Characterization of a Newly Discovered Picornavirus From a Patient With Diarrhea

描述（由申请人提供）：该项目的总体目标是完成我们在儿童腹泻样本中发现的一种新型小核糖核酸病毒的表征。腹泻是全球第三大传染性死亡原因，每年造成约200万人死亡和14亿例非致命性疾病。据估计，40%的腹泻病例病因不明。据保守估计，每年至少有50万人死于病因不明的儿童腹泻，因此，确定一种新的病原体，即使只是其中一小部分，对人类健康和基础科学都具有重要意义。我们最近使用大规模测序对腹泻样本进行了病毒宏基因组分析。在澳大利亚墨尔本收集的一名急性腹泻儿童的粪便中，鉴定出一个395 bp的序列，其中55%的氨基酸与一种已知的小核糖核酸病毒蛋白相同，这表明该样本中存在一种新型小核糖核酸病毒，我们将其称为wupv。该项目的目标是完成这种新型小核糖核酸病毒的序列，确定在其他标本中发现的程度，确定WUPV是否与腹泻有关，并产生传染性克隆或培养病毒。基因组序列将通过从粪便样本中提取的RNA中随机扩增cDNA末端（RACE）来完成。在获得完整序列后，将生成一个具有传染性的克隆。此外，我们将尝试在各种不同的细胞系中培养病毒。为了确定WUPV是否存在于其他样本中，我们将使用不同的特定引物对筛选3个国际和当地收集的粪便。为了证明WUPV是否与腹泻有关，我们将在一组腹泻粪便和对照粪便上使用相同的特异性引物，这些粪便在年龄、地理和季节上与每个腹泻样本相匹配。我们新发现的小核糖核酸病毒的流行病学和生物学特征将证实或驳斥其与胃肠炎全球疾病负担的关联和潜在贡献。

DESCRIPTION (provided by applicant): The overall goal of this project is to complete our characterization of a novel picornavirus that we discovered in a pediatric diarrhea sample. Diarrhea is the third leading infectious cause of death worldwide and causes approximately 2 million deaths and 1.4 billion non-fatal episodes yearly. It is estimated that 40% of diarrhea cases are of unknown etiology. A conservative estimate indicates that at least 500,000 annual deaths are caused by pediatric diarrhea of unknown etiology, and therefore the identification of a novel agent responsible for even a fraction of these episodes would be of great significance to human health and fundamental science. We recently conducted a viral metagenomic analysis of diarrhea samples using mass sequencing. In a stool collected in Melbourne, Australia from a child with acute diarrhea, one 395 bp sequence read was identified that had 55% amino acid identity to a known picornavirus protein, suggesting that a novel picornavirus was present in this sample, which we have termedWUPV. The goal of this project is to complete the sequence of this novel picornavirus, determine the extent to which it is found in additional specimens, determine if WUPV is associated with diarrhea, and generate an infectious clone or culture the virus. The sequence of the genome will be completed by 5' random amplification of cDNA ends (RACE) from RNA extracted from the stool sample. After the full sequence has been obtained an infectious clone will be generated. Additionally, we will attempt to culture the virus in a variety of different cell lines.To determine if WUPV is present in other samples we will use different pairs of specific primers to screen 3 international and local collections of stools. To demonstrate if WUPV is associated with diarrhea we will use these same specific primers on a cohort of diarrhea stools and control stools that are age-, geographic- and season- matched to each of the diarrhea specimens. Epidemiological and biological characterization of our newly discovered picornavirus will confirm or refute its association with, and potential contribution to, the global disease burden of gastroenteritis.