应用dsRNA测序技术检测草鱼呼肠孤病毒的混合感染

Detection of the co-infection of different grass carp reovirus strains using dsRNA sequencing technology

从草鱼(Ctenopharyngodon idellus)出血病疑似病料提取物感染的草鱼肾细胞(CIK)中提取病毒基因组dsRNA,电泳显示出水生呼肠孤病毒基因组的典型特征。应用简化的FLAC(full-length amplification of cDNA)技术扩增得到病毒的4个全长基因组片段进行测序分析。核酸序列BLAST分析表明:其中3个基因组片段与GCRV-873第5、9、10片段高度同源,另外1个基因组片段与GCRV-HZ08第11片段高度同源;因此推测病料中同时存在两种不同的病毒核酸,但也可能存在一种杂合病毒。根据已发表的GCRV-HZ08第5、9、10片段设计了3对引物对分离的病毒总RNA进行RT-PCR检测并测序,结果显示这3个片段与GCRV-HZ08第5、9、10片段均具有99%同源性,表明是由于混合感染而存在两株不同病毒的核酸dsRNA,两株病毒分别命名为GCRV-JX01和GCRV-JX02。首次运用dsRNA测序法检测出了GCRV病毒的混合感染,为草鱼呼肠孤病毒的流行病学和防控提供了一种新的技术选择。

Viral genomic dsRNAs were extracted from Ctenopharyngodon idellus kidney（CIK） cells inoculated by diseased grass carp visceral organs with hemorrhagic symptom,and analyzed by agarose gel to show the typical genomic pattern of Aquareovirus.The purified viral pathogen caused typical cytopathic effects in CIK cells.A simplified dsRNA sequencing technology modified from FLAC（full-length amplification of cDNA） method was employed to sequencing the partial viral dsRNA genomic fragments.Four full-length dsRNA genomic fragments of the amplified cDNA fragments were successfully sequenced.Phylogenetic analysis was performed to identify two different viral strains in our sample,which indicated that there were two GCRV strains,named as JX01 and JX02 respectively,causing disease in grass carp in Jiangxi province.The data further suggested that we could monitor the pandemic viral strains using our simplified dsRNA sequencing technology.Our research thus provides a new technical method for the prevention,control and epidemiology of grass carp reovirus disease.