A novel naturally ORF3a-Deficient Porcine Transmissible Gastroenteritis Virus Field Strain HN-2012: Isolation, Pathogenicity and Reverse Genetics System Development

Transmissible gastroenteritis virus (TGEV) is an enteric coronavirus causing severe diarrhea with high mortality in suckling piglets. In this study, a field TGEV strain, designated HN-2012, was isolated from the intestinal contents of diarrheic piglets in Henan Province, China. Genomic analysis identified a continuous 140-nucleotide deletion in HN-2012 resulting in the lack of the accessory protein 3a (ORF3a) compared with other TGEV Miller strains. Experimental infection of piglets with TGEV HN-2012 induced severe diarrhea and lung injury. Simultaneously, high viral loads were detected in both the small intestines and lung tissues, demonstrating that TGEV HN-2012 exhibits dual tropism for enteric and respiratory epithelia. Furthermore, a full-length cDNA infectious clone of TGEV HN-2012 was constructed using a yeast artificial chromosome system via transformation-associated recombination. The recombinant virus was successfully rescued and exhibited similar replication kinetics comparable to the parental virus. In summary, we isolated a novel, naturally TGEV ORF3a deletion strain associated with severe enteric disease and lung injury in piglets, and establishes an efficient reverse genetic system of TGEV. This work providing a valuable tool for investigating the TGEV evolutionary and pathogenesis mechanisms.

猪传染性胃肠炎病毒（Transmissible gastroenteritis virus, TGEV）属于肠道冠状病毒，可导致哺乳仔猪出现严重腹泻并伴随较高死亡率。本研究从中国河南省腹泻仔猪的肠道内容物中，分离得到一株野毒TGEV毒株，命名为HN-2012。基因组分析显示，相较于其他TGEV Miller株，HN-2012毒株存在一段连续140个核苷酸的缺失，致使其辅助蛋白3a（ORF3a）缺失。对仔猪进行TGEV HN-2012实验感染后，可诱导其出现严重腹泻与肺损伤。同时，在小肠与肺组织中均检测到高病毒载量，表明TGEV HN-2012对肠道上皮和呼吸道上皮均具有双重嗜性。此外，本研究通过转化相关重组技术，利用酵母人工染色体系统构建了TGEV HN-2012的全长cDNA感染性克隆，成功拯救获得重组病毒，其复制动力学特征与亲本病毒基本一致。综上，本研究分离得到一株新型天然缺失ORF3a的TGEV毒株，该毒株可导致仔猪出现严重肠道疾病与肺损伤，并建立了一套高效的TGEV反向遗传操作系统，为探究TGEV的进化与致病机制提供了极具价值的研究工具。