鹅细小病毒鹅胚成纤维细胞适应病毒株的培育及序列分析

Genomic sequence analysis of a goose embryo fibroblast adapted goose parvovirus

为培育高滴度的鹅细小病毒(GPV)细胞适应病毒株,本研究将原代鹅胚成纤维细胞(GEF)进行连续传代培养,并传至31代次,传代的GEF具有典型的成纤维细胞形态,培养48 h后形成单层细胞。选取不同代次的GEF接种GPV YN分离株,并在GEF中连续传代至F20代,病毒滴度由102.5TCID50/0.1 m L达到106.16TCID50/0.1 m L,表明GPV已适应GEF,并在感染GEF后的72 h产生明显细胞病变。对细胞适应毒F20和亲本病毒F0代进行基因序列比对结果显示,F20与F0存在133个核苷酸差异位点,主要集中在5'和3'非编码区。氨基酸序列共出现22个变异位点,主要集中在VP3蛋白中。这些变异与病毒对细胞培养的适应过程有关,其机理有待进一步研究。

To prepare high virus titer of the cell culture adapted goose parvovirus （GPV）,the primary goose embryo fibmblast （GEF） was prepared and continuingly passed for 31 passages,and the YN isolate of GPV was adapted in the GEF for 20 passages.In addition,the virus titers were increased steadily from 1025 TCID50/0.1 mL of the initial virus （F0） to 10616 TCID500.1 mL of the cell adapted virus （F20） which shown the significant cytopathic effect in GEF after 72 hours post inoculation.Furthermore,the whole genomic sequences of the F0 and the F20 virus were sequenced,respectively,and the alignment analysis showed that the mutations were found 133 for nucleotides and 22 for amino acids between the two viruses.Of which the nucleotide mutations located in 5＇ and 3＇ non-coding regions and the amino acids mutations located in VP3 protein region,which could be the essential mutations for the virus adaption in GEF.