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。
Viruses are considered to be one of the main causes of children's acute respiratory tract infections (ARI) and central nervous system (CNS) infections. Although a variety of detection technologies have been applied, most of the infections remain unknown. Unlike the conventional methods, ultra high-throughput pyrosequencing can unbiasedly detect all the nucleic acids in a clinical specimen. Thus, it can be used simultaneously for pathogen detection and new pathogen discovery. This study aimed at applying the high-throughput pyrosequencing to characterize the viral communities in respiratory samples collected from ARI children of unknown etiology and healthy children, and the cerebrospinal fluid (CSF) samples collected from children with unexplained acute CNS infections. So as to present a preliminary chart of the viromes in healthy and diseased populations, guiding the discovery of unexpected disease-associated viruses, and to establish an effective virus detection and surveillance platform.
One hundred and five pooled nasopharyngeal aspirates (NPA) collected from229ARI children with suspected viral infections of unknown etiology,20pooled pharyngeal swabs collected from97healthy children and40pooled CSF samples collected from109children with suspected viral CNS infections but tested negative in the common viruses screening were enrolled in the present study for separate pyrosequencings. DNase&RNase treatments in combination with filtration were used to remove the non-viral nucleic acids in each pooled sample. Then the remaining filtrations were subjected to nucleic acids extraction and purification. A sequence-independent amplification (SIA) and multiple barcode primers were applied to amplify and label the nucleic acid products of the each pooled sample. Balanced mixture of each SIA amplicons was then subjected to454pyrosequencing, output data were retrieved and then analyzed by a pipeline of bioinformatic analysis to characterize the virus-like reads.
Virus enrichment followed by pyrosequencing resulted in2,642,142and668,403reads from the ARI and healthy children's samples, respectively. After a pipeline of bioinformatic analysis,9,178(of2,642,142) and1,205(of668,403) non-redundant reads were finally showed to be of viral origin. The virome of the ARI children's NPA samples contained virus-like reads relating to58distinct viruses which belong to23viral families. Most of the reads were homologous to the viruses of the families Anelloviridae, Parvoviridae, Coronaviridae, Paramyxoviridae, Polyomaviridae and Orthomyxoviridae. Vertebrate, invertebrate, plant and eukaryotic micro-organism virus homologous reads were found in the virome. For the samples of healthy children, the best blast hits of the virus-like reads could be classified into39distinct viruses, which covered vertebrate, invertebrate, plant, eukaryotic microorganism and archea viruses, belonging to22viral families. Unlike the ARI children's samples, the most frequently identified virus-like reads were related to the families Anelloviridae, Polyomaviridae, Caliciviridae, Herpesviridae, Virgaviridae and Circovirida. Except some reads showed a significant similarity to the known viruses, part of the virus-like reads identified in both viromes displaying a low identity to the known sequences in the databases, suggesting the existence of some putatively novel viruse-like reads in the viromes.
A total of962,920reads with an average length of312bp were obtained from the sequencing of CSF samples. Bioinformatic analysis identified1,061non-redundant reads displaying a potential viral origin. Blast analysis matched the non-redundant reads to54distinct viruses which represented25taxonomic families. Anelloviridae and Parvoviridae were the most frequently identified families in the homologous blast analysis of the virus-like reads. Vertebrate and invertebrate, plant, eukaryotic micro-organism and archea viruse-like reads were identified in the virome. Beside some human and vertebrate viruses homologous reads, a few reads related to the arboviruses, such as Brevidensovirus, Chloriridovirus and Densovirus, were also characterized. As the results of the respiratory samples, some of the virus-like reads only displayed a limited similarity to the known viral sequences in the databases.
Utilizing a viral metagenomics strategy, we gave a preliminary chart of the virome in the respiratory and CSF samples, and varied viral communities were revealed in different type of clinical samples and health conditions. The results provide a further advance in the understanding of the viral constituency in the respiratory and CSF samples and the potential role of some viruses in diseases.
Human parechoviruses (HPeVs), as a distinct entity and not rare pathogens among the diarrheal children, are still not well known to the clinicians. Epidemiological and clinical characteristics of HPeVs have been becoming the active research areas over recent years. As part of the routine survilliance, the current study was conducted to learn the latest prevalence of HPeVs in the hospitalized children with acute gastroenteritis in Lanzhou, China, and on this basis to learn the temporal changes in the HPeV prevalence when compared to our previous results. It was expected that this study would provide a more extensive and comprehensive understanding of HPeV in children with acute gastroenteritis in the region under investigation.
A real-time RT-PCR targeting the highly conserved5'UTR of the HPeV genome was applied to screen the HPeV in fecal samples which had been collected from289hospitalized children with acute gastroenteritis. Then the positive samples were subjected to a nested PCR for genotyping. Amplicons of the predicted size were recovered and sequenced, the obtained sequences of the HPeV-positive products were then phylogenetic analyzed to infer the genotypes. Enzyme immunoassay detections of the four common diarrheal viruses, i.e. rotavirus, astrovirus, calicivirus and adenovirus, were also conducted to screen for co-infections. The clinical data for the children were retrieved to learn the epidemical characteristics of the identified individual genotypes and to assess the association of the HPeV with acute gastroenteritis.
Of the289specimens,73(25.3%,73/289) were HPeV-positive. Nearly all the infections (70/73,95.9%) occurred in the first two years of life, and a larger proportion were between7to12months. The prevalence of HPeV was gender neutral. HPeV was more prevalent during July-September and November, with two peak periods in "August and November" and a minimum in "April through May". A total of56of73(76.7%) positive samples were successfully genotyped, HPeV genotypes1to4were identified. HPeV1was the most prevalent genotype, which was primarily identified from specimens collected during July-September (31/42,73.8%) with a peak in August. Most of the HPeV1positive children (37/42,88.1%) were younger than18months. It is noteworthy that HPeV2, a rarely reported genotype, was detected unexpectedly in this study. As far as we know, this is the first report of HPeV2prevalence in China to date. HPeV3was found in2.1%(6/289) fecal samples with an average age of9months. Infections of HPeV4were only seen in boys, the average age of the positive cases was12months. Within the nucleotide sequences an unexpected trinucleotide (GAT, nt241-243) insertion was found in all of the HPeV4sequences but not in the other genotypes. Co-infections were seen in64.4%(47/73) HPeV-infected subjects, and85.1%(40/47) of them were identified as dual infection. Rotavirus (29/47,61.7%) was the most frequently co-detected pathogen. Among the subpopulation not infected with any of the four common diarrheal viruses, HPeV showed a22.2%(26/117) positive rate. Co-infection was seen in each of the identified genotypes HPeV1to4. In addition to diarrhea, vomiting (42.3%,11/26) and respiratory symptoms (11.5%,3/26) were the other common symptoms presented in the children with HPeV-infection alone.
A comparable positive rate was revealed, while a temporal change of the seasonal distribution was noted when compared to our previous results. Changes in HPeV genotypes patterns, a three-month forward prevalence of HPeV1, a younger susceptible age to HPeV1than HPeV1, and a tendency of older children to be infected with HPeV4contrary to our previous report, were also revealed. HPeV2, a rarely reported genotype, was identified for the first time in China. In addition, an exclusive trinucleotide (GAT) insertion in the HPeV4nucleotide sequences was identified. The profile of the co-infections with other enteric related viruses were similar to our previous findings. Lack of direct association of HPeVs with acute gastroenteritis was confirmed in the current study.
引文
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