Vol.:(0123456789) 1 3 Archives of Virology https://doi.org/10.1007/s00705-020-04814-0 ANNOTATED SEQUENCE RECORD Identifcation and genome sequencing of RNA viruses in the eucalyptus snout beetle Gonipterus spp. (Coleoptera: Curculionidae) Leonardo A. Silva 1  · Carolina Jordan 2  · Vanessa Rafaela de Carvalho 2  · Carlos F. Wilcken 2  · Bergmann M. Ribeiro 1 Received: 6 June 2020 / Accepted: 18 August 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020 Abstract The genomes of two putative new RNA viruses (macula-like virus and bunya-like virus) were identifed in total RNA extracted from dead eucalyptus snout beetles (Gonipterus spp.) from a laboratory colony. However, only bunya-like virus was detected in feld-collected insects. The macula-like virus has a monopartite single-stranded RNA genome that contains three open reading frames (ORFs) encoding an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), protein with unknown function. The bunya-like virus genome was predicted to consist of two RNA segments: a large segment (L) encoding a single protein (RdRp) and a small segment (S) encoding a putative nucleocapsid protein. The genus Gonipterus (Coleoptera: Curculionidae) includes around 20 species of defoliating insects of Australian origin that are distributed worldwide. Two species of this genus were introduced in Brazil in the last century, Gonipterus platensis and G. pulverulentus. G. platensis, also known as eucalyptus snout beetle, has emerged as a major pest of Eucalyptus spp., causing important losses in wood produc- tion [1, 2]. Chemical insecticides have been widely used to control agricultural pests, but this method is costly and dangerous for non-target organisms in forestry. The use of biological control strategies to control insects reduces pollu- tion by chemical pesticides and consequently benefts envi- ronment and human health. Currently, the biological control of G. platensis is carried out by Anaphes nitens (Girault) (Hymenoptera: Mymaridae), a tiny wasp that parasitizes eggs of G. platensis, and by the entomopathogenic fungus Beauveria bassiana, but with limited efective control [2, 3]. There are few reports of viruses infecting beetles (Table S1). An RNA-sequencing approach using next-generation sequencing (NGS) was used to investigate the presence of viruses in dead insects from a colony of G. platensis main- tained at the Biological Control of Forest Pests Laboratory, São Paulo State University (UNESP), Brazil. Ten dead insects (200 mg) were macerated in 1X phosphate-buf- ered saline (PBS-1X) (137.0 mM NaCl, 2.7 mM KCl, 10.0 mM Na 2 HPO 4 , 2.0 mM KH 2 PO 4 , pH 7.4). The homogen- ate was fltered once through cheesecloth and centrifuged three times at 4,000 × g for 10 min for clarifcation. The supernatant was centrifuged at 63,000 × g for 75 min on 5 ml of 20% sucrose cushion in a polyallomer ultracentrifuge tube (Beckman), and the pellet was resuspended in PBS-1X. RNA was extracted using the QIAamp Viral RNA Mini Kit from QIAGEN according to the manufacturer’s instructions. RNA samples were then used to construct cDNA libraries, followed by sequencing using a 2 × 100-bp read length on a HiSeq™ 2000 platform (Illumina, San Diego, CA, USA) at Macrogen Inc. (Seoul, Republic of Korea). The gener- ated reads were trimmed and assembled de novo using CLC Genome Workbench 6.5.2 (CLC bio, QIAGEN). Contigs related to viruses were retrieved using BLASTx against an in-house viral RefSeq database. To extend the assembled sequences as far as possible, generated/trimmed reads were mapped back to their respective viral genomes using the software Geneious 11.1.5 [4]. Genome annotation was also Handling Editor: Massimo Turina. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00705-020-04814-0) contains supplementary material, which is available to authorized users. * Bergmann M. Ribeiro bergmann@unb.br 1 Laboratory of Baculovirus, Cell Biology Department, UnB- University of Brasília, 70910-900 Brasília, DF, Brazil 2 Plant Protection Department, FCA/UNESP-São Paulo State University, Campus of Botucatu, 18610-307 Botucatu, SP, Brazil