PDF(Pubmed)
Abstract：
BACKGROUND: Hemorrhagic enteritis, caused by Turkey Hemorrhagic Enteritis Virus (THEV), is a disease affecting turkey poults characterized by immunosuppression and bloody diarrhea. An avirulent THEV strain that retains the immunosuppressive ability is used as a live vaccine. Characterizing the splice map of THEV is an essential step that would allow studies of individual genes mediating its immunosuppressive functions. We used RNA sequencing to characterize the splice map of THEV for the first time, providing key insights into the THEV gene expression and mRNA structures.
METHODS: After infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection. Total RNA was extracted, and poly-A-tailed mRNA sequenced. Reads were mapped to the THEV genome after trimming and transcripts assembled with StringTie. We performed PCR of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions.
RESULTS: Researchers previously annotated the THEV genome as encoding 23 open reading frames (ORFs). We identified 29 spliced transcripts from our RNA sequencing data, all containing novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. During validation we identified five additional unique transcripts, a subset of which were further validated by 3\' rapid amplification of cDNA ends (3\' RACE). Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated.
CONCLUSIONS: Similar to human adenoviruses, all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters. The genes are expressed under temporal regulation and THEV also produces multiple distinctly spliced transcripts that code for the same protein. Studies of the newly identified potential proteins should be urgently performed as these proteins may have roles in THEV-induced immunosuppression. Also, knowing the splicing of THEV genes should be invaluable to future research focusing on studying THEV genes, as this will allow accurate cloning of the mRNAs.
METHODS: After infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection. Total RNA was extracted, and poly-A-tailed mRNA sequenced. Reads were mapped to the THEV genome after trimming and transcripts assembled with StringTie. We performed PCR of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions.
RESULTS: Researchers previously annotated the THEV genome as encoding 23 open reading frames (ORFs). We identified 29 spliced transcripts from our RNA sequencing data, all containing novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. During validation we identified five additional unique transcripts, a subset of which were further validated by 3\' rapid amplification of cDNA ends (3\' RACE). Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated.
CONCLUSIONS: Similar to human adenoviruses, all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters. The genes are expressed under temporal regulation and THEV also produces multiple distinctly spliced transcripts that code for the same protein. Studies of the newly identified potential proteins should be urgently performed as these proteins may have roles in THEV-induced immunosuppression. Also, knowing the splicing of THEV genes should be invaluable to future research focusing on studying THEV genes, as this will allow accurate cloning of the mRNAs.
摘要：
背景：出血性肠炎，由土耳其出血性肠炎病毒（THEV）引起，是一种影响火鸡家禽的疾病，其特征是免疫抑制和血性腹泻。保留免疫抑制能力的无毒THEV菌株用作活疫苗。表征THEV的剪接图谱是允许研究介导其免疫抑制功能的单个基因的必要步骤。我们首次使用RNA测序来表征THEV的剪接图，提供对THEV基因表达和mRNA结构的关键见解。
方法：用疫苗株感染火鸡B细胞系后，一式三份的样品在4-，12-，24-,感染后72小时。提取总RNA,和聚A尾mRNA测序。修剪后，将读数映射到THEV基因组，并将转录本与StringTie组装。我们进行了THEVcDNA的PCR，克隆了PCR产物，并使用Sanger测序来验证所有确定的剪接点。
结果：研究人员先前将THEV基因组注释为编码23个开放阅读框（ORF）。我们从RNA测序数据中鉴定出29个剪接的转录本，尽管一些外显子与一些先前注释的ORF匹配，但它们都包含新的外显子。我们的数据也证实了三个注释的拼接点。在验证过程中，我们确定了五个额外的独特转录本，其中一个子集通过3个cDNA末端的快速扩增(3个RACE)进一步验证。因此，我们报告THEV的基因组包含34个转录本,这些转录本具有所有注释ORF的编码能力.然而,我们根据框内上游起始密码子的鉴定或额外编码外显子的检测,发现之前注释的ORF中有6个是截短的ORF.我们还确定了三个具有更长或更短亚型的注释ORF，和七个新颖的未注释的ORF，可能会被翻译；尽管调查它们是否被翻译超出了本手稿的范围。
结论：与人类腺病毒相似，所有THEV转录物都被剪接并组织成5个转录单位，在其同源启动子的控制下。这些基因在时间调节下表达，THEV还产生编码相同蛋白质的多个明显剪接的转录本。应紧急进行新鉴定的潜在蛋白质的研究，因为这些蛋白质可能在THEV诱导的免疫抑制中起作用。此外，了解THEV基因的剪接对于未来研究THEV基因的研究应该是无价的，因为这将允许精确克隆mRNA。
方法：用疫苗株感染火鸡B细胞系后，一式三份的样品在4-，12-，24-,感染后72小时。提取总RNA,和聚A尾mRNA测序。修剪后，将读数映射到THEV基因组，并将转录本与StringTie组装。我们进行了THEVcDNA的PCR，克隆了PCR产物，并使用Sanger测序来验证所有确定的剪接点。
结果：研究人员先前将THEV基因组注释为编码23个开放阅读框（ORF）。我们从RNA测序数据中鉴定出29个剪接的转录本，尽管一些外显子与一些先前注释的ORF匹配，但它们都包含新的外显子。我们的数据也证实了三个注释的拼接点。在验证过程中，我们确定了五个额外的独特转录本，其中一个子集通过3个cDNA末端的快速扩增(3个RACE)进一步验证。因此，我们报告THEV的基因组包含34个转录本,这些转录本具有所有注释ORF的编码能力.然而,我们根据框内上游起始密码子的鉴定或额外编码外显子的检测,发现之前注释的ORF中有6个是截短的ORF.我们还确定了三个具有更长或更短亚型的注释ORF，和七个新颖的未注释的ORF，可能会被翻译；尽管调查它们是否被翻译超出了本手稿的范围。
结论：与人类腺病毒相似，所有THEV转录物都被剪接并组织成5个转录单位，在其同源启动子的控制下。这些基因在时间调节下表达，THEV还产生编码相同蛋白质的多个明显剪接的转录本。应紧急进行新鉴定的潜在蛋白质的研究，因为这些蛋白质可能在THEV诱导的免疫抑制中起作用。此外，了解THEV基因的剪接对于未来研究THEV基因的研究应该是无价的，因为这将允许精确克隆mRNA。
