doi:10.14720/aas.2020.116.2.1832 Original research article / izvirni znanstveni članek Discovery and molecular characterisation of the first ambidensovirus in honey bees Sabina OTT RUTAR 1, Dušan KORDIŠ 1, 2 Received Avgust 13, 2020; accepted December 13, 2020. Delo je prispelo 13. avgusta 2020, sprejeto 13. decembra 2020 Discovery and molecular characterisation of the first am- Odkritje in molekularna karakterizacija prvega ambidenso- bidensovirus in honey bees virusa pri čebelah Abstract: Honey bees play a critical role in global food Izvleček: Čebele igrajo ključno vlogo v svetovni proizvo- production as pollinators of numerous crops. Several stressors dnji hrane kot opraševalci številnih poljščin. Številni stresorji cause declines in populations of managed and wild bee species, povzročajo upad populacij gojenih in divjih vrst čebel, kot so such as habitat degradation, pesticide exposure and patho- degradacija habitata, izpostavljenost pesticidom in patogeni. gens. Viruses act as key stressors and can infect a wide range of Virusi delujejo kot glavni stresorji in lahko okužijo številne species. The majority of honey bee-infecting viruses are RNA viruses of the Picornavirales order. Although some ssDNA vi- vrste. Večina virusov, ki okužijo čebele, so RNA virusi iz reda ruses are common in insects, such as densoviruses, they have Picornavirales. Čeprav so nekateri ssDNA virusi pogosti pri not yet been found in honey bees. Densoviruses were however žuželkah, na primer densovirusi, jih pri čebelah doslej še niso found in bumblebees and ants. Here, we show that densoviruses našli. Densovirusi pa so bili najdeni pri čmrljih in mravljah. Po- are indeed present in the transcriptome of the eastern honey kazali smo, da so densovirusi prisotni v transkriptomu azijskih bee (Apis cerana) from southern China. On the basis of non- čebel (Apis cerana) z južne Kitajske. Na osnovi nestrukturnih structural and structural transcripts, we inferred the genome in strukturnih transkriptov smo ugotovili genomsko struktu- structure of the Apis densovirus. Phylogenetic analysis has ro Apis densovirusa. Filogenetska analiza je pokazala, da novi shown that this novel Apis densovirus belongs to the Scindoam- Apis densovirus spada v rod Scindoambidensovirus v poddru- bidensovirus genus in the Densovirinae subfamily. Apis denso- žini Densovirinae. Apis densovirus ima ambisense organizacijo virus possesses ambisense genome organisation and encodes genoma in kodira tri nestrukturne proteine in razcepljeni VP three non-structural proteins and a split VP (capsid) protein. (kapsidni) protein. Dostopnost skoraj celotnega genoma Apis The availability of a nearly complete Apis densovirus genome may enable the analysis of its potential pathogenic impact on densovirusa bo omogočila analizo njihovega potencialno pa- honey bees. Our findings can thus guide further research into togenega vpliva na čebele. Naše ugotovitve lahko privedejo do the densoviruses in honey bees and bumblebees. nadaljnjih raziskav densovirusov pri čebelah in čmrljih. Key words: honey bees; densovirus; genome organisation; Ključne besede: čebele; densovirus; organizacija genoma; molecular characterisation molekulska karakterizacija 1 INTRODUCTION species such as habitat degradation, pesticide exposure and pathogens (Goulson et al., 2015; Potts et al., 2010; Honey bees (Apis mellifera) play a critical role in Evans and Schwarz, 2011; McMenamin et al., 2016; Mc- global food production as pollinators of numerous crops Menamin and Genersch, 2015). Viruses act as key stress- (Klein et al., 2007; Fürst et al., 2014). Several stressors ors and can infect a wide range of species (Grozinger and cause declining populations of managed and wild bee Flenniken, 2019). Overt viral infections can result in a 1 Josef Stefan Institute, Department of Molecular and Biomedical Sciences, Ljubljana, Slovenia 2 Corresponding author, e-mail: [email protected] Acta agriculturae Slovenica, 116/2, 383–393, Ljubljana 2020 S. OTT RUTAR and D. KORDIŠ wide range of symptoms, including wing deformities, ily. It is interesting that the Bombus and Apis densovirus- discoloration, hair loss, bloated abdomens, trembling, es are not very similar and belong to different densoviral paralysis, and mortality (Chen and Siede, 2007). Honey genera. Although the Bombus densovirus is also present bee populations have become increasingly susceptible to endogenised in the Bombus impatiens genome, this was colony losses due to pathogenic viruses spread by para- not the case for the Apis densovirus. The availability of sitic Varroa mites (Martin et al., 2012). a nearly complete Apis densovirus genome may enable The majority of honey bee-infecting viruses are RNA the analysis of its potential pathogenic impact on honey viruses of the Picornavirales order (Chen and Siede, 2007; bees. Our findings can thus guide further research into Levitt et al., 2013; Brutscher et al., 2016; McMenamin and the densoviruses in honey bees. Flenniken, 2018; Beaurepaire et al., 2020). Common bee viruses include: the Dicistroviruses (Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), Acute bee paraly- 2 MATERIALS AND METHODS sis virus (ABPV), and Black queen cell virus (BQCV)); the Iflaviruses (Deformed wing virus (DWV), Kakugo virus, 2.1 DiscoverY of THE Apis ambidensovirus Varroa destructor virus-1/DWV-B, Sacbrood virus (SBV), IN public transcriptomic databases and Slow bee paralysis virus (SBPV)); and taxonomically unclassified viruses (Chronic bee paralysis virus (CBPV) Sequence database searches were finished in July and the Lake Sinai viruses (LSVs)) (reviewed in Chen and 2020. The protein queries were diverse densoviral NS1 Siede, 2007 and Brutscher et al., 2016). Recently identified and VP sequences. The database analysed was the Tran- positive sense single-stranded RNA viruses (+ssRNA) vi- scriptome Shotgun Assembly (TSA) at the National Cent- ruses include Bee macula-like virus (BeeMLV) in the Ty- er for Biotechnology Information (www.ncbi.nlm.nih. moviridae family (Galbraith et al., 2018), Apis mellifera gov). Comparisons were made using the TBLASTN pro- flavivirus and Apis mellifera nora virus 1 (Remnant et al., gram (Gertz et al., 2006), with the E-value cut-off set to 2017). Apis mellifera rhabdovirus and bunyavirus were re- 10−5 and default settings for other parameters. TBLASTN cently described (Remnant et al., 2017) and represent first searching was restricted to different taxa (Protostomia, bee-infecting negative sense single-stranded RNA viruses Hymenoptera, Apoidea and Apis). Apis cerana transcrip- (-ssRNA). tome (erroneously named Apis mellifera carnica) contains Honey bees are infected by a small number of DNA 52.177 contigs. Apis ambidensovirus sequences were viruses (Chen and Siede, 2007). Among double-stranded compared to reference protein sequences of all parvovi- DNA viruses two honey bee-infecting viruses have been ruses. DNA sequences of the Apis ambidensovirus were found. The Apis mellifera filamentous virus (AmFV) is translated with the Translate program (web.expasy.org/ from the Baculoviridae family and has been sequenced translate/). and characterized (Gauthier et al., 2015; Hartmann et al., 2015). The Apis cerana iridovirus from the Iridoviridae family has not yet been sequenced (Bailey et al., 1976; Bro- 2.2 AnalYsis of endogenous virus ELE- menshenk et al., 2010; Tokarz et al., 2011). Very recently, ments a number of single-stranded DNA viruses (ssDNA) as- sociated with Apis mellifera have been reported, belong- Endogenous copies of densoviruses were detected ing to circoviruses (Circoviridae) (Galbraith et al., 2018), using the TBLASTN algorithm against hymenopteran ge- genomoviruses (Genomoviridae) (Kraberger et al., 2019), nomes available in the Whole Genome Shotgun Database CRESS DNA viruses (Cressdnaviricota) (Kraberger et al., (WGS) and Sequence Read Archive (SRA) at the NCBI, 2019) and microviruses (Microviridae) that infect the using densoviral protein sequences as queries. The que- honey bee bacterial community (Kraberger et al., 2019). ries involved NS1, NS2, NS3 and VP protein sequences. Although some ssDNA viruses are common in in- Comparisons were made using the TBLASTN program sects, such as densoviruses (Parvoviridae) (Cotmore (Gertz et al., 2006), with the E-value cut-off set to 10−5 and et al., 2014; Pénzes et al., 2020), they have not yet been default settings for other parameters. found in honey bees. Densoviruses were however found in bumblebees and ants (Schoonvaere et al., 2018; Valles et al., 2013). Here, we show that densoviruses are indeed 2.3 Prediction of protein domains present in the Apis cerana transcriptome from southern China. Genome organisation and phylogenetic analysis In order to recognize potential protein domains in have shown that this novel Apis densovirus belongs to the the protein sequences analysed, we used NCBI CDD da- Scindoambidensovirus genus in the Densovirinae subfam- tabase (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), 384 Acta agriculturae Slovenica, 116/2 – 2020 Discovery and molecular characterisation of the first ambidensovirus in honey bees by applying a cut-off E-value of 0.01. All Apis and Bom- ies have found numerous endogenised densoviruses in in- bus densovirus proteins were compared against SMART sect genomes (Liu et al., 2011; Francois et al., 2016). Me- (smart.embl-heidelberg.de), InterPro (www.ebi.ac.uk/ tatranscriptomic analyses of major invertebrate lineages interpro/) and Pfam (pfam.xfam.org) protein domain da- have enabled the discovery of a