华东地区传染性法氏囊病病毒流行株的分离及其生物学特性分析

Isolation and characterization of biological properties of infectious bursal disease viruses from eastern China in recent years

为了解华东地区近年来传染性法氏囊病病毒(IBDV)的遗传变异现状,从该地区近三年来IBD疫苗免疫失败鸡群中分离到7株IBDV,遂被命名为J3、A4、S8、S9、A11、S14和J17。这7株IBDV均能直接适应于鸡胚培养,但对CEF、DF-1以及DF-EGFP-mi R-9三种细胞的适应性和细胞培养中的病毒效价有显著差异,较易适应于DF-EGFP-mi R-9细胞,A11毒株在DF-EGFP-miR-9细胞中的TCID_(50)高达1×10^(11)/0.1m L。用第3代鸡胚毒分别感染6周龄SPF雏鸡,均表现出临床症状和死亡情况,发病率为100%,死亡率在50%~70%之间,而用第20代细胞毒感染的SPF雏鸡,均未出现临床症状和死亡情况。将这7株IBDV分离毒VP2基因全长序列进行同源性分析,核苷酸和编码氨基酸序列的同源性分别为97.4%~99.6%和98.7%~99.8%。遗传进化分析显示,这7株IBDV分离毒均属于血清Ⅰ型,分布在三个相对独立的超强毒力IBDV(vvIBDV)分枝中。VP2特征性氨基酸位点分析,在第222、242、253、256、279、284、299、330、451位的氨基酸残基分别是A(S14毒株为L)、I、Q、I、D、A、S、S和L,位于第326~332位的七肽基序为SWSASGS,均具有IBDV强毒株的特征性氨基酸序列。在VP2基因高变区的第一个亲水区,J17毒株有212N,S14毒株有222L,不同于其他强毒株或弱毒株。上述研究结果表明,近年来在华东地区IBD免疫失败的鸡群中流行着超强毒力vv IBDV,毒株的生物学特性有明显差异,毒株的来源比较复杂,这对于IBD的防控面临着新挑战。

To characterize genetic variations of infectious bursal disease viruses（IBDV） in eastern China in recent years, we isolated 7 IBDV strains from IBD outbreaks in the vaccinated flocks between2013 and 2015. The IBDV isolates were named J3,A4,S8,S9,AII,SI4,and J17,respectively. All the isolates were directly cultured in embryonated chicken eggs. However,there were significant differences in adaptability and virus titers when the isolates were propagated using CEF,DF-I and DF-EGFP-miR-9. All the isolates were relatively easy to grow on DF-EGFP-miR-9 and all the titer reached as high as lO^11 TCID50/O. imL. Clinical signs were observed in six-week-old SPF chickens that were inoculated with each of the isolates,which were passaged 3 times in embryonated chicken eggs. The morbidity was as high as 100% and the mortality was from 50% to 70%.In comparison,no clinical signs and mortality were observed when SPF chickens were inoculated with each of the isolates that were passaged 20 times on cell cultures. Homology analysis of the complete VP2 sequences of the 7 isolates revealed that the nucleotide and amino acid sequence identities were 97.4%-99.6%and 98.7%--99.8^,respectively. Phylogenetic analysis showed that the 7 isolates belonged to serotype 11BDV,and were distributed in 3 relatively distant vvIBDV clades,respectwely. Analysis of critical amino acid sites of VP2 showed that the amino acid residues were A （L for S14）,I,Q,I,D,A,S,S,and L,at positions 222, 242,253,256,279,284,299,330, and 451,respectively. The heptapeptide sequence （SWSASGS） was found in all isolates at positions 326-332,a characteristic of vvIBDV strains. The isolate J17 had a unique 212N residue and the isolate S14 had a unique 222L residue in the first hydrophilic domain of the highly variable region（HVR）,which was different from other attenuated or vvIBDV strains. Our results indicate that vvIBDV is prevalent in vaccinated flocks in eastern China. The strains have different biological characteristics and complex origins, which poses new challenges in the control of IBDV.