     

人A组轮状病毒北京地方株VP4血清型特异片段编码基因的序列分析

轮状病毒是引起婴幼儿严重腹泻的重要病原,其第四基因编码主要中和抗原VP4,而VP4可裂解为VP8和VP5两个片段。VP8为抗原型特异性片段。克隆并测定了具有代表性的三个轮状病毒北京株VP4编码基因5′端（VPS＋VPS一部分）887个核苷酸序列并据此推导出其氨基酸序列。结果表明,相同血清型的地方株和标准株之间具有高度同源性（92％～966％）,不同血清型间则变异较大（70．5％～71％）。氨基酸最大变异处位于aa84～172,并对胰酶作用位点在致病性中的可能性进行了讨论。     

Synthesis of a ricin toxin B subunit-rotavirus VP7 fusion protein in potato

A gene encoding the outer capsid glycoprotein (VP7) of simian rotavirus SA11, was genetically linked to the amino terminus of the ricin toxin B subunit (RTB) isolated from castor-oil plant (Ricinus communis) seeds. To assess fusion protein expression in plant cells, the VP7::RTB fussion gene was transferred into potato (Solanum tuberosum) cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The fusion gene was detected in transformed potato genomic DNA by polymerase chain reaction DNA amplification methods. Immunoblot analysis with anti-SA11 antiserum as the primary antibody verified the presence of VP7::RTB fusion protein in transformed potato tuber tissues. The plant-synthesized fusion protein bound RTB membrane receptors as measured by asialofetuin-enzyme-linked immunosorbent assay (ELISA). The ELISA results indicated that the VP7::RTB fusion protein was biologically active and made up approx 0.03% of total soluble transformed tuber protein. The biosynthesis of receptor binding VP7::RTB fusion protein in potato tissues demonstrates the feasibility of producing monomeric ricin toxin B subunit adjuvant-virus antigen fusion proteins in crop plants for enhanced immunity.     

A组轮状病毒NSP1基因克隆表达及免疫学性质研究

轮状病毒（rotavirus, RV）非结构蛋白1（non structural protein 1, NSP1）在病毒与宿主的相互作用中发挥着重要的功能。运用基因克隆和表达技术在大肠杆菌中表达了TB-Chen株RV NSP1蛋白,进行了NSP1的免疫学性质和RV感染细胞中NSP1蛋白的合成与分布以及NSP1的系统进化和基因分型研究。结果表明,大肠杆菌BL21(DE3)能高效表达重组NSP1蛋白（rNSP1）,rNSP1表达量约占菌体总蛋白的34.4%。rNSP1能诱导免疫豚鼠产生特异性血清抗体。Western blot及免疫荧光检测结果表明,抗rNSP1血清抗体能特异性识别自身蛋白,对SA11、Wa株的NSP1蛋白有交叉反应性;免疫荧光结果还表明,SA11感染的MA104细胞中合成的NSP1蛋白在细胞质中区域化聚集形成辐射状排列的颗粒状结构,而Wa株的NSP1不能形成此样结构。至今发现的A组RV至少可以分为16个不同的NSP1基因型,TB-Chen株NSP1为A2型。不同基因型有独特的敏感宿主范围,同一基因型可能感染不同种动物,同一种动物也可能感染不同基因型。基因型A4型和A16型仅在鸟类病毒株中出现;而且鸟类中只有A4型和A16型。研究结果为进一步研究NSP1蛋白质的结构功能及其应用开发奠定了很好的基础。     

胶体金法与RT-PCR法测定A群轮状病毒及G血清型分型

目的了解婴幼儿A群轮状病毒腹泻的流行病学特征。方法收集浙江省苍南县人民医院2009年1-12月份的婴幼儿腹泻粪便标本,采用胶体金免疫层析法和逆转录-聚合酶链反应法进行轮状病毒抗原检测和血清型分型,分析G血清型分型。结果 828份婴幼儿腹泻粪便轮状病毒阳性率为33.45%。在轮状病毒腹泻患儿中,1岁以内占49.82%,2岁以内占89.89%。本地区轮状病毒腹泻呈现季节高峰,11月至次年1月为轮状病毒腹泻流行期。毒株分型显示G3型为流行优势株（51.6%）,其次是G1型（26.6%）;另有13.7%为混合感染,包括G1＋G3型（10.5%）、G2＋G3型（2.4%）和G1＋G9型（0.8%）。结论 2009年浙江省苍南县婴幼儿轮状病毒腹泻的主要血清型是G3和G1,G3为优势毒株。 更多还原     

Determinants of the specificity of rotavirus interactions with the alpha2beta1 integrin

The human α2β1 integrin binds collagen and acts as a cellular receptor for rotaviruses and human echovirus 1. These ligands require the inserted (I) domain within the α2 subunit of α2β1 for binding. Previous studies have identified the binding sites for collagen and echovirus 1 in the α2 I domain. We used CHO cells expressing mutated α2β1 to identify amino acids involved in binding to human and animal rotaviruses. Residues where mutation affected rotavirus binding were located in several exposed loops and adjacent regions of the α2 I domain. Binding by all rotaviruses was eliminated by mutations in the activation-responsive αC-α6 and αF helices. This is a novel feature that distinguishes rotavirus from other α2β1 ligands. Mutation of residues that co-ordinate the metal ion (Ser-153, Thr-221, and Glu-256 in α2 and Asp-130 in β1) and nearby amino acids (Ser-154, Gln-215, and Asp-219) also inhibited rotavirus binding. The importance of most of these residues was greatest for binding by human rotaviruses. These mutations inhibit collagen binding to α2β1 (apart from Glu-256) but do not affect echovirus binding. Overall, residues where mutation affected both rotavirus and collagen recognition are located at one side of the metal ion-dependent adhesion site, whereas those important for collagen alone cluster nearby. Mutations eliminating rotavirus and echovirus binding are distinct, consistent with the respective preference of these viruses for activated or inactive α2β1. In contrast, rotavirus and collagen utilize activated α2β1 and show an overlap in α2β1 residues important for binding.     

轮状病毒与细胞表面受体相互作用的分子机制研究

目的轮状病毒是导致婴幼儿重症腹泻的重要病因。轮状病毒感染宿主细胞是一个多因素参与的复杂过程,包括病毒表面两种外壳蛋白与细胞表面唾液酸、整合素、热应激同源蛋白70等多种受体分子的相互作用。就轮状病毒与细胞受体相互作用的分子机制作了简要论述。     

建湖县城区2008—2011年轮状病毒减毒活疫苗接种副反应监测分析

目的了解轮状病毒活疫苗接种后副反应发生的情况,为该疫苗安全接种提供依据。方法对2008—2011年间每年的6月1日到8月31日在建湖县城区免疫规划门诊接种的婴幼儿登记数据进行分析。结果接种5 139人,报告副反应114人,报告率2.218%。报告发生率,一般副反应109例,发生率为2.121%;异常副反应5例,发生率为0.097%。结论接种轮状病毒活疫苗异常副反应发生率低,接种疫苗安全。