Identification and analysis of a novel negevirus isolated from a leaf beetle, <i>Aulacophora lewisii</i>

doi:10.3785/j.issn.1008-9209.2022.06.293

Journal of Zhejiang University (Agriculture and Life Sciences)

2022, Vol. 48

Issue (6): 744-752 DOI: 10.3785/j.issn.1008-9209.2022.06.293

Research articles

Identification and analysis of a novel negevirus isolated from a leaf beetle, Aulacophora lewisii

Haiqiang WANG(

),Yan ZHANG,Zhuangxin YE,Jichong ZHUO,Chuanxi ZHANG,Junmin LI,Jianping CHEN(

)

State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products/Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Rural Affairs and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, Zhejiang, China

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Abstract

negevirus (nege-like virus) is a newly proposed virus taxon of insect-specific RNA viruses and has been found in various insect species. In this study, the complete genome of a novel nege-like virus, named as “Nbu Aulacophora lewisii nege-like virus 1” (NbuALNV-1), was identified from a single leaf beetle, Aulacophora lewisii. NbuALNV-1 is a positive-sense single-stranded RNA virus with the genome of 9 832 nucleotide (nt) in length (excluding the polyA tail), with a 5′ untranslated region (UTR) of 292 nt and a 3′ UTR of 77 nt, and six open reading frames (ORFs). Five conserved domains of negeviruses were predicted to be encoded by the genome of NbuALNV-1, consisting of the viral methyltransferase domain, RNA virus helicase domain, and RNA-dependent RNA polymerase (RdRp) domain in ORF1, and the DISB-ORF2_chro domain and SP24 domain in the other two ORFs. Phylogenetic analysis of RdRp amino acid sequences of NbuALNV-1, together with Hubei Wuhan insect virus 9, Aphis glycines virus 3, and Fort Crockett virus showed that they formed as an “unclassified” clade and closely related to members in plant viruses of the family Kitaviridae. Furthermore, typical characteristics of virus-derived small interfering RNAs were observed by NbuALNV-1-derived small interfering RNAs, indicating that NbuALNV-1 might induce the host RNA interfering antiviral immune response. In summary, a nege-like virus was identified in A. lewisii, and NbuALNV-1 is the first negevirus discovered in the insect order Coleoptera.

Key words： negeviruses a novel virus identification small interfering RNA Aulacophora lewisii

Received: 29 June 2022 Published: 25 December 2022

CLC:

S 433.5

Corresponding Authors: Jianping CHEN E-mail: 18735424758@163.com;jianpingchen@nbu.edu.cn

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	Haiqiang WANG
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	Chuanxi ZHANG
	Junmin LI
	Jianping CHEN

Cite this article:

Haiqiang WANG,Yan ZHANG,Zhuangxin YE,Jichong ZHUO,Chuanxi ZHANG,Junmin LI,Jianping CHEN. Identification and analysis of a novel negevirus isolated from a leaf beetle, Aulacophora lewisii. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(6): 744-752.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.06.293 OR https://www.zjujournals.com/agr/Y2022/V48/I6/744

黑守瓜中一种新negevirus的鉴定和分析

negevirus（nege-like virus）是病毒学领域新增的一个分类单元，主要包括在节肢动物中鉴定的一类昆虫特异性病毒。本研究利用宏转录组技术从黑守瓜（Aulacophora lewisii）中鉴定到一种新的negevirus，命名为Nbu Aulacophora lewisii nege-like virus 1（NbuALNV-1）。NbuALNV-1为正义单链RNA病毒，全基因组包含9 832个核苷酸（nucleotide, nt）（不包括多聚腺苷酸尾），5 $′$ 、3 $′$ 非翻译区长度分别为292、77 nt。NbuALNV-1基因组包含6个开放阅读框，预测具有5个典型的negeviruses保守结构域，包括病毒甲基转移酶结构域、RNA病毒解旋酶结构域、RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase, RdRp）结构域、DISB?ORF2_chro结构域和SP24结构域。本研究利用NbuALNV-1和已报道的negeviruses的RdRp氨基酸序列基于最大似然法构建的系统发育分析表明，NbuALNV-1与Hubei Wuhan insect virus 9、Aphis glycines virus 3和Fort Crockett virus形成了一个独立分支，且该分支和北岛病毒科（Kitaviridae）的植物病毒在进化上具有相关性。此外，本研究对黑守瓜中NbuALNV-1病毒来源的干扰小RNA进行分析，发现这些干扰小RNA具有典型的病毒来源干扰小RNA的特征，表明NbuALNV-1能诱导宿主的RNA干扰抗病毒免疫反应。综上所述，本研究鉴定并分析了黑守瓜中的一种新的negevirus——NbuALNV-1，也是首次在鞘翅目昆虫中发现negevirus。

关键词： negeviruses, 新病毒鉴定, 干扰小RNA, 黑守瓜

引物名称 Primer name	引物序列（5 $′$ →3 $′$ ） Primer sequence (5 $′$ →3 $′$ )	引物用途 Usage of primer
长引物 Long primer	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	5′/3′ RACE扩增
短引物 Short primer	CTAATACGACTCACTATAGGGC	5′/3′ RACE扩增
NbuALNV-1_3′ RACE_F	GTCGTCTTGAATCACTGACAGAG	NbuALNV-1基因组3′末端 RACE扩增
NbuALNV-1_5′ RACE_R	ACATACTTAACCTACAGAATAACCT	NbuALNV-1基因组5′末端 RACE扩增
NbuALNV-1_cDNA_1F	GGGATAAACATAAATTTTAC	NbuALNV-1基因组中间序列扩增
NbuALNV-1_cDNA_1R	AGTACGAACACTACAACCGACAT
NbuALNV-1_cDNA_2F	CCCATGCTTTGCGTTTACTTTCA
NbuALNV-1_cDNA_2R	CACCACCAACGCATTTAGGA
NbuALNV-1_cDNA_3F	CGCGGGTACTCCTAAAATTG
NbuALNV-1_cDNA_3R	CGTTAGAGAGAGCTTGGGGC
NbuALNV-1_cDNA_4F	CCTGAAAGATTGGGGCGGTA
NbuALNV-1_cDNA_4R	AGCATGGAACGGCAACATCA
NbuALNV-1_cDNA_5F	CTGGTGGTGGTGTCGACGGTTATTGCTGTG
NbuALNV-1_cDNA_5R	TTCCAAACAAAACGACTAGAACGAG
NbuALNV-1_cDNA_6F	CTTGGTATTTTCAGGAAGCTGGTTT
NbuALNV-1_cDNA_6R	TGAAAAACGGACAGGTACG

Table 1 Primer information

Fig. 1 Genome structure and reads coverage of NbuALNV-1A. Genome RNA of NbuALNV-1; B. Reads coverage of the genome of NbuALNV-1.

Table 2 Homology of RdRp amino acid sequences among NbuALNV-1 and reported negeviruses

Fig. 2 Homology alignment analysis based on RdRp amino acid sequences of nege/nege-like virus and NbuALNV-1

Fig. 3 Phylogenetic tree of RdRp amino acid sequences of NbuALNV-1 and reported nege/nege-like virus based on the maximum likelihood

Fig. 4 Analysis of NbuALNV-1 derived vsiRNA


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[1]	Shen Enhui, Liu Yang, Ye Chuyu, Fan Longjiang. Recent studies on non-coding small RNAs in plants[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2014, 40(4): 370-378.

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