新型鸭呼肠孤病毒的分离鉴定及其σC基因遗传进化分析

Isolation and identification of the novel duck reovirus and phylogenetic analysis of its σC gene

新型鸭呼肠孤病毒(NDRV)病,是一种以肝脾不规则坏死、出血斑/点和心肌、腔上囊出血为主要特征的新疫病。为研究某养鸭企业不同年份分离的NDRV病株的生物学特性,本研究将2019年从25份樱桃谷鸭脏器组织中分离的3株病毒以及2016年和2017年分离的2株病毒同时进行了生物学特性比较研究。RT-PCR及基因测序结果表明,该5株分离病毒均为NDRV,且纯净性良好。体外复制研究表明,该5株分离病毒均能100%致死鸡胚,且在Vero细胞中增殖良好;除2017年的分离株外,其余4株分离病毒均能在CEF中产生明显的细胞病变。σC基因同源性及遗传进化分析显示,5株NDRV分离株与其它NDRV病毒株之间具有较高的同源性(90.0%~98.2%),而与番鸭呼肠孤病毒(MDRV)同源性较低(29.6%~46.1%),再次证明5株分离病毒均为NDRV,但2017年分离株NDRV-SD19/6202与其余4株病毒的遗传距离稍远。σC基因编码氨基酸序列分析结果显示,以CEF适应病毒为代表的NDRV-SD19/6201株与CEF非适应病毒NDRV-SD19/6202株之间存在13个氨基酸差异位点,但这些差异位点是否是引起两株病毒复制特性差异的关键氨基酸还需要进一步研究。综上,本研究结果表明来源于同一养殖企业不同年份分离的NDRV病毒株之间生物学特性存在一定差异,提示持续开展该病流行病学调查的必要性,同时为了解NDRV的遗传进化特征和基因变异情况提供参考。

The novel duck reovirus(NDRV)disease is a new infectious disease characterized by irregular liver and spleen necrosis,hemorrhagic spots and lesions in the myocardium and bursa.In order to study the biological characteristics of NDRV strains isolated from a duck farm in different years,3 strains isolated from 25 cherry valley duck in 2019 and 2 strains isolated in 2016 and 2017 were analyzed in this study.The results of RT-PCR and sequence analysis showed that 5 isolates were all NDRVs and no foreign viruses contamination.The results of replication in vitro indicated that all five isolates had 100%mortality to chicken embryos,and proliferate well in Vero cells.Except for the isolates(NDRV-SD19/6202)from 2017,the remaining four isolates could induce significant cytopathic effects in CEF cells.The phylogenetic analysis ofσC genes showed that these five isolates shared higher homology(90.0%-98.2%)with NDRVs,while they shared lower homology(29.6%-46.1%)with Muscovy duck reovirus(MDRVs),which proves that the five isolates are NDRV again,but the isolate NDRV-SD19/6202 has a far genetic distance from the other four strains.Amino acid sequence analysis results ofσC showed that there were 13 amino acid differences between the NEFV-SD19/6201 represented by the CEF-adapted virus strain and the NEFV-SD19/6202 non-adapted CEF virus strain.However,whether these amino acids cause differences in the replication characteristics of the two strains of viruses requires further study.Our data indicated that there were some differences in the biological characteristics of NDRV strains isolated from different years of the same duck farm,suggesting the necessity of continuing the epidemiological investigation of the disease.This study provides a theoretical reference for understanding the genetic evolution and variation of NDRV.