Children's Respiratory Tract, Central Nervous System And The Associated Diarrhea Disease Virus Infection Pathogenic Study

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.