IBDV流行强毒HQ-b株与其细胞适应毒生物学特性比较及VP2、VP5基因序列分析

为了解IBDV流行强毒HQ-b株囊毒与其细胞适应毒间生物学特性差异及2毒株毒力变化与VP2、VP5基因变异的关系,对2毒株的细胞适应性、致病性等进行比较,同时对其VP2、VP5基因序列进行分析。结果表明,HQ-b株囊毒对CEF、CEK、CELi、DF-1和Vero均不适应,而细胞适应毒HQ株仅不能适应Vero细胞、且批内及批间毒价稳定。致病性结果显示HQ-b株对4周龄SPF鸡致死率高达80%,是真正的超强毒,而细胞适应毒致死率已降为0%。对VP2基因高变区研究表明,HQ-b株具备IBDV超强毒株的分子特征,即222A、256I、294I和299S;其细胞适应毒HQ株除222A→P、256I→V、294I→L和299S→N外,在VP2公认的毒力位点253(Q→H)、279(D→N)、284(A→T)位氨基酸也发生改变,导致细胞适应毒具备经典弱毒株的分子特征,即222P、256V、279N、284T、294L和299N。对VP5基因研究表明:流行强毒HQ-b株也具有IBDV超强毒株的分子特征;其细胞适应毒VP5基因有12个位点碱基突变并导致9处氨基酸变异,尤其是ORF区第2个碱基由"T"突变为"C"后,导致细胞适应毒VP5的N端丢失了4个氨基酸,这种突变与现有的疫苗毒完全一致。本研究提供了超强毒HQ-b培育、驯化后致病性和细胞适应性转变的分子机理,也丰富了IBDV分子流行病学的理论。

Comparison of Biological Characteristics and Sequence Analysis of VP2 and VP5 Genes of Infectious Bursal Disease Virus Field Strain HQ-b and Its Cell-adapted Strain HQ

The objective of this study was to investigate the difference of biological characteristics between infections bursal disease virus(IBDV) field strain HQ-b and its cell-adapted virus strain HQ,and the relationship between the virulence change and mutation of VP2 and VP5 genes.Cell culture characteristics,pathogenicity to SPF chicken,sequences of VP2 and VP5 genes of the two strains were analyzed and compared.The results showed that the field strain HQ-b can not be adapted to grow in CEF,CEK,CELi,DF-1 and Vero cell lines.Cell-adapted virus strain HQ can be adapted to above cells except for Vero cell line.TCID50 titers in cultured cells were stable in different lot.It was indicated that the Field Strain HQ-b was a very virulent IBDV strain(vvIBDV).Mortality of 4-week-old SPF chicken infected with HQ-b and HQ was 80% and 0% respectively.The results of VP2 sequence(the hv regions) analyses showed that the Field Strain HQ had vvIBDV conserved amino acids(aa): 222 A,242 I,256 I,294 I and 299 S.The 222th(A→P),256th(I→V),294th(I→L),299th(S→N),253th(Q→H),279th(D→N) and 284th(A→T) aa of HQ were all changed,which caused it having attenuated strain conserved aa: 222 P,256 V,279 N,284 T,294 L and 299 N.The analyses results of VP5 sequence showed that the HQ-b had molecular characteristics of vvIBDV.HQ virus had 12 mutation sites and 9 of them made the corresponding aa change,especially,the mutation from T to C at the second bp of VP5 gene open-reading-frame of HQ virus caused lose first "ATG" and 4 aa at the N-terminal of VP5 protein.The mutations were very similar to vaccine strains.The research provides the molecular mechanism of alteration of culture properties and pathogenicity for vvIBDV HQ-b after adaption and enriches the molecular epidemiological theory for IBDV.