一株鸭源禽4型副粘病毒的分离鉴定及遗传进化特征分析

Isolation and identification of an avian 4 paramyxovirus and analysis of its genetic evolution

目的对不明原因死亡的家鸭进行病原鉴定及分离,对其全基因序列进行遗传信息系统发育分析,初步揭示病原的致病性和传播特点,为进一步研究禽4型副粘病毒的致病与传播机制提供科学依据。方法采集病死鸭脏器,经处理后接种SPF鸡胚,分离具有血液凝集特性的病原体。通过随机聚合酶链反应,用锚定特异引物对模板进行非特异性扩增。扩增产物经纯化后克隆、测序,登录NCBI数据库进行BLAST比对以确定分离毒株种类;通过Sanger测序技术对病毒进行全基因组测序,采用MEGA7.0软件进行序列比对,通过邻近归并法(Neighbor-Joining)构建种系发生进化树分析其基因特征。结果分离到1株血液凝集特性的病原体(SDD01),通过电镜观察、随机聚合酶链反应及测序确定为禽4型副粘病毒。PCR扩增得到其-NP-P-M-F-HN-基因全长序列以及L基因的完整CDS区。F基因氨基酸基序分析显示,蛋白切割位点为DVQPR↓F,MDT>144h,ICPI为0。系统发育分析毒株位于东半球谱系中Ib进化分支,与APMV-4/Mallard/Hubei/2014等野鸟源病毒株同源性为99.4%。结论在死亡家鸭脏器中分离到禽4型副粘病毒。该病毒对鸡的致病性较低,系统发育分析其F基因与野鸟源禽4型副粘病毒同源性为99.3%~99.4%,表明家鸭感染的禽4型副粘病毒可能由野鸟引入,并可在家禽和野鸟之间循环传播。

Objective To isolate and identify apathogen from a duck that died of unknown causes and to sequence its entire genome in order to analyze its genetic characteristics,to perform a phylogenetic analysis to reveal the characteristics of its transmission,and to test its pathogenicity in order to assess its virulence to thus provide a scientific basis for further study of the pathogenicity and mechanism of transmission of avian paramyxovirus type 4. Methods cDNA of the pathogen was amplified with specific primers.The products of amplification were cloned and sequenced using apolymerase chain reaction（PCR）.A BLAST search of the NCBI database was performed to identify the pathogen.Whole genome sequencing of the pathogen was performed using Sanger sequencing.Phylogenetic analysis was performed in MEGA 7.0 using the neighbor-joining method with 1,000 bootstrap replicates.In addition,the pathogenicity of the virus was determined using MDT and ICPI. Results A pathogen,SDD01,that caused hemagglutination was isolated from the dead duck.Based on electron microscopy and sequencing of the products of amplification,the SDD01 isolate was identified as avian paramyxovirus type 4.The full-length sequence of genes in the order-NP-P-M-F-HN-gene and the complete CDS region of the L gene were successfully amplified and sequenced.Amino acid motif analysis of the F gene indicated that the site of protein cleavage was DVQPR ↓ F,MDT144 h,and ICPI was 0.Phylogenetic analysis revealed that the SDD01 strain is in lineage 1 bfound in the Eastern hemisphere and that the strain has the highest homology with the isolate APMV-4/Mallard/Hubei/2014. Conclusion Avian paramyxovirus type 4 was isolated from a dead domestic duck.The virulence of the virus to chickens was low.Phylogenetic analysis indicated that the complete F gene of the SDD01strain of APMV-4shared 99.3%-99.4%nucleotide similarity with a strain from wild birds,indicating that the virus can be transmitted between wild birds and backyard waterfowl.