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1 Four novel detected in Magellanic Penguins (Spheniscus magellanicus) in 2 Chile 3 4 Running title: Novel picornaviruses in Magellanic Penguins 5 6 Juliette Hayer1, Michelle Wille2, Alejandro Font3, Marcelo González-Aravena3, Helene Norder4,5, 7 Maja Malmberg1,6 8 9 10 11 1 - Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, 12 Uppsala, Sweden; 13 2 - Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and 14 Environmental Sciences and School of Medical Sciences, The University of Sydney, Sydney, 15 Australia 16 3 - Instituto Antártico Chileno, Plaza Muñoz Gamero 1055, Punta Arenas, Chile; 17 4- Department of Infectious Diseases/Virology, Institute of Biomedicine, Sahlgrenska Academy, 18 University of Gothenburg, Sweden 19 5- Region Västra Götaland, Sahlgrenska University Hospital, Department of Clinical Microbiology, 20 Gothenburg, Sweden 21 6 - Department of Biomedical Sciences and Veterinary Public Health, Swedish University of 22 Agricultural Sciences, Uppsala, Sweden 23 24 Co-corresponding authors: Juliette Hayer [email protected] ; Maja Malmberg 25 [email protected] 26 27 Word count (abstract): 219 28 Word count (text): 4377 29

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30 Abstract 31 32 Members of the Picornaviridae comprise a significant burden on the poultry industry, causing 33 diseases such as gastroenteritis and hepatitis. However, with the advent of metagenomics, a number 34 of picornaviruses have now been revealed in apparently healthy wild birds. In this study, we 35 identified four novel belonging to the family Picornaviridae in healthy Magellanic Penguins 36 (Spheniscus magellanicus), a near threatened species found along the coastlines of temperate South 37 America. We collected 107 faecal samples from 72 individual penguins. Twelve samples were 38 initially sequenced by high throughout sequencing with metagenomics approach. All samples were 39 subsequently screened by PCR for these new viruses, and approximately 20% of the penguins were 40 infected with at least one of these viruses, and seven individuals were co-infected with two or more. 41 The viruses were distantly related to members of the genera Hepatoviruses, Tremoviruses and 42 unassigned viruses from Antarctic Penguins and Red-Crowned Cranes. Further, they had more than 43 60% amino acid divergence from other picornaviruses, and therefore likely constitute novel genera. 44 That these four novel viruses were abundant among the sampled penguins, suggests Magellanic 45 Penguins may be a reservoir for several picornaviruses belonging to different genera. Our results 46 demonstrate the vast undersampling of wild birds for viruses, and we expect the discovery of 47 numerous avian viruses that are related to Hepatoviruses and Tremoviruses in the future. 48 49 Importance 50 Recent work has demonstrated that Antarctic penguins of the genus Pygoscelis are hosts for an 51 array of viral species. However, beyond these Antarctic penguin species, very little is known about 52 the viral diversity or ecology in this highly charismatic avian order. Through metagenomics we 53 identified four novel viruses belonging to the Picornaviridae family in faecal samples from 54 Magellanic Penguins. These highly divergent viruses, each possibly representing novel genera, are 55 related to members of the Hepatovirus, genera, and unassigned picornaviruses 56 described from Antarctic Penguin and Red-crowned Cranes. By PCR these novel viruses were 57 shown to be common in Magellanic Penguins, indicating that penguins may play a key role in their 58 epidemiology and evolution. Overall, we encourage further sampling to reveal diversity, 59 ecology, and evolution in these unique avian taxa. 60 61 Keywords: Sphenisciformes, penguins, Picornaviridae, viral metagenomics, Hepatovirus 62

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63 Introduction 64 65 Penguins (Order: Sphenisciformes) are unique in the avian world. Through evolution, they have 66 acquired numerous physiological adaptations to exploit and thrive in marine environments. 67 Penguins are found throughout the temperate regions of the Southern Hemisphere, ranging from the 68 Antarctic continent to as far north as the Galapagos Islands. Despite population declines of many 69 penguin species, largely due to climate change and overfishing of their prey, little is known about 70 the pathogens and parasites harboured by these birds. Indeed, infections with various 71 microorganisms are known to play a role in reducing avian populations, such as substantial declines 72 of native Hawaiian birds due to avian malaria, negative survival effects of albatross due to avian 73 cholera, and an negative survival effect of a number of North American passerine species due to 74 infection with West Nile Virus (1-4). 75 76 To date, studies of viral presence and prevalence among penguins has been limited, opportunistic, 77 and related to sick birds in nature (eg (5-7)) or in rehabilitation centres (eg (8)). Further, studies of 78 viruses in penguins are highly biased towards the charismatic Antarctic Penguins, with serology 79 studies dating back to the 1970’s (9). Due to technological limitations, i.e. both serology and PCR 80 based studies allow for the assessment of only described viruses, very little progress has been made 81 revealing the viral communities in these species. This has changed dramatically with the rise of 82 metagenomics and metatranscriptomics (9, 10), wherein novel and highly divergent viral species 83 may be described. As a result, since 2015, more than 25 different novel viruses have been described 84 in Antarctic and sub-Antarctic penguins (9-11). These viruses include members from the 85 , Astroviridae, , , , , 86 , Orthomyxovirdae, , , Picornaviridae, 87 Picobirnaviridae and (9, 10, 12-15). Beyond Antarctica, evaluation of penguins for 88 viruses is haphazard, although many of the same viral families have been detected through both 89 virology and serology studies of Magellanic Penguins (Spheniscus magellanicus), African Penguins 90 (Spheniscus demersus), Little Blue Penguins (Eudyptula minor), and Galapagos Penguins 91 (Spheniscus mendiculus) (6-8, 16-23). It is clear from these studies that the viral diversity of 92 penguins is vastly underappreciated, and thus the role of penguins as potential hosts for an array of 93 viruses are yet to be revealed. 94 95 Through metagenomics, a number of novel picornaviruses have recently been described in samples 96 from wild birds. These viruses fall into the genera Megrivirus, Sapelovirus, Avihepatovirus, and 97 other highly divergent viruses with unassigned genera (24-29). In penguins, seven picornaviruses

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98 have been described: Ross Virus, Scott Virus, Amundsen Virus (Hepatovirus/Tremovirus-like), 99 Shirase Virus (Sinicivirus-like), Wedell Virus (Pigeon B-like), Pingu Virus 100 (Gallivirus-like) and Penguin Megrivirus, all of which have been isolated from apparently healthy 101 Antarctic penguins (10, 13, 30). Until recently, avian picornaviruses were exclusively associated 102 with morbidity and mortality in poultry and other birds (pigeons and passerines) (31, 32). Through 103 an increased effort in investigating the virome in healthy wild birds, we are beginning to rewrite the 104 narrative of many viral families. There is mounting evidence to suggest that picornaviruses are not 105 exclusively disease causing, but rather that many picornavirus species detected in wild birds are not 106 associated with any signs of disease. 107 108 In this study we aimed to identify and characterise viruses of Magellanic Penguins. We sampled 109 faeces from Magellanic Penguins breeding on the Magdalena island, an important breeding colony 110 for this species, situated in southern Chile, and we used a combination of high-throughput 111 metagenomic sequencing, followed by PCR screening to disentangle virus diversity and prevalence. 112 The detection of four novel viruses at high prevalence in penguins without obvious disease, 113 provides further evidence that penguins are reservoir hosts for a multitude of viruses belonging to 114 different virus families and genera, including numerous viruses yet to be revealed. Finally, this 115 finding has important evolutionary implications for the emergence/evolution of the picornavirus 116 lineage comprising Hepatovirus and Tremoviruses. 117 118 Results 119 120 Sequence data and assembly 121 We performed metagenomics sequencing of 12 faecal samples collected from Magellanic Penguins 122 in a colony on Magdalena Island, Chile. 123 High-throughput sequencing of the sample produced 25,357,939 reads (range 517,072-3,558,413 124 reads) with a mean length of 229 bp representing a total of 5.86 gigabases of sequence data. After 125 quality control and filtering using PRINSEQ 24,027,073 reads (range 463,974-3,438,3911 reads) 126 remained (5.75 Gb). Of these we assembled an average of 10,228 contigs, of which 4% comprised 127 viral contigs (Table S1). Reads have been deposited to the European Nucleotide Archive (ENA), 128 accession number PRJEB40660. 129 For all samples, about 25% of the reads could be taxonomically classified. For 7 samples, greater 130 than 50% of the reads could be classified. The majority of these reads were microbial, i.e. bacterial 131 or viral; however, a number of penguin (host) reads were also identified. For 2 samples, is_034_015 132 and is_034_016 viral reads were found in 33.8% and 17.3% of all reads. A vast majority of those

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133 were classified as viruses belonging to the Picornaviridae family, and three previously not 134 described viral genomes were assembled. From sample is_034_018, 33% (397 out of 1,179 contigs) 135 were classified as viral, of which 313 were classified as Picornaviridae. 136 Overall at the contig level, more than 25% of de novo assembled contigs were successfully 137 classified.Eight samples had greater than 50% of the contigs classified as bacterial, viral or 138 chordates. 139 140 Four novel, divergent picornaviruses in Magellanic Penguins 141 142 We revealed four novel picornaviruses in faecal samples from Magellanic Penguins: Sphenifaro 143 virus, Sphenigellan virus, Sphenimaju virus, Sphenilena virus. These viruses were recovered from 3 144 different genomic libraries corresponding to 3 samples from individuals. Two viruses (Spenifaro 145 virus and Sphenilena virus) were identified in a single sample, i.e. from the same penguin. Overall, 146 the abundance (proportion of reads based on read mapping back to assemblies) of each virus was 147 low (<1%) with the exception of Sphenimaju virus, which comprised 58.57% of all reads in the 148 sample (is_034_015). The sample is_034_016 that contained two different viruses (Sphenifaro virus 149 and Sphenilena virus) had low abundance for both viruses. Each of these viruses comprised 0.29% 150 and 0.21% of the reads in the sample, respectively (Table 1). 151 152 These new viruses, recovered from the same penguin colony at the same time, are highly divergent 153 from each other, sharing only 32-43% similarity at the amino acid level, suggesting that they 154 represent not only four different species but potentially four different genera. Three of the viruses 155 were most similar to members in the genus Hepatovirus in the Picornaviridae family when the 156 genomes were analysed by Blastx. However, at the amino acid level the similarity was low, ranging 157 from 35-37% (Table 1). The fourth virus, Sphenifaro virus, was most similar to an unassigned virus 158 described in Red-crowned Cranes (Grus japonensis) when using Blastx, although as with the Blast 159 results of the other viruses, the amino acid percentage identity was low (37.91%). By phylogenetic 160 analysis these viruses were found in the same major lineage as members of the Hepatovirus and 161 Tremovirus genera, in addition to newly described viruses from Antarctic Penguins (10) and Red- 162 crowned Cranes (29) (Figure 1, Figure S1). However, as with the blast results, the viruses revealed 163 here shared <30% amino acid similarity in the P1 region to other viruses in this lineage. Further, all 164 four viruses had long branch lengths in the phylogeny, all this together suggest that each of these 165 new picornaviruses may represent new genera in the Picornaviridae family demonstrating the vast 166 undersampling of viruses in this part of the tree. 167

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168 Phylogenetic analysis reveals that the most closely related fish picornaviruses fall into a sister clade 169 to the avian and mammalian viruses of the Hepatovirus, Tremovirus and the recently described 170 picornaviruses from Antarctic penguins, red-crowned cranes and the new viruses described here 171 (Fig 1). Further, the viruses described in this study have less than 36% (22-36%) amino acid 172 similarity to picornaviruses from fish across the P1. As such, the evidence suggests that the viruses 173 revealed here are most likely bona fide avian viruses and do not derive from the prey. 174 175 To investigate the frequency of these four new viruses in other samples sequenced by high 176 throughput sequencing, all libraries were mapped against each of these new viral genomes. In the 177 sample containing Sphenimaju virus (is_034_015), there were over 5,000 reads mapping to the 178 Sphenigellan virus. In the sample with both Sphenifaro virus and Sphenilena virus (is_034_016), 179 there were over 1,000 reads mapping the Sphenigellan virus and Sphenimaju virus. Overall, there 180 were 4 libraries with no evidence of any of the novel viruses described here, 2 with reads against 181 only one of the novel viruses, and 6 libraries with evidence of more than one of these viruses (Table 182 S2). 183 184 Using InterProScan domain prediction and our knowledge of Picornaviridae genomic features, we 185 could predict the 3 main regions P1, P2, P3 for three of the viruses (Figure 1B). For Sphenilena 186 virus, it was not possible to predict the exact P2 region location. Mature peptides 1AB, 1C and 1D 187 could also be located, as well as 2C, 3C and 3D peptides for all 4 viruses. On the contrary, peptides 188 2A, 2B, 3A and 3B could not be predicted from the 4 polyproteins, however, most of the viral 189 functional domains could be identified (Table S3 – S6): the viral coat, the helicase, the NTPase and 190 the RNA dependant RNA-polymerase. The 4 assembled viral genomes and their annotations have 191 been deposited to ENA, accession numbers: ERZ1667848, ERZ1667849, ERZ1667850, 192 ERZ1667851. 193 194 The four new picornaviruses are common among Magellanic penguins 195 To reveal the prevalence of the new viruses identified in this study, 107 faecal samples were 196 screened by PCR for these 4 viruses. The samples were from 72 individual Magellanic Penguins, 197 one Rockhopper penguin (Eudyptes chrysocome chrysocome), and three Kelp gulls (Larus 198 dominicanus). Overall, at least one of these 4 viruses was detected in 28 samples (25%; 95% 199 confidence interval [CI] 18-34%). All positive samples were from Magallanic Penguins, of which 200 22 were positive for at least one of the 4 viruses (21%, 95 CI 14-30%). The prevalence was highest 201 for Sphenilena virus with 11 PCR positive samples. Sphenifaro virus, Sphenigellan virus and 202 Sphenimaju virus were found in in 7, 7, and 9 Magellanic Penguin individuals, respectively. There

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203 was no statistically significant difference in prevalence between the viruses (X2 =3.01, df=3, 204 p=0.3886) (Fig 3). Interestingly, samples from 7 individuals contained more than one of these 205 viruses. Two individuals were positive for Sphenifaro virus and Sphenigellan virus, one individual 206 for Sphenifaro virus and Sphenimaju virus, three individuals were positive for Sphenimaju virus 207 and Sphenilena virus, finally, one sample contained 3 viruses: Sphenifaro, Spehnigellan, and 208 Sphenilena virus. 209 210 Sequencing the ~460bp PCR product demonstrated sequence variability for each of the new viruses. 211 There was an average pairwise identity of 91.6%, 96.5%, 98% and 91% for Sphenifaro, 212 Sphenigellan, Sphenimaju, and Sphenilena virus, respectively. The pairwise similarity for 213 Sphenimaju was higher (i.e. less diversity) than that for the other species, despite having a 214 comparable number of PCR products (n=9) (Fig 4) 215 216 217 Table 1: Metadata for the four novel hepato-like viruses revealed in this study Sample Virus Name Top Blastn Blastn Lengt Number Number of Name hit percent h of reads reads age in (proportion of identity sample reads in sample) is_034_0 Sphenifaro AUW34301 37.91% 7201 916,871 2,687 (0.29%) 16 virus Picornaviridae bp red crowned crane is_034_0 Sphenigellan YP_00916403 38.76% 7384 1,090,48 1,238 (0.11%) 18 virus 0 Phopivirus bp 9 is_034_0 Sphenimaju YP_00917921 38.37% 7441 2,689,81 1,575,605 15 virus 6 Bat bp 3 (58.57%) hepatovirus is_034_0 Sphenilena YP_00921578 35.83% 7048b 916,871 1,919 (0.21%) 16 virus 0 Tupaia p hepatovirus A 218 219

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220 221 Figure 1: Genomic features of the novel picornaviruses revealed in this study (A) Phylogeny of the P1 mature 222 peptide of select genera of the Picornaviridae. We included members of lineage VI and V, as defined by (32), and the 223 tree was rooted based on this lineage divergence. In addition to avian genera, we also included members of the 224 Limnipivirus and Potamipivirus known to infect fishes and Parechoviruses known to include mammals. Viruses 225 described here are adjacent to a filled circle. Genera and clades dominated by fish viruses have been indicated by a fish 8 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.26.356485; this version posted October 28, 2020. 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 4.0 International license.

226 silhouette downloaded from phylopic.com and distributed by a Creative Commons attribution. Penguin silhouette was 227 developed by M. Wille. Bootstrap values >70% are shown. The scale bar indicates the number of amino acid 228 substitutions per site. A tree including all Picornavirus genera found in birds as is presented in Figure S1 (B) Genome 229 arrangement for the four novel picornaviruses described in this study. Domains we were able to identify are indicated 230 by filled box and place according to the amino acid position. Domains were identified using InterProScan. Detailed 231 results of domain searches are presented in Table S3-S6. (B) Amino acid identity (percentage similarity) of the viruses

232 revealed in this study and select reference sequences of the domains 2CNTPase, 3CPeptidase and 3DRdRp 233 234

235 236 Figure 2: Diversity of four novel picornaviruses revealed through PCR. (A) Prevalence of each novel virus 237 revealed in this study across samples collected from Magellanic Penguin individuals. The point estimate is presented as 238 a filled circle and error bars correspond to 95% confidence intervals (B) Maximum likelihood tree of ~400bp PCR 239 products sequenced from 20 positive samples. The tree was midpoint rooted for clarity only. Scale bar represents 240 number of nucleotide substitutions per site. Viruses sequences assembled following metagenomic sequencing are 241 indicated with a filled circle. 242 243 Discussion 244 In this study we aimed to reveal the viral diversity of Magellanic Penguins sampled in Chile. By 245 using high throughput sequencing we found four novel viruses belonging to the family 246 Picornaviridae. These viruses are highly divergent and share less than 40% of the amino acid 247 sequence in the P1 region with members of Hepatovirus and Tremovirus that they most closely 248 resemble. The vast majority of the viral reads from most libraries mapped to these novel viruses 249 indicate that these viruses were the dominant species in the faecal virome of the penguins. Further, 250 through PCR screening we showed that over 20% of the sampled penguins in this study were 251 shedding at least one of these viruses, and many individuals were co-infected with two or three of 252 these new viruses. The viruses revealed here, in addition to novel and unassigned viruses from 253 Antarctic Penguins (14) and Red-crowned Cranes (29) likely constitute at least three novel genera. 254 This finding of very divergent avian picornaviruses forming one branch together with members of

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255 the genera Tremovirus and Hepatovirus shows on the need to further classify the genera with the 256 Picornaviridae family into subfamilies or lineages, and illuminates that there are many more 257 picornaviruses to be identified, especially from this part of the Picornaviridae phylogeny. 258 259 The charismatic Sphenisciformes have long been a target for virus surveillance, with early studies 260 initiated in Antarctic Penguins in the 1970’s (9, 33). These studies were of course limited to 261 screening for only described viruses, which until recently were viruses of poultry such as influenza 262 A virus, Newcastle Disease virus, infectious bursal disease virus (e.g. (9, 33-36). As a consequence 263 of this focus on poultry relevant virus surveillance, until very recently, few viruses had been 264 described in penguin species, globally. While our viral catalogues are expanding, without detailed 265 virus ecology studies, it remains unclear the role that penguins may play as hosts to an array of 266 viruses. This is particularly true for viruses detected in penguins in rehabilitation centres (8). The 267 high prevalence of the four novel viruses identified in this study, and the high frequency of co- 268 infections identified, indicates that these penguins are likely an important reservoir hosts for these 269 viruses. To understand the host range of these viruses, we would strongly encourage for the 270 sampling and surveillance of not only other populations of Magellanic Penguins, but also other 271 penguin species found in South America, and globally. Evidence for connectivity of penguin 272 species and populations as hosts for viruses is sparse, however studies from avian avulavirus 273 provide some clues. First, we have now seen repeated detection of Avian avulavirus 17, 18, 19 in 274 three Antarctic Penguin species and across a number of colonies along the Antarctic peninsula. 275 More interesting was the detection of avian avulavirus 10, first in Rockhopper Penguins from the 276 Falkland/Malvinas Island (15), and more recently in Antarctic Penguins (14). This data suggests 277 that there is capacity of viruses to be shared across multiple penguin species and locations. How 278 these viruses may fit into the larger migratory flyways used by other birds in South America, such 279 as Red Knot (Calidris canutus), is very unclear (37, 38). In addition to ecological questions, of 280 importance would be to understand the route of transmission of these viruses. Given the detection in 281 faeces, and the fact that faecal-oral route of transmission is very common in RNA viruses of wild 282 birds, such as influenza A virus and avian avaulaviruses, we may speculate that these viruses are 283 transmitted by the faecal oral route. This, however, would need to be confirmed by dedicated 284 studies. Taken together, we would argue for, not only more sampling and metagenomic studies in 285 these hosts, but also more consistent and repeated sampling to reveal both virus diversity and virus 286 ecology. 287 288 Metagenomic tools have rapidly allowed for the expansion of described avian picornaviruses, but 289 also redefined our understanding of the impact that these viruses have on their hosts. Prior to 2010,

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290 almost all described avian picornaviruses caused disease in their hosts. This includes duck hepatitis 291 virus (genus Avihepatovirus) (39), hepatitis virus (genus Megrivirus) (40) and avian 292 sapelovirus (genus Sapelovirus) (41) which cause hepatitis in domestic ducks and turkeys, avian 293 encephalomyelitis virus (genus Tremovirus) causing a neural disorder encephalomyelitis (42) in 294 domestic gallinaceous birds, and a number of viral genera causing gastroenteritis in domestic 295 galliforms (31). Indeed, a recent metagenomic study revealed that a with gastroenteritis was 296 shedding 6 picornaviruses, simultaneously, although it is unclear which, if any, of these viruses was 297 causing the disease (31). There is no doubt that the viruses isolated from poultry cause disease, 298 however metagenomic studies have now revealed more than 20 novel picornavirus species in wild 299 birds, and on all occasions the sampled birds had no signs of disease (10, 13, 24-29), with the 300 exception of Poecevirus causing avian keratin disorder (43). In this study, we demonstrated not only 301 a high viral load (large proportion of reads), but also that a number of penguins were co-infected 302 with at least two of the new picornaviruses described here without any obvious signs of disease. 303 Similarly, in a metagenomic study, Wille et al. (2018) found Red-necked Avocets (Recurvirostra 304 novaehollandiae) to be co-infected with nine different viruses, including 3 highly divergent 305 picornaviruses. This suggests that wild birds are able to tolerate high viral loads and diversity in the 306 absence of overt disease (44, 45). One hypothesis for the discordance in disease signs in wild birds 307 as compared to poultry is that wild birds have a long history of host-pathogen co-evolution (24, 46). 308 Mass produced and ducks, which suffer from disease when infected with an array of virus 309 species, are a relatively new host niche in evolutionary time (47, 48). 310 311 With more viral metagenomic studies being undertaken in under sampled hosts, such as fishes, 312 many viral families once thought to exclusively infected birds and mammals now include members 313 infecting fish and amphibians (49). Indeed, a number of picornaviruses have now been described in 314 fish, and these viruses belong to genera falling within lineages previously dominated by viruses of 315 mammmals and birds. The Limnipvirus and Potamipivirus genera are a case in point; these viruses 316 fall within lineage IV (as defined by (32)) which includes a number of mammalian and avian 317 infecting viral genera such as Avihepatovirus and Parechovirus (49-51). Shi et al. further described 318 a number of fish viruses falling sister to established lineage IV viruses (Hepatovirus and 319 Tremovirus) (49). Given that the viruses we characterized in this study were all identified in birds 320 and are not in clades comprising fish viruses strongly suggests that we have detected avian viruses, 321 rather than viruses of the diet of penguins. This distinction is especially important to clarify in 322 samples collected from cloacae or faeces. These viruses of fish further demonstrate important 323 evolutionary patterns in the lineage V viruses. That the phylogeny of the Hepatovirus, Tremovirus, 324 and unassigned avian viruses and fish viruses generally follows the phylogeny of the hosts from

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325 which they are sampled suggests that there has been a process of virus-host co-divergence, that 326 likely extends hundreds of millions of years. It further suggests that we may expect to find 327 numerous avian picornaviruses that fall sister to the Hepatovirus and Tremovirus genera. It has been 328 estimated that over 99% of viruses are still to be described (52), and therefore we anticipate a large 329 number of picornaviruses are yet to be identified particularly from the incredible diversity of wild 330 birds. 331 332 Material and Methods 333 334 Ethics Statement 335 The project was ethically approved by the Chilean National Forestry Corporation (CONAF) for the 336 region of Magallanes, Chile (RESOLUCIÓN No :517/2015). 337 338 Study site 339 Sampling was carried out on the Magellanic penguin colony situated on the Magdalena Island in the 340 Strait of Magellan in Chilean Patagonia (-52°55′10″S 70°34′34″W), from 19 - 21 November 2015. 341 The colony comprises approximately 63,000 breeding pairs of penguins as the last count in 2007 342 (53). 343 344 Freshly deposited faecal samples (n=107) were collected from birds comprising 72 Magellanic 345 penguins, 1 Southern Rockhopper penguin and, 3 Kelp Gulls. Some penguin faeces (n=30) were 346 sampled on more than once. An additional 2 samples were collected from the soil around the 347 colony. All samples were collected using sterile plastic tools and placed in 1 ml RNAlater 348 (ThermoFisher) and stored at room temperature for up to 72h prior to storage in -80 °C. For eight 349 penguins, a duplicate sample was stored dry in sterile tubes without RNAlater for approximately 5h 350 at 8 °C prior to storage in -80 °C. Samples remained at -80 °C until processing. 351 352 Sample preparation 353 An initial 12 faecal samples from Magellanic penguins were prepared for viral metagenomic 354 sequencing. Eight of the samples had been stored in RNAlater (ID:s 1,11,34,36,51,81,87,88) and 4 355 samples were stored in dry tubes without RNAlater (NR2, NR4, NR5, NR6). Dry samples were 356 treated with 20 U RNase I (Epicentre) prior to RNA extraction. 357 358 All samples were homogenized in 800μl PBS using OmniTip Homogenizer (Omni International 359 Inc) for 15 sec and then placed on ice for 30 sec followed by 15 sec homogenization. The

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360 homogenate was centrifuged at 4,000 rpm for 10 minutes, thereafter the supernatant was transferred 361 to a 0.65μm filter (Millipore) and centrifuged at 12,200 rpm for 4 minutes. The filtrate was further 362 transferred to a Ultrafree-CL GV 0.22 μm Sterile filter column and centrifuged at 4,800 rpm for 4 363 minutes. Thereafter RNA was extracted using Trizol and choloroform and the RNA was cleaned up 364 using RNAeasy (Qiagen) according to protocol. Qubit HS RNA assay was used for quantification. 365 366 The extracted RNA was amplified using Ovation RNA-Seq v2 (NuGEN) according to the 367 manufacturer’s recommendations. The final product was purified with GeneJET PCR purification 368 kit (ThermoFisher) followed by Qubit HS DNA assay for quantification. 369 370 A negative PBS control samples was included throughout the laboratory workflow. 371 372 Library preparation and sequencing 373 Sequencing libraries were constructed using the AB Library Builder System (Ion Xpress™ Plus and 374 Ion Plus Library Preparation for the AB Library Builder™ System protocol, ThermoFisher) and 375 size selected on the Blue PippinTM (Sage Science). Library size and concentration were assessed 376 by a Bioanalyzer High Sensitivity Chip (Agilent Technologies) and by the Fragment Analyzer 377 system (Advanced Analytical). Template preparation was performed on the Ion Chef™ System 378 using the Ion 520 & Ion 530 Kit-Chef (ThermoFisher). Samples were sequenced on 530 chips using 379 the Ion S5™ XL System (ThermoFisher). 380 381 Bioinformatics analysis 382 The obtained reads were trimmed by quality in 5’ and 3’ and filtered by mean quality using 383 PRINSEQ (v 0.20.4) with a PHRED quality score of 20 (54). The good quality reads were used to 384 produce de-novo assemblies with Megahit version 1.1.1 (55). A taxonomic classification of the 385 obtained contigs was performed by running Diamond (v 0.9.6) (56) against the non-redundant 386 protein database (release April 2019) and using the LCA algorithm from Megan 6 (v6.11.7) (57) to 387 visualise the classification of each contig. A taxonomic classification at the read level was also 388 performed using Diamond (blastx + LCA, using the output format 102). The classified output table 389 was converted into a Kraken report to allow visualisation with the R package Pavian (58). 390 391 Comparative genomics and phylogenetic 392 We interrogated the 4 contigs representing near full length picornavirus genomes (>6000bp). Gene 393 prediction was performed using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) and protein

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394 domains prediction using InterProScan (59). Reads were subsequently mapped back to viral 395 contigs using the Burrows Wheeler Aligner BWA-MEM (60). 396 397 Amino acid sequences of the polyprotein were aligned using MAFFT with the E-INS-I algorithm 398 (61). Reference genomes for the Picornaviridae tree were limited to those genera containing avian 399 (and mammalian) genera as per Wille et al. 2019b. Gaps and ambiguously aligned regions were 400 stripped using trimAL (62). The most appropriate amino acid substitution model was then 401 determined for each data set, and maximum likelihood trees were estimated using IQ-TREE (63). 402 403 Annotation and file conversion for submission of annotated viral genome to ENA 404 For generating EMBL flat files to submit the annotated viral genome to ENA, we have used several 405 tools. First, we used Prokka (64), for generating a GFF file with the polyprotein coding sequence 406 (CDS) with some manual modification if required. 407 Using scripts from AGAT (Another Gff Analysis Toolkit; https://github.com/NBISweden/AGAT, 408 version v0.5.0), the corrected GFF files were standardised with ‘agat_convert_sp_gxf2gxf.pl’ and 409 proteins were extracted using ‘agat_sp_extract_sequences.pl’. The extracted polyproteins were then 410 run through InterProScan online and TSV files containing the domain annotations were 411 downloaded. The InterProScan annotation could be integrated into the GFF file using 412 ‘agat_sp_manage_functional_annotation.pl’. In order to submit the annotated viral genomes to 413 ENA, the GFF files were converted into EMBL file using EMBLmyGFF3 (65). 414 415 Ascertaining the prevalence of four novel picornaviruses 416 RNA was extracted from the 107 faecal samples using QIAamp Fast DNA Stool mini kit (Qiagen), 417 followed by cDNA synthesis using the High-Capacity cDNA Reverse Transcription (Applied 418 Biosystems). Custom primers were designed for each of the four hepato-like viruses revealed 419 (Table S7). For Sphenifaro and Sphenigellan viruses a nested PCR approach was employed, and for 420 Sphenimaju and Sphenilena viruses a semi-nested approach was employed. The 50 μl PCR reaction

421 mix contained 1xTaq Buffer, 2.25mM of MgCl2 (Applied Biosystems), 0.2mM of dNTP (Roche), 1 422 U of Taq Polymerase (Roche), 1.5mM of each primer and 5μl of cDNA. The same reaction mix 423 was used in the nested PCRs with 5μl of the first PCR product as template. The PCR reactions were 424 run with primary denaturation at 94°C for 4 minutes followed by 40 cycles of denaturation at 94°C 425 for 20 seconds, annealing at 54°C for 30 seconds followed by polymerization at 72°C for 90 426 seconds. The PCR products were visualized on 1.5% agarose gel electrophoresis and amplified 427 products were purified using QIAquick PCR purification kit (Qiagen) according to manufacturer’s 428 protocol. Purified amplicons were sent to Eurofins Genomics (Germany) for Sanger sequencing.

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429 430 Viral prevalence was calculated using the bioconf() package and statistically evaluated using a Chi 431 squared test in R v 3.5.3 integrated into RStudio 1.1.463. 432 433 Acknowledgments 434 435 Financial support was obtained from the DEANN project, Marie Curie Action funded by the 436 European Commission – Grant agreement ID: 612583 and The Swedish Research Council for 437 Environment, Agricultural Sciences and Spatial Planning, Formas (grant number 221-2012-586). 438 439 We acknowledge personnel at the Chilean National Forestry Corporation (CONAF) and assistance 440 by the National Genomic Infrastructure (NGI), which is a part of the Science for Life Laboratory 441 (SciLifeLab), as well as the Swedish National Infrastructure for large Scale Sequencing (SNISS) 442 initiatives. NGI is financially supported by SciLifeLab, Knut and Alice Wallenberg foundation, and 443 the Swedish Research Council. 444 445 We acknowledge the SLU Bioinformatics Infrastructure (SLUBI) for providing computing 446 resources and supporting JH. 447 448 MW is supported by an Australian Research Council DECRA Fellowship (DE200100977). 449 450 We acknowledge the support with molecular methods provided by Alexandra Corduneanu at 451 University of Agricultural Sciences and Veterinary Medicine in Romania and Fernando Pinto 452 Swedish at University of Agricultural Sciences in Sweden.

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