马传染性贫血病毒白细胞弱毒疫苗株及其亲本毒驴强毒株前病毒基因组比较分析

Comparison of Proviral Genomes between the Chinese EIAV Donkey Leukocyte-attenuated Vaccine and Its Parental Virulent Strain

为阐明马传染性贫血白细胞弱毒疫苗株(EIAVDLV)的致弱和免疫保护机理,对EIAVDLV121及其亲本驴强毒株(EIAVDV117)前病毒全基因组序列进行了测定,并结合准种理论,分析了EIAV疫苗致弱过程中基因组进化特点。利用LA-PCR技术对EIAVDV117和EIAVDLV121的前病毒基因组分两段进行扩增,分别获得4个和10个前病毒全基因组序列。EIAVDV117前病毒基因组平均为8236bp,G+C含量38.0。EIAVDLV121前病毒基因组平均8249bp,G+C含量37.3。两者的前病毒基因组平均差异率为2.8。其中S2、LTR和env基因差异较大,分别为4.1、3.9和3.1。此外,S2、S3和env推导的氨基酸的差异明显,分别为10.4、5.6和4.8(gp90为6.8)。EIAVDLV121各基因的异质性均显著高于EIAVDV117。研究发现体外培养的EIAVDLV121至少有5种类型的LTR混合存在。在gp90推导的氨基酸序列上,EIAVDV117比EIAVDLV121平均多2个N-糖基化位点,总数为19,其中3个为EIAVDV117特有。EIAVDLV121有1个疫苗株特有N-糖基化位点。研究结果为进一步探讨马传染性贫血弱毒疫苗生物学特性提供信息。

The donkey leukocyte-attenuated vaccine of equine infectious anemia virus （EIAV） was the first lentiviral vaccine that induced solid protection from the infection of virulent strains. To elucidate the mechanism of increased immunogenicity and attenuated virulence of the vaccine, the proviral genomic DNA of an EIAV vaccine strain, EIAVDLV121 was analyzed and compared with the genome of a parental virulent strain EIAVDV117. Full length viral genomic DNAs were amplified as two segments by LA-PCR and were cloned. Because of the genomic diversity of retroviral quasispecies, 10 full-length sequences of EIAVDLV121 and 4 full-length sequences of EIAVDV117 from randomly picked clones were analyzed. Results showed that the average length of the complete nucleotide sequence of EIAVDLV121 was 8,236bp and EIAVDV117 was 8,249bp, with the inter-strain diversity of 2.8%. As for individual genes between the vaccine and virulent strains, the differences in nucleotide sequence of S2, LTR and env were significantly higher than the other genes, with the diversity of 4.1%, 3.7 0% and 3. 10%, respectively. Considerable variations in deduced amino acid sequences were found in S2, S3 and env. The diversities were 10.4%, 5.6% and 4.8%, respectively. Furthermore, the LTR of EIAVDLV121 consisted of at least 5 subtypes grouped by their nucleotide sequences. There were two additional N-linked glycosylation sites in the deduced amino acid sequence of EIAVDV117 gp90 than that of EIAVDLV121. Among glycosylation sites in the gp90 of virulent strain, 3 were found unique only in EIAVDV117, of which 2 were located in the principle neutralizing domain （PND）. In addition, there was one EIAVDLV121-specific glycosylation site, which was positioned in the PND, too. Taken together, it is clear that greatly increased genomic diversity exists in the EIAV vaccine strain, which provides important information for the further study on biological characters of the Chinese EIAV attenuated vaccine.