山东省某地鹦鹉喙羽病病毒的全基因组序列及对SPF鸡胚的致病性探究  

Whole-Genome Sequences of Psittacine Beak and Feather Disease Virus Isolated in Shandong Province, China and Preliminary Study on the Pathogenicity of Specific Pathogen Free Chicken Embryo

作　　者：吴少鹏 宋艳 孙淑红 WU Shaopeng;SONG Yan;SUN Shuhong(College of Animal Science and Technology,ShandongAgricultural University,Tai an,271000,China)

机构地区：[1]山东农业大学动物科技学院

出　　处：《野生动物学报》2019年第3期670-678,共9页CHINESE JOURNAL OF WILDLIFE

基　　金：国家重点研究发展计划(2017YFD0500100);国家森林与野生动物保护专项基金(2130211)

摘　　要：2017年4月,山东省某鹦鹉场发生疑似喙羽病感染,鹦鹉出现异形喙和羽毛脱落,甚至急性猝死。目的:了解该地区鹦鹉喙羽病病毒( Psittacine beak and feather disease,PBFD )的流行现状、分子生物学特征和遗传变异规律,为PBFDV的防控提供参考依据。方法:收集怀疑有喙羽病病毒( PBFDV )感染的非洲灰鹦鹉的新鲜肝脏组织和羽毛样本,根据PBFDV全基因组序列设计特异性引物,从患病灰鹦鹉肝脏和羽毛组织中扩增PBFDV的全基因组序列,克隆到pMD19-T载体中,经测序获得PBFDV全长cDNA序列,利用DNAstar软件对全基因组进行遗传变异分析。由于目前尚未发现适合PBFDV繁殖的培养细胞或胚胎,我们尝试使用患病鹦鹉的羽毛研磨液注入7日龄SPF鸡胚的卵黄膜中。结果:分离到3株PBFDV ( GenBank登录号:MH279594,MH180298和MH190788 ),全基因组序列测序后,发现它们彼此具有93.3%—99.9%的相似性,并且它们也与GenBank中的一些参考序列具有86.1%—98.8%的相似性。卵黄囊注射病毒液后胚胎发育变缓,48—72 h内鸡胚死亡,全身出血。接着我们收集鸡胚的肝组织的研磨液在鸡胚上传代至第5代,均能使鸡胚死亡。提取来自每一代鸡胚肝组织的DNA都可以通过PCR扩增出PBFDV的目的条带。结论:以上实验结果表明,成功从患病鹦鹉体内克隆出PBFDV的全基因,并完成了序列分析,同时PBFDV接种鸡胚卵黄膜后成功复制。我们针对在该地区新出现的病毒从分子方面进行相关研究,分析其遗传变异特征,以研究该病毒与其他地区喙羽病病毒之间的亲缘关系,这些数据为研究该病毒的遗传变异和流行病学提供分子生物学依据。In April 2017,a few parrots in a farm had abnormally shaped beaks and feathers,and even experienced acute sudden death in Shandong Province.This study was conducted to understand the prevalence,molecular biological characteristics and genetic variation of psittacine beak and feather disease ( PBFD ) in a parrot farm of Shandong Province,and provide reference for disease prevention and control.We collected fresh liver tissue and feather samples of African grey parrots suspected of PBFDV infection,and designed a pair of specific primers based on the PBFDV genome-wide sequence to detect PBFDV in the feathers and liver of the diseased parrots.The whole genome sequence of PBFDV was amplified,cloned into pMD19-T vector,and the full-length cDNA sequence of PBFDV was obtained by sequencing and then analyzed by DNAstar software.No cultured cells or embryos suitable for PBFDV reproduction were found.We injected the yolk membrane of 7-day-old SPF chicken embryos with the feather slurry of the diseased parrot.Three strains of PBFDVs were isolated ( GenBank accession numbers:MH279594,MH180298 and MH190788 ).The whole genome sequence of the three strains shared 93.3%-99.9% similarity and they also shared 86.1%-98.8% similarity with other reference sequences in GenBank.After the injection of virus solution,the embryonic development of chicken was slowed and embryos died after 48-72 h,due to systemic hemorrhage.We then collected the grinding fluid of the liver tissue of the chicken embryo and passed it on to the fifth generation,after which all embryos died.The target band of PBFDV was amplified by PCR in DNA extracted from liver tissue of each generation of chicken embryo.Our experimental results showed that the complete gene of PBFDV was successfully cloned from the diseased parrot and sequence analysis was completed.At the same time,PBFDV was successfully replicated in chicken embryos.We conducted a molecular study on this emerging virus and analyzed its genetic variation to study the genetic relationship between the virus an

关 键 词：喙羽病病毒 全基因 遗传分析 

分 类 号：S855.3[农业科学—临床兽医学]