苹果茎痘病毒在山楂中的首次鉴定与序列分析

 山楂是我国重要的果树,但山楂病毒的相关研究较少。本研究通过高通量测序及生物信息学分析首次在山楂样品中发现苹果茎痘病毒(apple stem pitting virus,ASPV)。利用RT-PCR和cDNA末端快速扩增技术对ASPV山楂分离物全长序列进行扩增,结果表明,ASPV山楂分离物基因组全长由9 290个核苷酸组成,包含5个开放阅读框(open reading frames,ORFs)。ORF1编码与病毒复制相关的蛋白-RNA依赖的RNA聚合酶(RNA dependent RNA polymerase,RdRp),其在氨基酸水平上与ASPV苹果和梨分离物的RdRp序列相似性分别为72.8%~80.9%和81.7%~92.8%;ORFs 2~4表达与病毒移动相关的三基因块(triple gene block 1~3,TGB 1~3),其与印度苹果N分离物(LM999967)的氨基酸序列相似性最高,为92.9%~98.2%;ORF5编码外壳蛋白(coat protein,CP),与苹果和梨分离物CP的序列相似性较低,分别为64.8%~88.4%和69. 8%~92.2%,高于目前凹陷病毒属病毒新种的划分界限。将ASPV山楂分离物的全长核苷酸序列与其他ASPV分离物的全长序列进行比对,构建系统发育树,结果表明,ASPV山楂分离物与巴西苹果分离物BR-Brae2的亲缘关系最近,但两者核苷酸序列相似性较低(81.5%),说明与其他分离物相比该分离物具有较大的变异性。综上,本研究初步验证了山楂是ASPV的重要自然寄主,并首次获得了ASPV山楂分离物的全长基因组序列,为山楂病毒病诊断和防控策略的制定提供参考。

Identification and sequence analysis of apple stem pitting virus from hawthorn tree

Hawthorn is a kind of important fruit crop in China, while the associated researches on hawthorn viruses are very limited. We performed high-throughput sequencing and bioinformatics analysis on the hawthorn leaves and identified apple stem pitting virus (ASPV) in hawthorn samples for the first time. The whole genome of ASPV hawthorn isolate was obtained by RT-PCR. Bioinformatics analysis showed that the complete genome consists of 9 290 nucleotides (nt), including five open reading frames (ORFs). The ORF1 of the ASPV hawthorn isolate which encodes replication-associated protein RNA dependent RNA polymerase (RdRp) shared 72.8%-80.9% and 81.7%-92.8% with those isolates from apples and pears at nt and amino acid levels; ORFs 2-4 contains a triple gene block (TGB 1-3), and the amino acid sequence shows the highest similarity to ASPV Indian Apple N isolate (LM999967) of 92.9%-98.2%; ORF5 encodes the coat protein (CP) and has a relatively low similarity compared with CPs of apple and pear isolates (64.8%-88.4% and 69.8%-92.2%, respectively), which is higher than the current demarcation of new virus species. The whole nucleotide sequence of ASPV hawthorn isolate was aligned with other isolates and phylogenetic tree was constructed, which showed that ASPV hawthorn isolate was most closely related to BR-Brae2 isolate from apple in Brazil despite a relatively low nucleo-tide sequence identity (81.5%), indicating that the isolate had great variability. Above all, this study preliminarily confirmed that hawthorn is an important natural host of ASPV, and we obtained the full-length genome sequence of ASPV isolate for the first time, which provided references for diagnosis, prevention and control strategy formulation of hawthorn viruses.