bioRxiv preprint doi: https://doi.org/10.1101/2020.03.07.982207; this version posted March 8, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. 1 Meta-transcriptomic analysis of virus diversity in urban wild birds 2 with paretic disease 3 4 Wei-Shan Chang1†, John-Sebastian Eden1,2†, Jane Hall3, Mang Shi1, Karrie Rose3,4, Edward C. 5 Holmes1# 6 7 8 1Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and 9 Environmental Sciences and School of Medical Sciences, University of Sydney, Sydney, NSW, 10 Australia. 11 2Westmead Institute for Medical Research, Centre for Virus Research, Westmead, NSW, 12 Australia. 13 3Australian Registry of Wildlife Health, Taronga Conservation Society Australia, Mosman, 14 NSW, Australia. 15 4James Cook University, College of Public Health, Medical & Veterinary Sciences, Townsville, 16 QLD, Australia. 17 18 †Authors contributed equally 19 20 #Author for correspondence: 21 Professor Edward C. Holmes 22 Email: [email protected] 23 Phone: +61 2 9351 5591 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.07.982207; this version posted March 8, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. 24 25 Abstract: 248 words 26 Importance: 109 words 27 Text: 6296 words 28 Running title: Virome of urban wild birds with neurological signs. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.07.982207; this version posted March 8, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. 29 Abstract 30 Wild birds are major natural reservoirs and potential dispersers of a variety of infectious 31 diseases. As such, it is important to determine the diversity of viruses they carry and use this 32 information to help understand the potential risks of spill-over to humans, domestic animals, and 33 other wildlife. We investigated the potential viral causes of paresis in long-standing, but 34 undiagnosed disease syndromes in wild Australian birds. RNA from diseased birds was extracted 35 and pooled based on tissue type, host species and clinical manifestation for metagenomic 36 sequencing. Using a bulk and unbiased meta-transcriptomic approach, combined with careful 37 clinical investigation and histopathology, we identified a number of novel viruses from the 38 families Astroviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, 39 Flaviviridae, and Circoviridae in common urban wild birds including Australian magpies, 40 magpie lark, pied currawongs, Australian ravens, and rainbow lorikeets. In each case the 41 presence of the virus was confirmed by RT-PCR. These data revealed a number of candidate 42 viral pathogens that may contribute to coronary, skeletal muscle, vascular and neuropathology in 43 birds of the Corvidae and Artamidae families, and neuropathology in members of the 44 Psittaculidae. The existence of such a diverse virome in urban avian species highlights the 45 importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban 46 environments. This information will be increasingly important for managing disease risks and 47 conducting surveillance for potential viral threats to wildlife, livestock and human health. More 48 broadly, our work shows how meta-transcriptomics brings a new utility to pathogen discovery in 49 wildlife diseases. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.07.982207; this version posted March 8, 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-ND 4.0 International license. 50 Importance 51 Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of 52 novel diseases in domestic animals and human populations. Using unbiased RNA sequencing we 53 identified highly diverse viruses in native birds in Australian urban environments presenting with 54 paresis. This investigation included the clinical investigation and description of poorly 55 understood recurring syndromes of unknown etiology: clenched claw syndrome, and black and 56 white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, 57 this study describes methods that can effectively and efficiently characterize emergent disease 58 syndromes in free ranging wildlife, and promotes further surveillance for specific potential 59 pathogens of potential conservation and zoonotic concern. 60 61 Keyword: paresis, neurological syndrome, wildlife, birds, meta-transcriptomics, virus 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.07.982207; this version posted March 8, 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-ND 4.0 International license. 62 Introduction 63 Emerging and re-emerging infectious diseases in humans often originate in wildlife, with free- 64 living birds representing major natural reservoirs and potential dispersers of a variety of zoonotic 65 pathogens (1). Neurological syndromes, such as paresis, are of particular concern as many 66 zoonotic viral pathogens carried by wild birds with the potential to cause neurological disease are 67 also potentially hazardous to poultry, other livestock and humans. Examples of this phenomenon 68 include Newcastle disease virus (NDV, Avulavirus 1; family Paramyxoviridae), West Nile virus 69 (WNV, family Flaviviridae) and avian influenza viruses (family Orthomyxoviridae) (2). 70 Importantly, with growing urban encroachment, the habitats of humans, domestic animals and 71 wildlife increasingly overlap. A major issue for the prevention and control of wildlife and 72 zoonotic diseases is how rapidly and accurately we can identify a pathogen, determine its origin, 73 and institute biosecurity measures to limit cross-species transmission and onward spread. With 74 these ever changing environments, wildlife are also at risk from a conservation perspective, as a 75 number of emerging viral pathogens (WNV, Usutu virus, avian poxvirus, avian influenza, 76 Bellinger River snapping turtle nidovirus) have had adverse population level impacts (3-8). 77 Therefore, a comprehensive understanding of the diversity of the viral community, and the 78 ecology of microbes associated with urban wildlife mass mortality and emergent disease 79 syndromes, will improve our capacity to detect pathogens of concern and lead to improved 80 conservation and public health intervention (3). 81 82 Meta-transcriptomic approaches (i.e. total RNA-sequencing) have revolutionized the field of 83 virus discovery, transforming our understanding of the natural virome in vertebrates and 84 invertebrates (9, 10). This method relies on the unbiased sequencing of non-ribosomal RNA, and 85 has been used to identify novel viral species in seemingly healthy animals. In Australia, for 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.07.982207; this version posted March 8, 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-ND 4.0 International license. 86 example, meta-transcriptomic approaches have been used with invasive cane toads (Rhinella 87 marina) (11), waterfowl (12, 13), fish (14) and Tasmanian devils (Sarcophilus harrisii) (15). 88 These approaches have also been applied diagnostically in a disease setting, including in 89 domestic animals such as cats (Felis sylvestris) (16), dogs (Canis lupus familiaris) (17), cattle 90 (Bos taurus) with respiratory diseases (18-20), and pythons (Pythonidae) with neurological signs 91 (21). Notably, meta-transcriptomics has also been used to identify bacterial diseases, such as 92 tularemia infections in Australian ring-tailed possums (Pseudocheirus peregrinus) (22). Hence, 93 metagenomic approaches provide the capacity to rapidly and comprehensively map microbial 94 and viral communities and examine their interactions, improving our understanding of animal 95 health and zoonoses. 96 97 Investigations into wildlife diseases are often neglected and under-resourced. Consequently, 98 while many outbreaks and syndromes are reported, until recently limited molecular screening has 99 been performed to characterize the etiology where a novel organism is present. In Australia, 100 several neglected and undiagnosed disease outbreaks have been described in wild avian species 101 including those of suspected viral etiology. Notable examples include two syndromes termed 102 "clenched claw disease” (23-28) and “black and white bird disease” (29-31) that affect rainbow 103 lorikeets (Trichoglossus moluccanus) and several species of passerines, respectively. 104 105 Clenched claw syndrome (CCS) has been recognized as a form of paresis