〔Med. Entomol. Zool. Vol. 71 No. 3 p. 225‒243 2020〕 225 REFERENCE DOI: 10.7601/mez.71.225 Original Article RNA virome analysis of hematophagous Chironomoidea ies (Diptera: Ceratopogonidae and Simuliidae) collected in Tokyo, Japan Daisuke K*, 1), Katsunori M1), 2), Astri Nur F1), Michael A-B1), Yukiko H1), Toshihiko H1), Yoshio T1), Kyoko S1) and Haruhiko I 1) * Corresponding author: [email protected] 1) Department of Medical Entomology, National Institute of Infectious Diseases, 1‒23‒1 Toyama, Shinjuku-ku, Tokyo 162‒8640, Japan 2) Kyushu Research Station, National Institute of Animal Health, NARO, 2702 Chuzan-cho, Kagoshima City, Kagoshgima 891‒0105, Japan (Received: 26 June 2020; Accepted: 3 August 2020) Abstract: e development of sequencing technologies, in recent years, gives novel insights into the diversity of viruses in arthropods. Human pathogenic or possible pathogenic arthropod-borne viruses (arboviruses) including novel viruses from mosquitoes and ticks have been found by RNA virome analysis using a high-throughput sequencer. However, virome studies for other blood-sucking arthropods like biting midges as well as black ies are relatively scarce. In this study, to nd viruses in hematophagous Chironomoidea ies, we performed RNA virome analyses of eld-caught female Culicoides arakawae and Simulium aureohirtum as a pilot study. In the analyses, six novel viruses belonging to ve virus taxa were detected, showing that RNA virome analysis using the next- generation sequencer was a strong method for understanding the viruses in both biting midges and black ies. is study indicated that C. arakawae and S. aureohirtum, which are not a popular vector for human pathogenic viruses, have a variety of viruses which are as many as other important vectors including mosquitoes and ticks. Furthermore, RNA virome analysis of a variety of blood-sucking insects will aid in not only discovering novel arboviruses but also understanding novel importance for arboviral vectors. Key words: virome, metagenome, biting midge, black y, insect-speci c virus, Jingmenvirus I (reviewed in Mullen and Murphree, 2019). e genus Culicoides is the main taxon within the family e blood-sucking property in arthropods is needed from the aspect of disease vectors for both humans for disease transmission to occur in animal hosts. and animals. Oropouche fever due to Oropouche Among hematophagous insects, ies in Diptera such orthobunyavirus is a Culicoides-borne human as mosquitoes and tsetse ies are important vectors viral disease which is endemic in the entire Latin of several types of pathogens (viruses, protozoans, America (Mullen and Murphree, 2019). Except for and larial nematodes) and causes serious public human diseases, Culicoides midges pass on a variety health problems in several areas of the world (Durden of pathogenic viruses to domesticated animals and Mullen, 2019). Among the dipteran insects, the (Mullen and Murphree, 2019). In Japan, for instance, blood-sucking properties have been evolved in an Akabane disease caused by Akabane virus and independent manner several times (Wiegmann et transmitted by Culicoides midges is a serious issue al., 2011). Many famous disease vectors (e.g., biting for livestock ruminants with stillbirth and congenital midge, black y, mosquito, and sand y) are known in malformations, etc. (Yanase, 2009). suborder Nematocera in Diptera. e biting midges More than 2,200 species of black ies have and black ies are in close association taxonomically been descried worldwide (reviewed in Adler and within Nematocera since the families Ceratopogonidae McCreadie, 2019). Black ies are well known vectors (including biting midges) and Simuliidae (including of human onchocercasis caused by the larial black ies) are categorized into the same superfamily, nematode Onchocerca, which is endemic in several Chironomoidea. countries in the central belt of Africa and in tropical e Ceratopogonidae are widespread with America (reviewed in Adler and McCreadie, 2019). 6,267 surviving described species in 123 genera In Japan, 11 human cases of zoonotic onchocercasis 226 Med. Entomol. Zool. due to O. dewittei japonica Uni, Bain & Takaoka al., 2001) with primer A and primer B designated by have been reported so far (Fukuda et al., 2019), and Xiong and Kocher (1991). Moreover, the sequence Simulium bidentatum (Shiraki) was pointed out as of the DNA barcoding region of the cytochrome c the vector species of this nematode (Fukuda et al., oxidase subunit I (COI) gene was ampli ed with the 2010). Moreover, some species in Simuliidae were primer set LCO1490 and HCO2198 (Folmer et al., regarded as the vectors for an avian blood parasite, 1994). e amplicons were puri ed and as well as Leucocytozoon lovati (Sato et al., 2009). In contrast, sequenced as reported previously (Kobayashi et al., vesicular stomatitis virus is just known as a virus that 2018). e specimens were stored at -80°C until the is transmitted by a black y in the Americas (reviewed following analyses. in Adler and McCreadie, 2019). e development of sequencing technologies, in Next-generation sequencing recent years, gives novel insights into the diversity A basic technique of next-generation sequencing of viruses in nature (e.g., Shi et al., 2016a, 2018). (NGS) was the same as reported previously Present-day studies have shown that quite diverse (Kobayashi et al., 2020). Shortly, the pooled female viruses present in arthropods (e.g., Li et al., 2015; Shi C. arakawae and S. aureohirtum were homogenized et al., 2016a) and several studies indicated human with the medium. e supernatant of the centrifuged pathogenic or possible pathogenic arthropod-borne homogenates was passed through a sterilized 0.45 µm viruses (arboviruses) including novel viruses by RNA lter. To digest DNA and RNA derived from host virome analysis of hematophagous arthropods (Tokarz insects, nuclease treatment was conducted. Nuclease et al., 2014, 2018; Moutailler et al., 2016; Bouquet et cocktail [14 units of TURBO DNase (Invitrogen), 12.5 al., 2017; de Souza et al.,2018; Brinkmann et al., 2018; units of Baseline-ZERO DNase (epicentre), and 5 µg Harvey et al., 2019a; Temmam et al., 2019; Faizah et of RNase A (Nippon gene)] was added to the 380 µL al., 2020; Kobayashi et al., 2020). Additionally, a lot ltrate and incubated at a temperature of 37°C for 1 of viruses have been discovered from mosquitoes by hour. RNA extraction was carried out by ISOGEN II virome analyses so far (reviewed in Atoni et al., 2019). (Nippon gene), and cDNA was synthesized with the Almost all studies were principally conducted on use of NEBNext RNA rst-strand and second-strand mosquitoes and ticks, and the studies for other blood- synthesis modules (New England Biolabs). Eventually, sucking arthropods are relatively scarce (Temmam et library preparation steps were done with the use of al., 2016; Harvey et al., 2019b; Modha et al., 2019). TruSeq Nano DNA library prep kit (illumina) or In this manuscript, to quest as well as characterize NEBNext Ultra II End Repair/dA-Tailing Module viruses in biting midges and black ies, we have (New England Biolabs) and NEBNext Ultra II Ligation accomplished the RNA virome analysis of eld-caught Module (New England Biolabs). e prepared library female C. arakawae (Arakawa) and S. aureohirtum was ampli ed as needed by PCR enzyme and primer Brunetti as a pilot study. In the course of the analyses, cocktail which are supplied with the TruSeq nano six various types of virus-like sequences were found, DNA library prep kit or NEB Next Ultra II Q5 Master and further genetic and phylogenetic analyses were Mix (New England Biolabs). e puri ed library was done for the characterization of virus properties. assessed with the use of the MiniSeq system (Illumina) with the MiniSeq Mid Output kit (300 cycles) M M (Illumina). e acquired reads were imported into the Collection and identication of biting midges and CLC Genomics Workbench version 12 (Qiagen), and black ies de novo assembly was conducted. e possible viral Biting midges and black ies were collected in sequence was pointed out by BLAST searches from the the continual mosquito surveillance in the National resulting contigs. Institute of Infectious Diseases which is located at Shinjuku, Tokyo, Japan (Tsuda and Hayashi, 2014). Conrmation of the endogenous viral elements of e collection methods were reported previously detected viruses (Tsuda and Hayashi, 2014). In brief, a battery- Endogenous viral elements (EVEs) of various operated CDC-like suction trap with 1 kg dry ice was viruses were found in diverse arthropods (Shi et used for the collection, and the trap was utilized for al., 2016a). For the possibilities of EVEs of detected 24 hours. Collected biting midges were identi ed by viruses to be con rmed, viral genome-speci c primer morphology. Contrarily, molecular identi cation was sets were designated based on the resultant contigs attempted for the identi cation of the species of black (Table 1). RNA and DNA were extracted from the ies with the use of genomic DNA that is extracted by ltrate and then subjected to RT-PCR and PCR with alkaline lysis (Rudbeck and Dissing, 1998) from their the use of the primers (Table 2), the same method one or two legs. e mitochondrial 16S ribosomal previously described (Kobayashi et al., 2020). Internal RNA (rRNA) gene was utilized for this experiment controls in this experiment were ampli ed using the in accordance with the previous study (Otsuka et primer sets for the 28S rRNA gene of C. arakawae Vol. 71 No. 3 2020 227 Table 1. List