Complete Genome Analysis of a Novel Picorna-Like Virus From a Ladybird Beetle, Cheilomenes Sexmaculata

The ladybird beetle Cheilomenes sexmaculata (family Coccinellidae, order Coleoptera), is a common insect predator of agricultural pests. In this study, the full genome sequence of a novel picorna-like virus, temporarily named “Cheilomenes sexmaculata picorna-like virus 1” (CSPLV1), was identied from C. sexmaculata. The full-length sequence of CSPLV1 was 11,384 nucleotide (nt) in length (excluding the polyA tail) with one predicted open reading frame (ORF) encoding 3727 amino acids, a 13 nt 5' untranslated region (UTR) and a 187 nt 3' UTR. The ORF of CSPLV1 consisted of four distinct domains including an RNA virus helicase domain (3029-3319 nt), a peptidase domain (5555-6121 nt), an RNA-dependent RNA polymerase domain (7154-8101 nt) and a picorna-like coat protein domain (8606-9283nt). Phylogenetic analysis based on the conserved RdRP sequence showed that CSPLV1, together with Wuhan house centipede virus 3, Hypera postica associated virus 1 and Diabrotica undecimpunctata virus 1, formed as an unclassied group which is closely related to the clade Solinviviridae. To the best of our knowledge, CSPLV1 is the rst picorna-like virus revealed in C. sexmaculata.


Introduction
Picornaviruses are a group of positive-sense single-stranded RNA viruses, and the order picornavirales comprises Caliciviridae, Dicistroviridae, I aviridae, Marnaviridae, Picornaviridae, Polycipiviridae, Secoviridae, and Solinviviridae. Picornaviruses possess a genome with a large protein attached to the 5' end but no overlapping open reading frames (ORFs), and all the RNAs are translated into a polyprotein before processing [2]. Furthermore, using the RNA-dependent RNA polymerase (RdRP) of these viruses by phylogenetic analysis keeps pace with the assignments for the established picorna-like virus family [2][3][4][5]. Hence, RdRP sequence is suitable for analyzing the diversity of picorna-like viruses. The picorna-like viruses have been reported in numerous insects, such as Drosophila (Drosophila C virus, DcV) [6], Apis mellifera (sacbrood virus) [7], and Culex spp.(Culex picorna-like virus) [8]. For Coleoptera, several picorna-like viruses has been reported from different host insect families, such as Scarabaeidae (oryctes rhinoceros) [9], Chrysomelidae (Aulacophora lewisii) [10] and Coccinellidae (Harmonia axyridis) [11]. Cheilomenes sexmaculata, another member of the family Coccinellidae, is an important aphidophagous predator for the biocontrol of agricultural pests, which is widely distributed in Asia [12]. In this study, the complete genome sequence of a novel picorna-like virus was identi ed and characterized in C. sexmaculata.
Colonies of ladybird beetles were collected from cucumber leaves in August 2019 in Jinhua, Zhejiang, China. Total RNAs were extracted from a single ladybird beetle using TRIzol reagent (Invitrogen, Waltham, MA, USA). The Nanodrop 2000 spectrophotometer (Thermo Scienti c, Waltham, MA, USA) was later employed to determine the concentrations of RNA samples for subsequent NGS analysis. The RNA library (paired-end sequencing, 150 bp) was performed on the Illumina HiSeq 4000 platform (Illumina, San Diego, CA, USA). Afterwards, the Trinity software (Version 2.8.5) with default parameters was used for the de novo assembly with the preliminary treated clean reads. Thereafter, the assembled contigs were searched in the Barcode of Life Data (BOLD) system (http://www.boldsystems.org/) to acquire the accurate cytochrome coxidase I (COI) sequence of the ladybird beetle species. Then, a BlastN search against the nucleotide (nt) database in National Center for Biotechnology Information (NCBI) was conducted to con rm the species of the beetles. Our result indicated that COI sequence of the collected beetles was exactly same to that of C. sexmaculata (GenBank Number: KM207085.1).
To identify the potential viral-like sequences in the transcriptome of C. sexmaculata, the assembled contigs were searched against the entire viral reference database in NCBI (https://www.ncbi.nlm.nih.gov/genome/viruses) by using BlastX. As the result, a candidate picorna-like viral sequence (11,369 nt) with a relatively high abundance (53.39X coverage) was discovered from the assembled transcripts of C. sexmaculata. To avoid false positive match, this sequence was also aligned against the NCBI nt and non-redundant protein databases. Subsequently, the candidate sequence was further con rmed by reverse transcription PCR (RT-PCR) and Sanger sequencing., The rapid ampli cation of cDNA ends (RACE) assay was performed to determine the extreme 5' and 3' terminal sequences of the identi ed virus using the SMARTer® RACE 5'/3' kit (Takara, Beijing, China) and further con rmed by Sanger sequencing. The primers used in this study are listed in Supplementary Table S1. This picorna-like virus was tentatively named "Cheilomenes sexmaculata picorna-like virus 1" (CSPLV1), and its complete genome sequence was submitted to GenBank with the accession number of OK632508 (Supplementary File S1).
The full-length genome sequence of CSPLV1 was 11,384 nt in length (excluding the polyA tail) with only one predicted ORF (14-11,197 nt) encoding 3727 amino acids, a 13 nt 5' untranslated region (UTR) and a 187 nt 3' UTR. In addition, based on InterProScan conserved domain prediction (http://www.ebi.ac.uk/interpro), the ORF of CSPLV1 contained four distinct domains, including an RNA virus helicase domain (Hel) (3029-3319 nt), a Peptidase domain (Pep) (5555-6121 nt), an RNA-dependent RNA polymerase domain (RdRP) (7154-8101 nt) and a picorna-like coat protein domain (8606-9283 nt) (Fig. 1A). The relatively abundant reads across CSPLV1 genome was indicated in Fig. 1B. Moreover, to evaluate the potential replication of CSPLV1 in C. sexmaculata, reverse strand of CSPLV1was detected by strand speci c RT-PCR. Primers 1/5 and primers 2/5 PCR products using viral antisense cDNA as a template were obtained the anticipated size. Sanger sequencing of these PCR products con rmed that the sequences of PCR products were consistent with those indicated in the CSPLV1 genome (Fig. 1C). Next, phylogenetic analysis was performed using the conserved RdRp amino acid sequences of CSPLV1 and the previously reported viruses in the Picornavirales. Sequences were obtained from NCBI GenBank and aligned using MAFFT The results of phylogenetic tree analysis clearly indicated that CSPLV clustered with Wuhan house centipede virus 3 (WHCV3) (YP_009342325.1) (Fig. 2).
In conclusion, based on analysis of the full-length genome sequence and phylogenetic analysis, CSPLV1, a novel unclassi ed picorna-like virus, is identi ed from the ladybird beetle, C. sexmaculata. To the best of our knowledge, it is the rst picorna-like virus reported in C. sexmaculata.   . Detection of the minus strand of CSPLV1 in C. sexmaculata by reverse transcription PCR using primers shown in (A). Primer 1 or primer 6 was used to generate cDNA product from viral antisense or sense strand, respectively. Primers 1/5 and primers 2/5 PCR products were ampli ed using antisense cDNA as a template. Primers 4/6 and primers 3/5 PCR products were ampli ed using sense cDNA as a template. Primers 1/5 PCR product was produced using an antisense cDNA produced by primer 2 as a negative control (NC).

Figure 2
Phylogenetic analysis of CSPLV1 and previously reported viruses in the Picornavirales based on the conserved RdRP amino acid sequences. Bootstrap values are placed over each node of the tree (when>50). Scale bars represent percentage divergence. The CSPLV1 identi ed in this study is indicated with red font.

Supplementary Files
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