A novel negevirus isolated from Aedes vexans mosquitoes in Finland

Negeviruses are insect-specific enveloped RNA viruses that have been detected in mosquitoes and sandflies from various geographical locations. Here, we describe a new negevirus from Northern Europe, isolated from pool of Aedes vexans mosquitoes collected in Finland, designated as Mekrijärvi negevirus (MEJNV). MEJNV had a typical negevirus genome organization, is 9,740 nucleotides in length, and has a GC content of 47.53%. The MEJNV genome contains three ORFs, each containing the following identified conserved domains: ORF1 (7,068 nt) encodes a viral methyltransferase, an FtsJ-like methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase, ORF2 (1,242 nt) encodes a putative virion glycoprotein, and ORF3 (660 nt) encodes a putative virion membrane protein. A distinctive feature relative to other currently known negeviruses is a 7-nucleotide-long overlap between ORF1 and ORF2. MEJNV shares the highest sequence identity with Ying Kou virus from China, with 67.71% nucleotide and 75.19% and 59.00% amino acid sequence identity in ORF 1 and ORF 2, respectively. ORF3 had the highest amino acid sequence similarity to Daeseongdong virus 1 and negevirus Nona 1, both with 77.61% identity, and to Ying Kou virus, with 71.22% identity. MEJNV is currently the northernmost negevirus described. Our report supports the view that negeviruses are a globally distributed, diverse group of viruses that can be found from mosquitoes in a wide range of terrestrial biomes from tropical to boreal forests.

RNA helicase, and an RNA-dependent RNA polymerase (RdRp), ORF2 encodes glycoproteins, and ORF3 encodes virion membrane proteins [1][2][3]. The recently proposed "Negevirus" taxon consists of two clades, named "Nelorpivirus" and "Sandewavirus" [3,5], and the taxon is related to plant viruses of the genera Cilevirus, Higrevirus and Blunervirus [1,7]. In recent years, the "Negevirus" taxon has been expanding with several new negeviruses being reported. The geographical origins of mosquito-associated negeviruses include various terrestrial biomes, mainly temperate, subtropical, and tropical forest areas. Here, we report a novel negevirus isolated from Aedes vexans mosquitoes from a boreal coniferous forest area in Finland.
Mosquitoes were collected for an arbovirus study in August 2018 in Mekrijärvi (62°45′43″N, 30°57′32″E), Eastern Finland [8]. One mosquito pool consisting of 26 individual female mosquitoes caused a strong cytopathic effect (CPE) in Aedes albopictus C6/36 cells. The voucher specimen for this mosquito pool was identified morphologically [9] as Aedes vexans. RNA was extracted from virus isolation supernatant collected 10 days after infection, using TRIzol Reagent (Thermo Fisher). A sequencing library was prepared using an NEBNext Ultra II RNA Library Prep Handling Editor: T. K. Frey. Kit (New England Biolabs), quantified using an NEBNext Library Quant Kit for Illumina (New England Biolabs), and sequenced on a MiSeq platform using a MiSeq Reagent Kit v2 with 150-bp paired-end reads. Raw sequence reads were trimmed, and low-quality (quality score < 30) and short (< 50 nt) sequences were removed using Trimmomatic [10]. Thereafter, de novo-assembly was conducted using Mega-Hit, followed by re-assembly against the de novo assembled consensus sequence using the BWA-MEM algorithm [11] implemented in SAMTools version 1.8 [12]. A total of 357,745 reads mapped to the largest contig, with mean coverage of 7,624 (range, 146-17,546).
A genomic sequence of 9,740 nt was identified as a negevirus using an NCBI BLASTx search, and the isolate was designated as "Mekrijärvi negevirus" (MEJNV) (Gen-Bank accession number MT522375). Three ORFs were identified in the MEJNV genome using NCBI ORFfinder (https ://www.ncbi.nlm.nih.gov/orffi nder/) ( Fig. 1), flanked by untranslated regions (unverified) of 509 nt (5'UTR) and 162 nt (3'UTR) in length. MEJNV was most similar to Ying Kou virus strain YK1714 (NC_040636.1) originating from Culex pipiens pallens mosquitoes in China, with 67.71% nucleotide sequence identity. The genome size and the detected ORFs were similar, and the GC content (GC%) of 47.53% was slightly higher when compared to previously described negeviruses (avg. GC% = 41.11%). The 3'terminus of ORF1 and the 5'terminus of ORF2 were found to overlap by seven nucleotides, and this is a distinct feature of the MEJNV genome. Conserved protein domains were identified using NCBI CD-search (https ://www.ncbi.nlm.nih.gov/ Struc ture/cdd/wrpsb .cgi) (Fig. 1). The identified domains and their positions within the ORFs align with those of previously described negeviruses [1] (Fig. 1). The evolutionary relationship of MEJNV to other related negeviruses was assessed by constructing a phylogenetic tree based on ORF1 amino acid sequences of negevirus strains (n = 43). The database sequences were aligned using MAFFT version 7.453 [13] and used to compute a midpoint-rooted maximumlikelihood tree with 1,000 bootstrap replicates in IQ-TREE version 1.6.12 [14], using the ModelFinder [15] and ultrafast bootstrap [16] algorithms (Fig. 2). ModelFinder suggested LG+F+R10 as the optimal substitution model.  Funding Open access funding provided by University of Helsinki including Helsinki University Central Hospital. This study was supported by grants from the Jane and Aatos Erkko Foundation, the Sigrid Jusélius Foundation, the Academy of Finland, the University of Helsinki Doctoral Programme in Interdisciplinary Environmental Sciences (DENVI), the Paulo Foundation and the Finnish Cultural Foundation the Kymenlaakso Regional Fund.

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Conflict of interest The authors declare no conflict of interest.
Availability of data and material The Mekrijärvi negevirus genome sequence has been deposited in the NCBI GenBank database under accession number MT522375.
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Fig. 2
Phylogenetic tree of negeviruses and related viruses (n = 43) computed from protein sequences of ORF1 containing the RNAdependent RNA polymerase (RdRp). The host, country of origin, and collection year are shown in square brackets. Unknown information is indicated by a hyphen. Sequences were aligned using MAFFT, and a maximum-likelihood tree with 1,000 bootstrap replicates was built using IQ-TREE. The tree is rooted at the midpoint