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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 1 Four novel Picornaviruses 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 1 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 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 viruses 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, Tremovirus 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 virus diversity, 59 ecology, and evolution in these unique avian taxa. 60 61 Keywords: Sphenisciformes, penguins, Picornaviridae, viral metagenomics, Hepatovirus 62 2 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. 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 Adenoviridae, Astroviridae, Caliciviridae, Circoviridae, Coronaviridae, Herpesviridae, 86 Paramyxoviridae, Orthomyxovirdae, Polyomaviridae, Papillomaviridae, Picornaviridae, 87 Picobirnaviridae and Reoviridae (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 3 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. 98 have been described: Ross Virus, Scott Virus, Amundsen Virus (Hepatovirus/Tremovirus-like), 99 Shirase Virus (Sinicivirus-like), Wedell Virus (Pigeon Picornavirus 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