da Silva Leonardo Assis, Basso Marcos Fernando, Ribeiro Bergmann Morais
Laboratory of Baculovirus, Cell Biology Department, University of Brasília (UnB), Campus Universitário Darcy Ribeiro, Brasília, DF, CEP 70910-900, Brazil.
BIOMOL/BIOTEC Laboratory, Mato Grosso Cotton Institute (IMAmt), Rondonópolis, MT, 78740-970, Brazil.
Arch Virol. 2023 Jan 4;168(1):29. doi: 10.1007/s00705-022-05672-8.
The cotton boll weevil (CBW; Anthonomus grandis; Coleoptera: Curculionidae) is considered the major insect pest of cotton, causing considerable losses in yield and fiber quality. An increase in the boll weevil population due to increasingly inefficient chemical control measures is of great concernamong cotton producers. The absence of conventional or transgenic cultivars with minimal resistance to CBW has stimulated the search for new molecular and biological tools for efficient control of this insect pest. In this study, we used a metagenomic approach based on RNA deep sequencing to investigate the presence of viruses and coding viral RNA in apparently healthy native adult CBW insects collected from cotton crops in Mato Grosso state, Brazil. Using an Illumina HiSeq 2000 paired-end platform, 138,798 virus-related reads were obtained, and a consensus sequence of a putative new virus, 10,632 nucleotides in length, was assembled. The sequences of the 5' and 3' untranslated regions (UTRs) were determined by rapid amplification of cDNA ends (RACE), followed by Nanopore sequencing. The complete genome sequence included a 5'-UTR (1,158 nucleotides), a 3'-UTR (561 nucleotides), and a single ORF of 8,913 nucleotides encoding a large polyprotein. Sequence analysis of the putative polyprotein showed several regions with high sequence similarity to structural and non-structural proteins of viruses of the family Iflaviridae. Pairwise alignments of polyprotein amino acid sequences showed the highest sequence identity (32.13%) to a partial polyprotein sequence of a putative iflavirus (QKN89051.1) found in samples from wild zoo birds in China. Phylogenetic analysis based on full polyprotein sequences of different iflaviruses indicated that this new picorna-like virus is most closely related to iflaviruses found in lepidopteran insects, and it was therefore tentatively named "Anthonomus grandis iflavirus 1" (AgIV-1). This is, to our knowledge, the first complete viral genome sequence found in CBW, and it could provide a basis for further studies about the infectivity and transmission of this virus and its possible association with symptoms or acute disease. AgIV-1 could potentially be used to develop biological or molecular tools, such as a viral vector to carry interfering RNA molecules for CBW control.
棉铃象甲（CBW；墨西哥棉铃象甲；鞘翅目：象甲科）被认为是棉花的主要害虫，会导致产量和纤维品质大幅下降。由于化学防治措施效率越来越低，棉铃象甲数量增加，这引起了棉花种植者的极大关注。缺乏对棉铃象甲具有最低抗性的常规或转基因品种，促使人们寻找新的分子和生物学工具来有效防治这种害虫。在本研究中，我们使用基于RNA深度测序的宏基因组学方法，调查从巴西马托格罗索州棉花作物中采集的看似健康的本地成年棉铃象甲昆虫中病毒的存在情况以及编码病毒的RNA。使用Illumina HiSeq 2000双端平台，获得了138,798条与病毒相关的reads，并组装出了一个推定的新病毒的共有序列，长度为10,632个核苷酸。通过cDNA末端快速扩增（RACE），随后进行纳米孔测序，确定了5'和3'非翻译区（UTR）的序列。完整的基因组序列包括一个5'-UTR（1,158个核苷酸）、一个3'-UTR（561个核苷酸）以及一个8,913个核苷酸的单一开放阅读框，编码一个大的多聚蛋白。对推定的多聚蛋白进行序列分析，发现几个区域与Iflaviridae科病毒的结构和非结构蛋白具有高度序列相似性。多聚蛋白氨基酸序列的两两比对显示，与在中国野生动物园鸟类样本中发现的一种推定的iflavirus（QKN89051.1）的部分多聚蛋白序列具有最高的序列同一性（32.13%）。基于不同iflaviruses完整多聚蛋白序列的系统发育分析表明，这种新的类微小核糖核酸病毒与在鳞翅目昆虫中发现的iflaviruses关系最为密切，因此暂时命名为“墨西哥棉铃象甲iflavirus 1”（AgIV-1）。据我们所知，这是在棉铃象甲中发现的第一个完整病毒基因组序列，它可为进一步研究这种病毒的感染性和传播及其与症状或急性疾病的可能关联提供基础。AgIV-1有可能被用于开发生物或分子工具，比如一种携带干扰RNA分子用于防治棉铃象甲的病毒载体。
