JVI Accepted Manuscript Posted Online 21 June 2017 J. Virol. doi:10.1128/JVI.00680-17 Copyright © 2017 American Society for Microbiology. All Rights Reserved. 1 High Resolution Meta-Transcriptomics Reveals the Ecological 2 Dynamics of Mosquito-Associated RNA Viruses in Western Downloaded from 3 Australia 4 5 Mang Shia, Peter Nevilleb,c, Jay Nicholsonb,c,d, John-Sebastian Edena,e, Allison Imriec*, 6 Edward C. Holmesa* 7 http://jvi.asm.org/ 8 aMarie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, 9 School of Biological Sciences and Sydney Medical School, The University of Sydney, 10 Sydney, Australia. 11 bEnvironmental Health Directorate, Public Health Division, Department of Health, 12 Government of Western Australia, Australia. on May 27, 2018 by UNIV OF WESTERN AUSTRALIA M209 13 cSchool of Biomedical Sciences, The University of Western Australia, Australia. 14 dCenter for Vectorborne Diseases, Department of Pathology, Microbiology and Immunology, 15 School of Veterinary Medicine, University of California, Davis, USA. 16 eCentre for Virus Research, The Westmead Institute for Medical Research, Sydney, Australia. 17 18 *Corresponding authors: 19 Edward C. Holmes – [email protected] 20 Allison Imrie – [email protected] 21 22 Word count: Abstract – 247, Importance – 119, Main Text – 4835 23 Running title: Ecology of the Mosquito Virome 1 24 ABSTRACT Mosquitoes harbour a high diversity of RNA viruses, including many that 25 impact human health. Despite a growing effort to describe the extent and nature of the Downloaded from 26 mosquito virome, little is known about how these viruses persist, spread, and interact with 27 both their hosts and other microbes. To address this issue we performed a meta- 28 transcriptomics analysis of 12 Western Australian mosquito populations structured by species 29 and geographic location. Our results identified the complete genomes of 24 species of RNA http://jvi.asm.org/ 30 viruses from a diverse range of viral families and orders, among which 19 are newly 31 described. Comparisons of viromes revealed a striking difference between the two mosquito 32 genera, with viromes of mosquitoes from the Aedes genus exhibiting substantially less 33 diversity and lower abundance than those of Culex genus, within which viral abundance on May 27, 2018 by UNIV OF WESTERN AUSTRALIA M209 34 reached 16.87% of the total non-rRNA. In addition, there was little overlap in viral diversity 35 between the two genera, although the viromes were very similar among the three Culex 36 species studied, suggesting that host taxon plays a major role in structuring virus diversity. In 37 contrast, we found no evidence that geographic location played a major role in shaping RNA 38 virus diversity, and several viruses discovered here exhibited high similarity (95-98% 39 nucleotide identity) to those from Indonesia and China. Finally, using abundance level and 40 phylogenetic relationships we were able to distinguish potential mosquito viruses from those 41 present in co-infecting bacteria, fungi, and protists. In sum, our meta-transcriptomics 42 approach provides important insights into the ecology of mosquito RNA viruses. 43 44 IMPORTANCE Studies of virus ecology have generally focused on individual viral 45 species. However, recent advances in bulk RNA sequencing make it possible to utilize meta- 46 transcriptomic approaches to reveal both complete virus diversity and their relative 47 abundance. We used such a meta-transcriptomic approach to determine key aspects of the 48 ecology of mosquito viruses in Western Australia. Our results show that RNA viruses are one 2 49 of the most important components of the mosquito transcriptome, and we identified 19 new 50 virus species from a diverse set of virus families. A key result was that host genetic Downloaded from 51 background plays a more important role in shaping virus diversity than sampling location, 52 with Culex species harbouring more viruses at greater abundance than those from Aedes 53 mosquitoes. http://jvi.asm.org/ on May 27, 2018 by UNIV OF WESTERN AUSTRALIA M209 3 54 Mosquitoes (Diptera: Culicidae) act as vectors for a number of disease agents that infect 55 humans and domestic animals, including malaria, dengue virus, Chikungunya virus, and Zika Downloaded from 56 virus. However, in addition to their role as transmission vectors, mosquitoes harbour a far 57 larger virome, including many viruses that are confined to these insects, such that they are 58 “insect-specific” (1, 2). Although these insect-specific viruses that have no direct impact on 59 public health, they may modulate the transmission of viruses that are pathogenic to http://jvi.asm.org/ 60 vertebrates (3). The development of metagenomic sequencing approaches has therefore led to 61 a re-evaluation of the mosquito virome, including the recent discovery of viruses in the 62 families Bunyaviridae (4-8), Rhabdoviridae (6, 9-11), Orthomyxoviridae (6, 12), Flaviviridae 63 (13-15), Mesoviridae (16), Reoviridae (8, 17), as well as in the unclassified Chuvirus (6) and on May 27, 2018 by UNIV OF WESTERN AUSTRALIA M209 64 Negevirus (18) groups. In addition, metagenomics surveys have discovered viruses in 65 families not previously known to infect mosquitoes, such as the Iflaviridae, Dicistroviridae, 66 Totiviridae, Chrysoviridae, and Narnaviridae (8, 19-22). Although these viruses have not 67 been isolated or characterized in vivo, their host association is supported by the presence of 68 related endogenous viruses in the genomes of various mosquito species (8). Hence, it is clear 69 that mosquitoes harbour a substantial viral diversity, the majority of which may not be 70 associated with vertebrates (1, 2). 71 Despite our expanding knowledge of the mosquito virome, there have been fewer 72 studies of ecological aspects of these viruses within their hosts (1). It has been suggested that 73 most of these newly discovered viruses share features that distinguish them from “classic” 74 human pathogens, including (i) an inability to infect vertebrates or vertebrate cell lines, (ii) a 75 high prevalence, (iii) prolonged host infection, and (iv) vertical transmission (1, 2, 23). Based 76 on these features, these mosquito viruses have been referred to as “commensal” microbes (3). 77 In reality, however, little is known about their natural infection status (e.g. abundance, 78 frequency of superinfection), host specificity in relation to different mosquito species, 4 79 geographic distribution and movement, and interactions with hosts and other microbes that 80 may be present within a specific host. Downloaded from 81 To reveal more of the natural ecology of mosquito RNA viruses we employed a meta- 82 transcriptomics approach to characterise the entire RNA environment excluding ribosomal 83 RNA (rRNA) within a mosquito sample. Meta-transcriptomics has several advantages over 84 approaches such as cell culture, consensus PCR, and metagenomics methods based on viral http://jvi.asm.org/ 85 particle purification (24, 25), and has proven successful in characterizing the RNA viromes of 86 diverse invertebrates (6, 8, 14, 20). Specifically: (i) it reveals the entire RNA virome, with 87 sufficient coverage to reconstruct complete viral genomes, including those from co-infecting 88 parasites; (ii) it provides a reliable quantification and assessment of both viral and host on May 27, 2018 by UNIV OF WESTERN AUSTRALIA M209 89 RNAs; and (iii) it is relatively simple, requiring minimal sample processing. Most 90 importantly, meta-transcriptomics provides more information than the genome sequence 91 alone, allowing a straightforward characterization of viral diversity and ecology. 92 To infer aspects of virome ecology among mosquito species sampled from different 93 geographic locations we characterized the total transcriptome of 12 mosquito populations, 94 comprising five species collected from four locations in Western Australia. In particular, we 95 determined the number, type, and abundance of each virus within the context of the host 96 transcriptome and that of other microbial symbionts/parasites, and addressed whether these 97 parameters varied by species and/or sampling location. 98 99 RESULTS 100 The mosquito virome. We characterized the total transcriptome of 12 mosquito pools, 101 representing five species of mosquitoes sampled from four geographic locations in Western 102 Australia (Fig. 1). RNA sequencing of ribosomal (r) RNA-depleted libraries resulted in 40-47 103 million reads per pool, which were assembled de novo into 159,861 to 225,352 contigs. 5 104 Subsequent blast analyses revealed the complete genomes of 24 species of RNA viruses, of 105 which 19 are newly described here. These virus species fell into a wide range of RNA virus Downloaded from 106 groups, including those that fell within existing families and orders, namely the 107 Bunyaviridae, Mononegavirales, Orthomyxoviridae, Narnaviridae, Mesoniviridae, 108 Partitiviridae, Reoviridae, Totiviridae, Chrysoviridae, as well as in several newly described 109 groups: Qinvirus (a highly divergent group of negative-sense RNA viruses (8)), the Partiti- http://jvi.asm.org/ 110 like viruses, the Luteo-like viruses and the Negev-like viruses (Table 1). Importantly, these 111 viruses were unlikely to represent endogenous viral elements (EVEs) as they were present as 112 complete genomes without any interruption by frame-shifts, nonsense mutations, repeat 113 sequences, reverse transcriptases, or other features that are common to EVEs. on May 27, 2018 by UNIV OF WESTERN AUSTRALIA M209 114 For each library, the number of virus species varied from 1 to 10 (Table 1). The 115 abundance (i.e. frequency) of each virus also varied from 0.013% to 16.87% of total non- 116 rRNA reads within the pool (Table 1). In comparison, the host gene RPL32, which is often 117 used as a reference gene in quantitative PCR assays, showed consistent abundance levels 118 across all libraries (from 0.034 – 0.065%, Table 2). This suggests that the huge variation in 119 viral number and abundance is unlikely to be an artefact of sample processing or nucleic acid 120 extraction.