鹅细小病毒弱毒株的培育及其致弱前后全基因组中核酸变异的分析

Goose parvovirus attenuation and sequence analysis for the mutations in genome DNA of parental virus and its attenuated strains

为制备鹅细小病毒(GPV)弱毒株并研究其传代致弱前后的基因变化情况,本研究将GPV YG株在鹅胚成纤维细胞(GEF)和鸭胚成纤维细胞(DEF)中交替传代培养至72代次,并且对F0、F24、F48和F72代次病毒进行PCR全基因组扩增和测序。序列比对结果显示,F72代与F0代相比,5'和3'非编码区存在163个核苷酸差异,多数集中于反向终端重复序列(ITR)中,5'和3'两端均有6段碱基插入;编码区氨基酸序列比对结果显示,与F0代次病毒相比,F72代次病毒共有23个氨基酸位点突变,18个位于VP蛋白,5个位于非结构蛋白。病毒致病性试验表明,随着传代次数的增加,其毒力呈减弱趋势,并且F72代次病毒进一步致弱。本研究对GPV致弱后基因变化情况的分析及动物致病性试验结果为GPV毒力致弱机制的研究奠定了基础。

To prepare attenuated goose parvovirus （GPV） and examine the genetic variations of the attenuated GPV, the GPV YG strain was passed alternately in duck embryo fibroblast and goose embryo fibroblast for 72 series passages and the full genomic DNA of the virus at F0, F24, F48 and F72 were sequenced. Comparing with the parental virus, a total of 163 nucleotides differences was found in the F72 of the virus, which mainly located within the inverted terminal repeat in both 5＇ and 3＇ noncoding regions with 6 insertions from 1 nucleotide to 15 consecutive nucleotides. In addition, the F72 had 23 amino acids mutations, of which 5 amino acids located in NS protein and 18 amino acids in VP protein, compared with the parental virus. Furthermore, the animal tests showed that the pathogenicity of the GPV was further attenuated in goslings at F72 passages. The results of genetic variation and animal test of the attenuated GPV provide important reference for further studies of GPV pathogenesis mechanism.