bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Meta-transcriptomic comparison of the RNA viromes of the mosquito 2 vectors Culex pipiens and Culex torrentium in northern Europe 3 4 5 John H.-O. Pettersson1,2,3,*, Mang Shi2, John-Sebastian Eden2,4, Edward C. Holmes2 6 and Jenny C. Hesson1 7 8 9 1Department of Medical Biochemistry and Microbiology/Zoonosis Science Center, Uppsala 10 University, Sweden. 11 2Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, 12 School of Life and Environmental Sciences and Sydney Medical School, the University of 13 Sydney, Sydney, New South Wales 2006, Australia. 14 3Public Health Agency of Sweden, Nobels väg 18, SE-171 82 Solna, Sweden. 15 4Centre for Virus Research, The Westmead Institute for Medical Research, Sydney, Australia. 16 17 18 *Corresponding author: [email protected] 19 20 Word count abstract: 247 21 22 Word count importance: 132 23 24 Word count main text: 4113 25 26 1 bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 27 Abstract 28 There is mounting evidence that mosquitoes harbour an extensive diversity of 'insect-specific' 29 RNA viruses in addition to those important to human and animal health. However, because 30 most studies of the mosquito virome have been conducted at lower latitudes there is a major 31 knowledge gap on the genetic diversity, evolutionary history, and spread of RNA viruses 32 sampled from mosquitoes in northern latitudes. Here, we determined and compared the RNA 33 virome of two common northern Culex mosquito species, Cx. pipiens and Cx. torrentium, 34 known vectors of West Nile virus and Sindbis virus, respectively, collected in south-central 35 Sweden. Following bulk RNA-sequencing (meta-transcriptomics) of 12 libraries, comprising 36 120 specimens of Cx. pipiens and 150 specimens of Cx. torrentium, we identified 40 viruses 37 (representing 14 virus families) of which 28 were novel based on phylogenetic analysis of the 38 RNA-dependent RNA polymerase (RdRp) protein. Hence, we found similar levels of virome 39 diversity as in mosquitoes sampled from the more biodiverse lower latitudes. Four libraries, 40 all from Cx. torrentium, had a significantly higher abundance of viral reads, spanning ~7– 41 36% of the total amount of reads. Many of these viruses were also related to those sampled on 42 other continents, indicative of widespread global movement and/or long host-virus co- 43 evolution. Importantly, although the two mosquito species investigated have overlapping 44 geographical distributions and share many viruses, approximately one quarter of the viruses 45 were only found at a specific location, such that geography must play an important role in 46 shaping the diversity of RNA viruses in Culex mosquitoes. 47 48 Importance 49 RNA viruses are found in all domains of life and all global habitats. However, the factors that 50 determine virome composition and structure within and between organisms are largely 51 unknown. Herein, we characterised RNA virus diversity in two common mosquito vector 52 species, Culex pipiens and Culex torrentium, sampled from northern Europe. Our analysis 53 revealed extensive viral diversity, including 28 novel viruses, and was comparable to the 54 levels of diversity found in other temperate and tropical regions globally. Importantly, as well 55 as harbouring RNA viruses that are closely related to other mosquito-derived viruses sampled 56 in diverse global locations, we also described a number of viruses that are unique to specific 57 sampling locations in Sweden. Hence, these data showed that geographical factors can play an 58 important role in shaping virome structure even at local scales. 59 2 bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 60 Introduction 61 The mosquito (Diptera; Culicidae) genus Culex comprises more than a thousand species, with 62 representatives found globally (1). Culex species are vectors of a number of important 63 pathogens including West Nile virus (WNV) (Flaviviridae), Japanese encephalitis virus (JEV) 64 (Flaviviridae) and Sindbis virus (SINV) (Togaviridae), as well as a variety of nematodes (1– 65 3). One of the most widespread Culex species is the Northern House mosquito, Cx. pipiens, 66 that is distributed across the northern hemisphere. In Europe and the Middle East it occurs 67 together with Cx. torrentium, another Culex species with females and larvae that are 68 morphologically identical to Cx. pipiens. These two species have overlapping distributions 69 and share larval habitats. However, Cx. torrentium dominates in northern Europe while Cx. 70 pipiens is more abundant in the south (4). Both species are vectors for a number of bird- 71 associated viruses that can cause disease in Europe; for example, WNV, that may cause a 72 febrile disease with encephalitis, and SINV that may result in long lasting arthritis (2, 5). Cx. 73 pipiens is one of the most common WNV vectors in both southern Europe and North 74 America, and Cx. torrentium is the main vector of SINV in northern Europe (2, 6). Infections 75 with these pathogenic viruses occur in late summer when the viral prevalence accumulates in 76 passerine birds, the vertebrate hosts of both of these viruses (7, 8). Despite their importance as 77 vectors, little is known about the detailed biology of Cx. pipiens and Cx. torrentium due to the 78 difficulties in species identification, which can only be reliably achieved through molecular 79 means. Much of the biology of these species, such as their larval habitat and feeding 80 preferences, is considered similar. However, one significant difference between the two 81 species is that while Cx. pipiens harbours a high prevalence of the intracellular bacteria 82 Wolbachia pipientis, it is seemingly absent in Cx. torrentium (9). 83 84 In recent years, studies utilizing RNA-sequencing (RNA-Seq, or 'meta-transcriptomics') have 85 revealed an enormous RNA virus diversity in both vertebrates and invertebrates (10, 11). 86 Mosquitoes are of particular interest as many are well-known vectors of pathogenic viruses. 87 Importantly, recent studies have shown that these pathogenic viruses represent only a fraction 88 of the total virome in the mosquito species investigated. Indeed, mosquitoes clearly carry a 89 large number of newly described and divergent arthropod-specific viruses, with 90 representatives from many genetically diverse virus families and orders, such as the 91 Flaviviridae, Togaviridae and the Bunyavirales (12–16). However, most studies have been 92 conducted on latitudes below 55°, such that there is a marked lack of data of the mosquito 93 viral diversity present in northern temperate regions where the composition of mosquito 3 bioRxiv preprint doi: https://doi.org/10.1101/725788; this version posted August 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 94 species as well as environmental parameters differ significantly from lower latitudes. Indeed, 95 for many forms of life, biodiversity increases towards the equator (17), and the species 96 richness of mosquitoes is greater in tropical regions than temperate regions (18). A central 97 aim of the current study was therefore to investigate whether viral diversity co-varies in the 98 same manner. Given that Cx. pipiens and Cx. torrentium are two common Culex species in 99 northern and central Europe, and known vectors of SINV and WNV, they were chosen for 100 RNA virome investigation and comparison by RNA-Seq. 101 102 Results 103 RNA virome characterisation 104 We characterised the RNA viral transcriptome of two mosquito species, Cx. pipiens and Cx. 105 torrentium, collected from central and southern Sweden (Suppl. table 1). After high- 106 throughput sequencing, a total of 569,518,520 (range 34,150,856–62,936,342) 150bp reads 107 were produced from 12 ribosomal RNA-depleted sequence libraries that were assembled into 108 153,583 (4,333–33,893) contigs. From all the contigs assembled, we identified 40 that 109 contained RdRp sequence motifs and hence indicative of viruses, belonging to 14 different 110 viral families/orders: Alphaviridae, Bunyavirales, Endornaviridae, Luteoviridae, 111 Mononegavirales, Nidovirales, Orthomyxoviridae, Partitiviridae, Picornaviridae, Reoviridae, 112 Totiviridae, Tymoviridae and representatives from the divergent Virgaviridae, Negeviridae- 113 and Qin-viruses. For each viral family/order, between one and five virus species were 114 identified and in total 28 novel RNA virus species were discovered here, which were named 115 based on geographical location. 116 117 The relative number of all virus reads, as mapped to contigs with RdRp-motifs, compared to 118 the total amount of non-ribosomal RNA reads per library varied between 0.1–36.6% (Table 119 1). Notably, libraries 2, 10, 11 and 12 from Cx. torrentium were characterised by a 120 significantly higher number of viral reads compared to non-viral reads (Figure 1, Table 1).