儿童呼吸道、中枢神经系统和腹泻病相关病毒感染的病原研究

发布时间：2018-05-23 18:05

本文选题：高通量测序 + 宏基因组　；参考：《北京协和医学院》2013年博士论文

【摘要】：病毒是引起儿童急性呼吸道感染和中枢神经系统感染的最主要的病原体。尽管目前应用了多种检测技术,临床上多数上述感染病例依然病原体不明。与以往传统方法不同,高通量测序技术可以无需预知病原体的基因组信息而无偏倚地检测临床标本中近乎所有的核酸背景,因而既可以用于病毒病原体的检测也有助于一些新的以往未知的病毒病原体的发现。本研究即旨在应用高通量测序技术对健康儿童和病原不明疑似急性病毒性呼吸道感染患儿的呼吸道标本,以及常见脑炎相关病毒筛查阴性的疑似急性病毒性中枢神经系统感染患儿的脑脊液标本进行分析,借助生物信息学方法了解上述各标本中病毒群体的组成以及差异,为后续进一步的探索疾病相关病毒病原体和新的未知病毒病原体提供前期研究基础；同时探索建立一种有效的病毒检测方法。共选择229份多种呼吸道病毒(包括部分常见的与近年新发现的呼吸道病毒)筛查均为阴性的疑似急性病毒性呼吸道感染患儿的鼻咽吸出物标本、97份健康儿童的鼻咽拭子标本、以及109份常见脑炎相关病毒筛查均为阴性的疑似病毒性中枢神经系统感染患儿的脑脊液标本。上述标本首先按照每2-3份原始标本混合成1份混合标本的方式进行预混,然后经0.45/0.22μm滤膜过滤和DNase/RNase处理,提取纯化核酸,进而借助于序列非依赖性的随机扩增技术对纯化的核酸进行随机扩增,并对扩增产物进行Barcode标记,等量混合扩增产物构建测序文库。进而完成454高通量测序,测序结果经生物信息学分析,确定标本中所有病毒同源序列。急性呼吸道感染患儿与健康儿童呼吸道标本的测序分别获得2,642,142个和668,403个序列。在去除冗余序列、低质量序列和屏蔽重复序列后,经BLASTn和BLASTx比对,两组儿童的标本分别获得9,178个和1,205个含完整Barcode序列的非冗余病毒同源序列。急性呼吸道感染患儿标本的检出序列与来自于23个病毒科的58种不同的病毒显示出不同程度的同源性,其中涵盖了脊椎动物和非脊椎动物病毒、植物病毒和真核微生物病毒样序列。最常检出的病毒科包括Anelloviridae、Parvoviridae、Coronaviridae、Paramyxoviridae、Polyomaviridae和Orthomyxoviridae等。健康儿童标本的检出序列则与分属于22个病毒科的39种病毒序列同源,涵盖了脊椎动物和非脊椎动物病毒、植物病毒、真核微生物病毒和古细菌病毒。与急性呼吸道感染患儿不同,健康儿童标本的检出序列主要与来自于Anelloviridae、Polyomaviridae、Caliciviridae、Herpesviridae、Virgaviridae和Circoviridae等病毒科的病毒成员同源。在两组人群的检出序列中,部分检出序列与数据库中的病毒序列之间呈现出较低的Identity值,表明在所检出的序列中可能含有一些来自于以往未知的病毒病原体的序列。脑脊液标本测序共获得962,920个序列,平均读长312bp。在经过同上述相同的生物信息学分析后,共获得1,061条含完整Barcode序列的非冗余病毒同源序列。这些非冗余序列的BLAST比对分析显示他们与来自于25个病毒科的54种不同的病毒的序列具有相似性。Anelloviridae和Parvoviridae病毒科是序列比对分析中最常检出的病毒科。这些检出的病毒样序列涵盖了脊椎动物和非脊椎动物病毒、植物病毒、真核微生物病毒和古细菌病毒同源序列。除检出了部分人和脊椎动物病毒样的序列,还同时检出多种虫媒病毒同源序列,如Brevidensovirus、Chloriridovirus和Densovirus等病毒的同源序列。与呼吸道标本的测序结果相同,部分序列与数据库中已知的病毒序列之间仅显示出较低的同源性。通过应用病毒宏基因组学研究方法,我们获得了儿童呼吸道标本和脑脊液标本中病毒群体的结构和组成情况,研究结果显示不同的临床标本以及不同的健康状态下病毒群体的组成存在一定的差异,本研究中所获得的序列信息同时也将为后续进一步研究和探索疾病相关病毒提供了前期研究基础。人副肠孤病毒(human parechovirus, HPeV)是一种目前尚未被临床工作者所熟知和了解,但在婴幼儿腹泻群体中并非鲜见的病原微生物,近些年有关其流行特征及与临床疾病的相关性颇受关注。作为常规监测的一部分,本研究的目的旨在了解近期HPeV在5岁以下急性腹泻住院患儿人群中的流行情况,结合我们以往的监测结果了解该病原体在本研究地区不同时期的流行特征的变化规律,以更全面的了解该病原体在本研究地区腹泻儿童群体中的流行情况。应用基于HPeV5'UTR保守区的实时RT-PCR筛检一整年期间内收集的289份急性腹泻住院患儿的粪便标本。阳性标本再经巢式PCR扩增VP3/VP1连接区,回收目的扩增产物并测序,系统进化分析确定基因型别。所有临床标本同时进行轮状病毒、杯状病毒、星状病毒和腺病毒的酶免分析,以确定HPeV阳性标本的合并感染情况。结合临床资料和基因分型结果,了解每种基因型别的流行特征,以及统计分析HPeV的感染与临床表征之间的相关性。 HPeV的全年检出率为25.3%(73/289),其中95.9%(70/73)的感染病例发生在2岁以下的儿童,无性别差异,HPeV的检出主要集中在7月-9月和11月,其中在8月份和11月份呈现两个高峰流行季节,而4月-5月则呈较低的流行。73例阳性标本中56例确定了基因型别,分别为HPeV1-4型。其中HPeV1为最主要的检出型别,流行主要集中在7月-9月之间,多数感染病例小于18月龄,无性别差异。与此同时,本研究首次在国内检出2例较为罕见的HPeV2型。HPeV3型的检出率为2.1%(6/289),感染患儿的平均年龄为9月龄。HPeV4型感染患儿的平均年龄12月龄,此外,核酸序列的比对显示该基因型别具有其他基因型别所不具有的独特的GAT (nt241-243)三核苷酸插入。64.4%(47/73)的HPeV感染患儿伴有合并感染,合并感染见于所检出的四种基因型别。其中85.1%(40/47)的合并感染属于双重感染,轮状病毒(29/47,61.7%)为最主要的合并感染病原体。在上述四种常见腹泻病毒筛查均为阴性的患儿群体中,HPeV的检出率为22.2%(26/117)。除了腹泻症状外,呕吐(42.3%,11/26)和呼吸道症状(11.5%,3/26)是HPeV单独感染患儿最为常见的临床表现。 HPeV的检出率与以往我们关于该地区的监测结果接近,但是流行的季节性与以往有所不同,此外,各基因型别也呈现出与过往不同的流行特征,表现为HPeV1的流行较往年提前了3个月,而在本研究中HPeV3型倾向于感染更加年幼的儿童,HPeV4型则更多的检出于年长的儿童。本研究首次在国内检出了较为罕见的HPeV2型感染病例,并在HPeV4的核苷酸序列中检出了该基因型别特有的GAT三核苷酸插入现象。合并感染的结果与以往的监测结果类似,本研究的数据再次证实HPeV的感染与临床表征之间并没有显著的相关性。
[Abstract]:Virus is the most important pathogen that causes acute respiratory infection and central nervous system infection in children. Although many detection techniques are used, most of these cases are still unknown. Unlike traditional methods, high throughput sequencing technology can not be used to predict the genome information of pathogens without bias. Detection of almost all nucleic acid backgrounds in clinical specimens, which can be used for detection of virus pathogens and the discovery of some new previously unknown viral pathogens. This study is intended to apply high throughput sequencing technology to respiratory specimens of healthy children and unsuspected acute viral respiratory infections in children. The cerebrospinal fluid specimens of children with acute viral central nervous system infection, which are negative for the common encephalitis related virus screening, are analyzed. By means of bioinformatics methods, the composition and differences of the virus groups in all the above specimens are understood and provided for further exploration of the disease related pathogens and the new unknown virus pathogens. At the same time, explore an effective virus detection method.
A total of 229 multiple respiratory viruses (including some common and newly discovered respiratory viruses) were screened for nasopharyngeal aspirates in children suspected of acute viral respiratory infection, 97 healthy children's nasopharyngeal swabs, and 109 common encephalitis related viruses were negative in the suspected virus. The cerebrospinal fluid specimens of the armature nervous system infected children were premixed in the way of mixing each 2-3 original specimens into 1 mixed specimens. The purified nucleic acid was extracted by 0.45/0.22 micron filter membrane filtration and DNase/RNase treatment, and then the purified nucleic acid was randomly expanded by random amplification of non dependent sequence. In addition, the amplified products were labeled with Barcode, and the same amount of mixed amplification products were constructed to construct the sequencing library. Then 454 high flux sequencing was completed. The sequencing results were analyzed by bioinformatics to determine all the virus homologous sequences in the specimens.
2642142 and 668403 sequences of respiratory tract specimens from children with acute respiratory infection and healthy children were sequenced respectively. After removing redundant sequences, low mass sequences and shielding repeated sequences, 9178 and 1205 intact Barcode sequences of non redundant viruses were obtained from two groups of children after BLASTn and BLASTx comparison. The detection sequence of children with acute respiratory infection showed different degrees of homology with 58 different viruses from 23 virus families, including vertebrate and invertebrate viruses, plant viruses and eukaryotic microorganism virus like sequences. The most frequently detected diseases include Anelloviridae, Parvoviridae, Coronavir. Idae, Paramyxoviridae, Polyomaviridae, Orthomyxoviridae and so on. The detection sequence of healthy children is homologous to the 39 virus sequences belonging to 22 families, including vertebrate and invertebrate virus, plant virus, eukaryotic microorganism virus and palaeobacteria virus. The detection sequence is homologous to the virus members from the Anelloviridae, Polyomaviridae, Caliciviridae, Herpesviridae, Virgaviridae and Circoviridae. In the detection sequence of the two groups, the partial detection sequence and the virus sequence in the database show a lower Identity value, indicating the sequence detected. It may contain some sequences from the previously unknown viral pathogens.
A total of 962920 sequences were sequenced in the cerebrospinal fluid samples. After the average reading length 312bp. was analyzed with the same bioinformatics analysis above, 1061 non redundant virus homologous sequences containing complete Barcode sequences were obtained. The BLAST alignment analysis of these non redundant sequences showed that they were with the sequence of 54 different viruses from 25 virus families. The similarity of.Anelloviridae and Parvoviridae is the most frequently detected sequence in sequence alignment. These detected sequences cover the sequence of vertebrate and invertebrate viruses, plant viruses, eukaryotic and palaeobacteria viruses, and the sequence of virus like parts of the vertebrates and vertebrates, The homologous sequences of a variety of OLV homologous sequences, such as Brevidensovirus, Chloriridovirus and Densovirus, are also detected at the same time. The same sequence is similar to that of the respiratory tract specimens, and the partial sequences only show a lower homology between the known virus sequences in the database.
By using the method of viral metagenomics, we obtained the structure and composition of the virus population in the children's respiratory and cerebrospinal fluid specimens. The results show that there are certain differences in the composition of the virus groups in different clinical specimens and in different health states. The sequence information obtained in this study is also the same. It will provide a preliminary research basis for further research and exploration of virus related diseases.
Human parechovirus (HPeV) is a kind of pathogenic microorganism which is not known and understood by clinical workers but is not rare in infantile diarrhea population. In recent years, its epidemic characteristics and its correlation with clinical diseases are concerned. As a part of regular monitoring, the purpose of this study is to understand the purpose of this study. In recent years, the prevalence of HPeV in hospitalized children with acute diarrhea under 5 years old, combined with our previous monitoring results to understand the changes in the epidemic characteristics of the pathogen in different periods of this area, in order to get a more comprehensive understanding of the prevalence of the pathogen in the diarrhoea children's body in this area.
The fecal specimens of 289 hospitalized children with acute diarrhoea collected during the whole year were screened by the real-time RT-PCR screening based on the conservative area of HPeV5'UTR. The positive specimens were amplified by nested PCR for the VP3/VP1 connection area, and the target amplified products were sequenced and sequenced. All clinical specimens were simultaneously rotavirus and goblet disease. Enzyme immunoassay for virus, stellate virus and adenovirus to determine the combined infection of HPeV positive specimens. Combined with clinical data and genotyping results, the epidemic characteristics of each genotype were understood and the correlation between HPeV infection and clinical characterization was statistically analyzed.
The annual detection rate of HPeV was 25.3% (73/289), of which 95.9% (70/73) infected cases occurred in children under 2 years of age, with no sexual difference. The detection of HPeV was mainly in July and November, in August and November, there were two peak epidemic seasons, and in April, 56 of the lower epidemic.73 positive specimens confirmed the genotype. HPeV1-4 type, respectively, HPeV1 is the most important detection type. The epidemic is mainly concentrated in July -9 months, most of the cases are less than 18 month old, and there are no sexual differences. At the same time, the detection rate of 2 cases of HPeV2 type.HPeV3 is 2.1% (6/289) for the first time in our country. The average age of the infected children is 9 month old.HPeV4. The average age of the infected children was 12 month old. In addition, the comparison of nucleic acid sequences showed that the unique GAT (nt241-243) trinucleotide (47/73) HPeV infection in children with other genotypes was associated with the combined infection, and the combined infection was found in the detected four genotypes. Among them, 85.1% (40/47) was associated with a sense of merger. 29/47,61.7% (rotavirus (29/47,61.7%) is the most common infection pathogen. In these four common children with negative diarrhea virus screening, the detection rate of HPeV is 22.2% (26/117). In addition to diarrhea symptoms, vomiting (42.3%, 11/26) and respiratory tract symptoms (11.5%, 3/26) are the most common cases of HPeV alone. Clinical manifestation.
The detection rate of HPeV is close to our previous monitoring results about the area, but the seasonal characteristics of the epidemic are different from that in the past. In addition, the genotypes also show a different epidemic characteristics, showing that the prevalence of HPeV1 is 3 months ahead of the previous year. In this study, the HPeV3 type tends to infect younger children, HPeV4 type. A more rare case of HPeV2 infection was detected in this study for the first time in China, and the GAT trinucleotide insertion of the genotype was detected in the nucleotide sequence of HPeV4. The results of the combined infection were similar to those of previous monitoring results. The data of this study confirmed the infection of HPeV again. There was no significant correlation between clinical manifestations.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.1

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