Virome Analysis Reveals Diverse and Divergent RNA Viruses in Wild Insect Pollinators in Beijing, China

Abstract

Insect pollinators provide major pollination services for wild plants and crops. Honeybee viruses can cause serious damage to honeybee colonies. However, viruses of other wild pollinating insects have yet to be fully explored. In the present study, we used RNA sequencing to investigate the viral diversity of 50 species of wild pollinating insects. A total of 3 pathogenic honeybee viruses, 8 previously reported viruses, and 26 novel viruses were identified in sequenced samples. Among these, 7 novel viruses were shown to be closely related to honeybee pathogenic viruses, and 4 were determined to have potential pathogenicity for their hosts. The viruses detected in wild insect pollinators were mainly from the order Picornavirales and the families Orthomyxoviridae, Sinhaliviridae, Rhabdoviridae, and Flaviviridae. Our study expanded the species range of known insect pollinator viruses, contributing to future efforts to protect economic honeybees and wild pollinating insects.

Keywords: honeybees; next-generation sequencing; novel RNA viruses; viral diversity; wild insect pollinators.

Figure 1

Genome structures of novel viruses. Conservative domains (represented by brown rectangles) were annotated by CDD, while ORFs (represented by gray rectangles) were annotated based on the results of ORFfinder and the structure of the most closely related viral genome. The Xiangshan tymo-like virus may have an unassembled ORF encode capsid protein, which is represented by a dotted line.

Figure 2

Phylogenetic relationship of novel viruses. The phylogenetic relationship of novel viruses was inferred from conserved RdRp amino acid sequences. Related viruses only include those accurately classified by ICTV. RdRp-conserved sequences of novel viruses and related viruses in the trees were identified by CDD. Midpoint rooting phylogenetic trees were built using the maximum likelihood method with a bootstrap value of 1000, and bootstrap values (>80%) were shown on the branches. The amino acid substitution model was annotated below each tree. The tree of Orthomyxoviridae was based on polymerase subunit PA amino acid sequences, and the tree of narnavirus was based on complete RdRp amino acid sequences.

Figure 3

(A) Detection of each segment of segmented RNA viruses. (a) Xiangshan tombus-like virus segments 1 and 2 correspond to lanes 1 and 2. (b) Xiangshan insect virus segments 1 and 2 correspond to lanes 1 and 2. (c) Xiangshan orthomyxo-like virus segments 1–6 correspond to lanes 1–6. All PCR products were further verified by DNA sequencing. (B) Detection of complementary strands of several novel viruses. XOLV (lanes 3, 4), XTLV (lanes 5, 6), XPLV1 (lanes 7, 8), XPLV2 (lanes 9, 10), XSLV (lanes 11, 12), XIV (lanes 13, 14), and XPLV4 (lanes 15, 16). Lanes 3, 5, 7, 9, 11, 13, and 15 were the respective controls. DNA sequencing of PCR products of lanes 4, 8, 12, and 16 shows these bands in the specific amplification of virus sequences.