摘要：

为探究蚜虫体内携带的病毒种类,采集江苏地区豆蚜田间种群,鉴定种类后,提取豆蚜总RNA,以片段化的mRNA为模板构建测序文库,利用宏病毒组测序技术检测分析豆蚜体内的病毒种类,并对获得的2个新病毒进行检测鉴定。结果显示,测序数据经拼接、比对和分类注释,最终共得到病毒序列177条,与24种病毒序列一致或同源性较高,包括5种植物病毒(属于5个病毒科)和19种昆虫病毒(涉及9个科和6种暂未分类病毒);对植物病毒中测序丰度最高的种类进行基因检测,获得序列经Blast比对,与其同源性较高的病毒均来自细胞质弹状病毒属,并且它们具有相同的保守区,确定该病毒为一种新的细胞质弹状病毒,暂命名为Aphis craccivora associated rhabdovirus(AcARV);基于病毒L蛋白系统进化分析显示,AcARV与水稻条纹花叶病毒(RSMV)构成最小分支,说明这2种病毒在进化上最为近缘;鉴定的多数昆虫病毒种类在蚜虫中鲜有报道;通过RT-PCR和蛋白免疫印迹在豆蚜体内均检测出昆虫病毒(HiPV)衣壳蛋白VP1,证实了HiPV对豆蚜的感染,这是首次在蚜虫体内发现HiPV,HiPV豆蚜分离物VP1基因与日本、江苏灰飞虱分离物核苷酸同源性分别为95.5%,99.0%。研究结果表明,豆蚜体内携带多种植物病毒,并含有一种新的弹状病毒AcARV,同时,豆蚜体内昆虫病毒种类非常丰富,HiPV可感染蚜虫。

关键词: 豆蚜, 病毒种类, 宏病毒组测序, 细胞质弹状病毒, HiPV病毒

Abstract:

In order to explore virus species in aphids,Aphis craccivora natural populations from Jiangsu were collected and identified,and then the fragmented mRNA from aphid total RNA was used as template to construct the sequencing library.The virus species in A.craccivora were analyzed by virome sequencing technology,and the two novel viruses were further identified.The results showed that after splicing,alignment and classification annotation of sequencing data,177 viral contigs were finally obtained,which were consistent or highly homologous with the sequences of 24 viruses,including 5 plant viruses(involving 5 families)and 19 insect viruses(involving 9 families and 6 unclassified viruses).The virus with the highest sequencing abundance among plant viruses was detected,and after Blast,these viruses with higher homology were all from Cytorhabdovirus,and they had the same conserved domain,so it was considered as a novel Cytorhabdovirus,temporarily named Aphis craccivora associated rhabdovirus(AcARV).The phylogenetic analysis based on viral L proteins revealed that AcARV and Rice stripe mosaic virus(RSMV)clustered into the closest clade,indicating that the two viruses were the closest in evolution.Most identified insect virus species were rarely reported in aphids.Capsid protein VP1 of insect virus Himetobi P virus(HiPV)was detected in A.craccivora via RT-PCR and Western Blot,which confirmed the infection of HiPV in A.craccivora,and it was the first report that HiPV was found in aphids.HiPV VP1 gene of A.craccivora isolate shared 95.5%,99.0% nucleotide identities with Japan and Jiangsu Laodelphax striatellus isolates.These results clarify A.craccivora bears a variety of plant viruses,including a new rhabdovirus AcARV.Meanwhile,insect virus species are abundant in A.craccivora,and HiPV can infect aphids.

Key words: Aphis craccivora, Virus species, Virome sequencing, Cytorhabdovirus, Himetobi P virus