重庆地区儿童病毒性腹泻病原分子流行病学研究

发布时间：2018-04-13 14:42

本文选题：病毒性腹泻 + 基因型　；参考：《重庆医科大学》2014年博士论文

【摘要】：急性胃肠炎引起的腹泻病仍然是儿童时期，特别是3岁以内婴幼儿常见的疾病。而病毒性腹泻在其中占有重要的地位。除轮状病毒外，诺如病毒、扎如病毒、肠道腺病毒和星状病毒引起的腹泻也较为常见。因此研究上述四种病毒型腹泻的发病规律、临床表现、流行病学特点，对该病诊断和治疗以及疫苗研发都具有重要的意义。本课题通过三部分的研究，初步阐明重庆地区NoV、SaV、EAdV、AstV四种病毒的分子流行病学特点、规律，完善重庆市儿童腹泻的流行病学资料，为该病的预防控制提供科学资料和依据。 1.四种病毒的流行病学研究方法：收集2011年12月至2012年11月期间重庆医科大学附属儿童医院门诊临床疑诊病毒性腹泻病患儿大便标本560例，用RT-PCR技术检测诺如病毒、扎如病毒、肠道腺病毒和星状病毒基因组核苷酸片段，测序基因分型后建立进化树。结果：收集的560份大便标本，男女比例为1.43:1，患儿年龄从22天到12.5岁。RT-PCR检测结果示诺如病毒阳性率为21.25%(119/560)，扎如病毒阳性率为2.68%(15/560)，肠道腺病毒阳性率为5.17%(29/560)，星状病毒阳性率为1.25%(7/560)。基因分型研究显示诺如病毒以GII.42006b为优势株，但新的变异株GII.4Sydney株也有发现，其次为GII.61.25%(7/560)，GII.30.71%(4/560)，GII.20.36%(2/560)，GII.70.36%(2/560)，GII.120.54%(3/560)，GII.140.36%(2/560)，GI.10.18%(1/560)，GI.30.54%(3/560)。扎如病毒以GI.1为优势株，占1.43%（8/560），其后依次为GII.10.54%(3/560),GI.20.18%(1/560),GI.40.18%(1/560),GIV.10.36%(2/560)。肠道腺病毒优势流行株为G41型，占3.21%（18/560）,此外G310.71%（4/560），G3与G12各一株0.18(1/560)，G70.54%(3/560)，G20.36%(2/560)。星状病毒优势株为HastV-1型，占0.71%(4/560)，其后为HastV-40.36%(2/560)，HastV-20.18%(1/560)。本次研究中共发现8例混合感染患儿。各病毒感染高峰期为秋冬季为主，男女性别无差异。结论：重庆地区儿童四种病毒性腹泻中以诺如病毒感染为主，GII.4为优势株，但新型变异株GII.4Sydney已经出现。其次为肠道腺病毒感染，以G41型为主。扎如病毒感染以GI.1为主，星状病毒以HAstV-1型为主。病毒性腹泻仍以秋冬季为发病高峰，男女发病无性别差异。 2.诺如病毒的重组分析方法：在119份诺如病毒阳性标本中随机抽取28份，以RT-PCR技术扩增病毒ORF1区衣壳蛋白编码序列，并测序确定基因分型。与基于RdRp区的分型结果比较，两次分型结果不一致的标本为可疑重组病毒株。扩增包括RdRp区到VP1区部分序列的长片段，测序后将结果输入重组分析软件Simplot和RDP分析，寻找可能的重组事件及重组位点，排除标本混合感染的干扰。结果：抽取的28份标本中，有两份标本两次分型结果不一致。CQ242基于RdRp区序列分型为GII.3，基于VP1区序列分型为GII.12；CQ426基于RdRp区序列分型为GII.4，基于VP1区序列分型为GII.6。将两份标本进行长片段扩增，CQ426测序失败，无法判定。CQ242测序成功，确定为重组株，重组类型为GII.3/GII.12。结论：在重庆地区诺如病毒感染性腹泻中，存在病毒重组的情况，重组类型为GII.3/GII.12，属于基因型间重组。因重组会导致病毒毒力、感染性、理化性质等方面的变化，须密切注意，警惕诺如病毒性腹泻爆发流行的可能。 3.诺如病毒衣壳蛋白的生物信息学分析方法：从NCBI的Protein数据库中下载重庆地区诺如病毒流行株GII.42006b的衣壳蛋白氨基酸序列AGS41450。通过瑞士生物信息学研究所的专业蛋白质分析系统(Expert Protein Analysis System，，ExPASy)，分析蛋白质的一级结构理化性质，二级结构的组成，并利用同源建模技术建立蛋白质的三维模型结果，预测该蛋白B细胞抗原表位。结果：诺如病毒GII.42006b衣壳蛋白由540个aa残基组成，总分子量58991.5，等电点为5.53，负电荷残基数47，正电荷残基数35。蛋白质半衰期约30小时，不稳定系数为45.3，脂肪系数为78.15，总平均亲水性为-0.195，为极弱性亲水蛋白。主要的跨膜段为107-124位aa残基，1-106位置的部分为膜内段，125-540为膜外段。同源建模模型显示531-540位aa残基突出伸向外部，且抗原性最强。B细胞表位预测结果同样显示此十位aa残基正是可能的B细胞表位位点。531-540位aa对应的核苷酸序列与NoV全序列比对，定位于全序列的6677-6707位核苷酸上，正好处于衣壳蛋白P区编码序列。结论：诺如病毒GII.42006b衣壳蛋白VP1的抗原性位点是第531-540位aa组成，对应的核苷酸编码序列为6677-6707，同源建模蛋白三维结构显示该段aa突起伸向外部，与电镜下NoV P区突起部位吻合。
[Abstract]:Diarrhoea caused by acute gastroenteritis is still a common disease in children , especially within 3 years . Viral diarrhea plays an important role in the pathogenesis of viral diarrhea . In addition to rotavirus , it is very important to study the pathogenesis , clinical manifestation and epidemiology of four kinds of virus diarrhea .

1 . Epidemiological study of four viruses

Methods : 560 patients with viral diarrhea were collected from December 2011 to November 2012 in Chongqing Medical University Affiliated to Chongqing Medical University . Using RT - PCR technique , we detected the nucleotide fragment of virus , ligation , adenovirus and star virus , and established the phylogenetic tree after sequencing .

Results : The positive rate of GII . 42006b was 21.25 % ( 1 / 560 ) , GII . 30.54 % ( 2 / 560 ) , GII . 20.18 % ( 1 / 560 ) , GII . 20.36 % ( 2 / 560 ) , GII . 70.36 % ( 2 / 560 ) , GII . 20.18 % ( 1 / 560 ) , GII . 30.54 % ( 2 / 560 ) , GII . 20.18 % ( 1 / 560 ) , GII . 30.54 % ( 2 / 560 ) , GII . 20.18 % ( 1 / 560 ) , GII .

Conclusion : The infection of GII . 4 is dominant in four kinds of viral diarrhea of children in Chongqing , but the new mutant GII . 4Sydney has appeared . Second , the infection of adenovirus type GII . 4Sydney is the main type of G41 type . The virus infection is mainly in the form of G41 . The virus diarrhea still takes HAstV - 1 type as the main . Viral diarrhea still takes autumn and winter as the onset peak , and there is no sex difference between male and female .

2 . recombination analysis of a virus such as a virus

Methods : 28 samples were randomly extracted from 119 non - viral positive samples , amplified by RT - PCR and sequenced . The results were compared with the typing results based on RdRp region . The samples with different typing results were suspected recombinant virus strains . After sequencing , the fragments of partial sequences of RdRp region to VP1 region were amplified , and the results were input into the recombination analysis software Simplot and RDP analysis to find possible recombination events and recombination sites to exclude the interference of mixed infection .

Results : Of the 28 samples , the results of two typing were inconsistent . CQ242 was classified as GII . 3 based on RdRp region sequence and GII . 12 based on the sequence of VP1 region .
CQ426 is based on the RdRp region sequence and is GII . 4 . Based on the sequence typing of the VP1 region to GII . 6 . Two specimens are subjected to long - fragment amplification , CQ426 sequencing fails , and it cannot be determined . CQ242 sequencing is successful , the recombinant strain is determined as recombinant strain , and the recombinant type is GII . 3 / GII . 12 .

Conclusion : In the case of infectious diarrhea in Chongqing , the recombinant type is GII . 3 / GII . 12 , which belongs to the recombination between genotypes . It is necessary to pay close attention to the possibility of the outbreak of viral diarrhea due to the changes of virus virulence , infectivity and physical and chemical properties .

3 . Bioinformatic Analysis of Nore Virus Capsid Protein

Methods : The amino acid sequence of capsid protein in Chongqing , such as GII . 42006b , was downloaded from its Protein database . By using Expert Protein Analysis System ( ExPASY ) , the composition of protein was analyzed by Expert Protein Analysis System ( ExPASY ) , the structure of secondary structure was analyzed , and three - dimensional model of protein was established by homologous modeling technique to predict the epitope of protein B cell antigen .

Results : The protein was composed of 540 aa residues , the total molecular weight was 58991.5 , the isoelectric point was 5.53 , the negative charge residual base 47 , the positive charge residual base 35 , the total average hydrophilicity was - 0.195 , the non - stability coefficient was 45.3 , the fat coefficient was 78.15 , the total average hydrophilicity was - 0.195 , and it was a very weak hydrophilic protein .

Conclusion : The antigenic sites of the capsid protein VP1 of the virus GII . 42006b are 5377 - 6707 , and the corresponding nucleotide coding sequence is 6677 - 6707 . The three - dimensional structure of the homologous modeling protein shows that the protrusion of the segment aa is extended to the outside , and is in agreement with the NoV P region of the NoV P region under the electron microscope .

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.1

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