4株广西鸭源坦布苏病毒分离及初步鉴定

Isolation and Identification of Duck Tembusu Virus from Guangxi

本实验经尿囊腔接种方式从SPF鸡胚和健康鸭胚分离到4株广西鸭源坦布苏病毒。采集4羽病鸭的卵泡膜和输卵管无菌处理后接种SPF鸡胚和健康鸭胚。收获24h后死亡的鸡胚和鸭胚的尿囊液。所分离到的病毒经传4代后，鸡胚和鸭胚的死亡时间集中在90～110h之间。根据GENBANK已有的坦布苏病毒序列设计一对特异性引物，结果收获的尿囊液RT-PCR产物能扩增出特异性片段，并且排除其他引起鸭产蛋下降的病毒。参照文献用另外一对引物扩增出坦布苏病毒的部分E基因序列，比对分析后，这4株鸭源坦布苏病毒之间的核苷酸相似性为99．2％～99．9％，与中国其他坦布苏病毒分离株的相似性介于95．8％-97．64％，与马来西亚分离株sitiawan株和MM1775株相似性介于86．4％～87．6％。绘制系统进化树发现，这4个分离株处于一个独立的小分支，说明广西的鸭源坦布苏病毒与中国其他地区的分离株存在一定的差异。

Four isolates of Duck Tambusu Virus were obtained from SPF chicken embryo and healthy duck embryo by allantoic cavity inoculation. Follicular theca and oviduct of the ducks were collected under aseptic condition, then injected to SPF chicken embryoes and healthy duck embryo by allantoic cavity inoculation. Allantoic fluid was collected from embryoes died after 24 h. After 4 passages, the isolates caused embryoes deaths between 90 and 110 h. Allantoic fluid were PCR positive for Duck Tambusu Virus and negative for other viruses causing egg drop of ducks. Partial E gene of the isolates was amplified, then analyzed and compared with the Tambusu Virus sequences, revealing that there was 99.2%-99.9% nucleotide identity among the 4 isolates, and they had 95.8%-97.64% similarity with other Tambusu Virus isolates from China, 86.4%-87.6% with sitiawan and MM1775 from Malaysia. Phylogenetic analysis showed that the 4 isolates were located in an independant sub-branch, suggesting that Duck Tambusu Virus isolates in Guangxi were different from the isolates from other area of China to some extent.