肉鸡源传染性法氏囊病新型重排毒株的分离鉴定及遗传进化分析

Isolation,identification and genetic evolution analysis on a new reassortant strain of infectious bursal disease virus from broilers

[目的]明确云南某肉鸡场疑似传染性法氏囊病病毒(IBDV)感染病例的病原类型及分子特征,以了解IBDV新型重排毒株在肉鸡中的流行情况、分子进化趋势,为制定有效的传染性法氏囊病(IBD)防控和疫苗选用策略提供参考依据。[方法]对疑似IBDV感染的病鸡法氏囊进行研磨处理后,采用巢式RT-PCR进行检测,后将IBDV阳性的法氏囊组织悬液接种于9日龄SPF鸡胚并进行病毒分离,对病毒的vVP2和VP1-b基因扩增并进行遗传变异分析。[结果]成功分离出一株新型IBDV重排毒株(命名为YN-YX221110),接种9日龄SPF鸡胚后,胚体整体呈现弥漫性出血,头、颈以及背部皮肤有出血点。基于vVP2基因序列,分离株YN-YX221110与新型变异株(nvIBDV)参考株的核苷酸相似性最高,为93.2%~97.9%,分离株YN-YX221110的vVP2基因222A特征性氨基酸位点与UK661、HK46等超强毒株(vvIBDV)参考株相同,249K、256V、279N、284A、294L、299S等位点与新型变异株(nvIBDV)参考株中的SHG19相同;在基于vVP2基因的遗传进化树中,分离株YN-YX221110与GX-QZ191002、SHG19等nvIBDV参考株聚为一支,属于A2d分支。基于VP1-b基因序列,分离株YN-YX221110与参考株中的经典毒株(cIBDV)类似株核苷酸相似性最高,为97.8%~98.4%,分离株YN-YX221110的vVP2基因特征性氨基酸位点240D、242D、287T、390L和393E与致弱毒株(attIBDV)参考株中的B87、Gt相同;在基于VP1-b基因序列的系统发育进化树中,分离株YN-YX221110与参考株Gt、B87和CEF94等cIBDV类似株中的attIBDV聚为一支,属于B1a分支。分离株YN-YX221110基因型为A2dB1a。[结论]成功分离得到肉鸡源IBDV分离株YN-YX221110,其A片段来源于nvIBDV,B片段来源于cIBDV类似株中的attIBDV,为新型IBDV重排毒株。

[Objective]The study aimed to determine the pathogen type and molecular characteristics of suspected infectious bursal disease vi-rus(IBDV)infections in a broiler farm in Yunnan in order to understand the prevalence and molecular evolution trends of novel reassorted IBDV strains in broilers and to provide a reference for formulating effective infectious bursal disease(IBD)prevention,control and vaccine selection strategies:[Method]Bursae of Fabricius from chickens suspected of IBDV infection were homogenized and tested using nested RT-PCR.Positive bursal tissue suspensions were inoculated into 9-day-old SPF chicken embryos for virus isolation.The vVP2 and VP1-b genes of the virus were amplified and analyzed for genetic variation.[Result]A novel reassorted IBDV strain(named YN-YX221110)was successful-ly isolated.After inoculation into 9-day-old SPF chicken embryos,the embryos showed diffuse hemorrhages,with bleeding spots on the head,neck,and back skin.Based on the vVP2 gene sequence,the YN-YX221110 isolate showed the highest nucleotide similarity with novel variant strains(nvIBDV)reference strains,ranging from 93.2%to 97.9%.The VP2 gene of the YN-YX221110 isolate shared characteristic amino acid positions 222A with the very virulent(vvIBDV)reference strains such as UK661 and HK46,and positions 249K,256V,279N,284A,294L and 299S with the novel variants IBDV(nvIBDV)reference strain SHG19.In the genetic evolutionary tree based on the vVP2 gene,the YN-YX221110 isolate clustered with nvIBDV reference strains such as GX-QZ191002 and SHG19,belonging to the A2d branch.Based on the VPI-b gene sequence,the YN-YX221110 isolate showed the highest nucleotide similarity with classical virulent(cIBDV)strains,ranging from 97.8%to 98.4%.The characteristic amino acid positions of the vVP2 gene in the YN-YX221110 isolate,240D,242D,287T,390L and 393E,were the same as those in attenuated(attIBDV)reference strains such as B87 and Gt;In the phylogenetic tree based on the VPI-b gene sequence,the YN-YX221110 isolate clustered with atIBDV strains such as Gt,B87 and CEF94 in the clBDV-like group,belonging to the Bla branch.The genotype of the YN-YX221110 isolate was A2dBla.[Conclusion]The broiler-derived IBDV isolate YN-YX221110 is successfully isolated,with segment A derived from nvIBDV and segment B from attIBDV in cIBDV-like strains,representing a novel reassorted IBDV strain.