meta-transcriptomic analysis of virus diversity in urban ... · 68 include newcastle disease virus...

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Meta-transcriptomic analysis of virus diversity in urban wild birds 1

with paretic disease 2

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Wei-Shan Chang1†, John-Sebastian Eden1,2†, Jane Hall3, Mang Shi1, Karrie Rose3,4, Edward C. 4

Holmes1# 5

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1Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and 8

Environmental Sciences and School of Medical Sciences, University of Sydney, Sydney, NSW, 9

Australia. 10

2Westmead Institute for Medical Research, Centre for Virus Research, Westmead, NSW, 11

Australia. 12

3Australian Registry of Wildlife Health, Taronga Conservation Society Australia, Mosman, 13

NSW, Australia. 14

4James Cook University, College of Public Health, Medical & Veterinary Sciences, Townsville, 15

QLD, Australia. 16

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†Authors contributed equally 18

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#Author for correspondence: 20

Professor Edward C. Holmes 21

Email: [email protected] 22

Phone: +61 2 9351 5591 23

.CC-BY-NC-ND 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.03.07.982207doi: bioRxiv preprint

https://doi.org/10.1101/2020.03.07.982207

http://creativecommons.org/licenses/by-nc-nd/4.0/

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Abstract: 248 words 25

Importance: 109 words 26

Text: 6296 words 27

Running title: Virome of urban wild birds with neurological signs. 28


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Abstract 29

Wild birds are major natural reservoirs and potential dispersers of a variety of infectious 30

diseases. As such, it is important to determine the diversity of viruses they carry and use this 31

information to help understand the potential risks of spill-over to humans, domestic animals, and 32

other wildlife. We investigated the potential viral causes of paresis in long-standing, but 33

undiagnosed disease syndromes in wild Australian birds. RNA from diseased birds was extracted 34

and pooled based on tissue type, host species and clinical manifestation for metagenomic 35

sequencing. Using a bulk and unbiased meta-transcriptomic approach, combined with careful 36

clinical investigation and histopathology, we identified a number of novel viruses from the 37

families Astroviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, 38

Flaviviridae, and Circoviridae in common urban wild birds including Australian magpies, 39

magpie lark, pied currawongs, Australian ravens, and rainbow lorikeets. In each case the 40

presence of the virus was confirmed by RT-PCR. These data revealed a number of candidate 41

viral pathogens that may contribute to coronary, skeletal muscle, vascular and neuropathology in 42

birds of the Corvidae and Artamidae families, and neuropathology in members of the 43

Psittaculidae. The existence of such a diverse virome in urban avian species highlights the 44

importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban 45

environments. This information will be increasingly important for managing disease risks and 46

conducting surveillance for potential viral threats to wildlife, livestock and human health. More 47

broadly, our work shows how meta-transcriptomics brings a new utility to pathogen discovery in 48

wildlife diseases. 49


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Importance 50

Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of 51

novel diseases in domestic animals and human populations. Using unbiased RNA sequencing we 52

identified highly diverse viruses in native birds in Australian urban environments presenting with 53

paresis. This investigation included the clinical investigation and description of poorly 54

understood recurring syndromes of unknown etiology: clenched claw syndrome, and black and 55

white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, 56

this study describes methods that can effectively and efficiently characterize emergent disease 57

syndromes in free ranging wildlife, and promotes further surveillance for specific potential 58

pathogens of potential conservation and zoonotic concern. 59

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Keyword: paresis, neurological syndrome, wildlife, birds, meta-transcriptomics, virus 61


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Introduction 62

Emerging and re-emerging infectious diseases in humans often originate in wildlife, with free-63

living birds representing major natural reservoirs and potential dispersers of a variety of zoonotic 64

pathogens (1). Neurological syndromes, such as paresis, are of particular concern as many 65

zoonotic viral pathogens carried by wild birds with the potential to cause neurological disease are 66

also potentially hazardous to poultry, other livestock and humans. Examples of this phenomenon 67

include Newcastle disease virus (NDV, Avulavirus 1; family Paramyxoviridae), West Nile virus 68

(WNV, family Flaviviridae) and avian influenza viruses (family Orthomyxoviridae) (2). 69

Importantly, with growing urban encroachment, the habitats of humans, domestic animals and 70

wildlife increasingly overlap. A major issue for the prevention and control of wildlife and 71

zoonotic diseases is how rapidly and accurately we can identify a pathogen, determine its origin, 72

and institute biosecurity measures to limit cross-species transmission and onward spread. With 73

these ever changing environments, wildlife are also at risk from a conservation perspective, as a 74

number of emerging viral pathogens (WNV, Usutu virus, avian poxvirus, avian influenza, 75

Bellinger River snapping turtle nidovirus) have had adverse population level impacts (3-8). 76

Therefore, a comprehensive understanding of the diversity of the viral community, and the 77

ecology of microbes associated with urban wildlife mass mortality and emergent disease 78

syndromes, will improve our capacity to detect pathogens of concern and lead to improved 79

conservation and public health intervention (3). 80

81

Meta-transcriptomic approaches (i.e. total RNA-sequencing) have revolutionized the field of 82

virus discovery, transforming our understanding of the natural virome in vertebrates and 83

invertebrates (9, 10). This method relies on the unbiased sequencing of non-ribosomal RNA, and 84

has been used to identify novel viral species in seemingly healthy animals. In Australia, for 85


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example, meta-transcriptomic approaches have been used with invasive cane toads (Rhinella 86

marina) (11), waterfowl (12, 13), fish (14) and Tasmanian devils (Sarcophilus harrisii) (15). 87

These approaches have also been applied diagnostically in a disease setting, including in 88

domestic animals such as cats (Felis sylvestris) (16), dogs (Canis lupus familiaris) (17), cattle 89

(Bos taurus) with respiratory diseases (18-20), and pythons (Pythonidae) with neurological signs 90

(21). Notably, meta-transcriptomics has also been used to identify bacterial diseases, such as 91

tularemia infections in Australian ring-tailed possums (Pseudocheirus peregrinus) (22). Hence, 92

metagenomic approaches provide the capacity to rapidly and comprehensively map microbial 93

and viral communities and examine their interactions, improving our understanding of animal 94

health and zoonoses. 95

96

Investigations into wildlife diseases are often neglected and under-resourced. Consequently, 97

while many outbreaks and syndromes are reported, until recently limited molecular screening has 98

been performed to characterize the etiology where a novel organism is present. In Australia, 99

several neglected and undiagnosed disease outbreaks have been described in wild avian species 100

including those of suspected viral etiology. Notable examples include two syndromes termed 101

"clenched claw disease” (23-28) and “black and white bird disease” (29-31) that affect rainbow 102

lorikeets (Trichoglossus moluccanus) and several species of passerines, respectively. 103

104

Clenched claw syndrome (CCS) has been recognized as a form of paresis in rainbow lorikeets in 105

eastern Australia, in which birds present recumbent with poor withdrawal reflexes and clenched 106

feet. Although the syndrome may be multifactorial, a proportion of the cases are characterized by 107

non-suppurative encephalomyelitis and ganglioneuritis, and these cases are suspected to have a 108

viral etiology. Similarly, a series of morbidity and mortality events termed black and white bird 109


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disease (BWBD) have occurred in Australian magpies (Gymnorhina tibicen), Pied currawongs 110

(Strepera graculina), Australian ravens (Corvus coronoides) and magpie larks (Gallina 111

cyanoleuca) along the Australian east coast (31). Diseased birds present in groups, either dead or 112

paretic. Although these emergent disease syndromes are suspected to be caused by viral 113

infections, they have been poorly described, and to date no viral pathogens have been identified 114

in culture, such that the cause and mechanisms of disease remain elusive. 115

By exploiting meta-transcriptomic approaches, validated with clinical manifestation and 116

histopathological findings of infection, we investigated the potential viral etiology in historically 117

archived outbreaks of birds fitting the syndrome descriptions associated with black and white 118

bird disease and clenched claw syndrome in Australia, along with other sporadic cases where 119

viral infection was suspected. In particular, we characterized CCS and BWBD and identified a 120

number of novel viruses from the families Astroviridae, Picornaviridae, Polyomaviridae, 121

Paramyxoviridae, and Parvoviridae in common urban wild birds. 122

Results 123

124

Clinical and histological description of rainbow lorikeets 125

To formulate a syndrome description for CCS, pathology records from the Australian Registry of 126

Wildlife Health from 451 rainbow lorikeets presenting between 1981 and 2019 were reviewed 127

for unexplained non-suppurative inflammation within the central nervous system. A total of 55 128

birds were found to match these search terms. The signalments, clinical signs and histological 129

lesions from these birds are summarized chronologically in Table S1. The index case was a 130

juvenile female rainbow lorikeet found in Mosman, NSW in November 1984, and the last known 131

case was recorded in an adult, female from the same location in May 2007. The majority of cases 132


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occurred in adult birds (28 adults, 20 juveniles, 7 age unspecified), including 24 males, 21 133

females and 10 gender unspecified. Although cases were distributed throughout the year, twice 134

as many CCS events occurred in October than any other single month. 135

136

Clenched feet was the most prevalent presentation (n = 40), followed by an inability to fly (n = 137

13), unspecified neurological signs (n = 8), paresis (n = 6), leg paralysis (n = 3), wing paralysis 138

(n = 2), head tilt (n = 3), tremors (n = 3), ascending or progressive neurological signs (n = 2), 139

head bob (n = 2), opisthotonus (n = 1), ataxia (n = 1), and rolling (n = 1) (Figure 1a,b). Body 140

condition was noted in 29 records and 11 birds were classified as being in good condition, 17 141

birds were considered thin or very thin, and one bird was emaciated. Less common presentations 142

included flying into a window (n = 1) and predation (n = 2). The most common cause of death 143

was euthanasia (n = 37). 144

145

Common microscopic lesions in CCS affected lorikeets are illustrated in Figure 1c-e and include 146

mild to severe perivascular cellular infiltrates ranging from one to eight cells deep, composed of 147

lymphocytes, plasma cells and smaller numbers of macrophages. Gliosis, spongiotic change of 148

the neuropil, Wallerian degeneration and nerve cell body degeneration to necrosis were 149

uncommon and restricted to those animals with moderate to severe inflammation. Non-150

suppurative inflammation within the central nervous system was more prevalent and more severe 151

within the caudal brainstem (28/54), cerebellum (30/54) and spinal cord (44/49), compared with 152

the cerebrum and anterior brain stem (12/54). Peripheral nervous system changes were also 153

noteworthy, as 5 of 47 birds examined had non-suppurative spinal ganglioneuritis, and 18 of 19 154

birds examined had non-suppurative neuritis, often with Wallerian degeneration (13/19). 155

156


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An adult, male red-collared lorikeet (Trichoglossus rubritorquis) from Queensland was also 157

found with a history of euthanasia following presentation with clenched feet, ataxia, and thin 158

body condition. Histological changes in this animal were consistent with CCS, including non-159

suppurative lesions that were mild in the cerebrum, and more moderate in the brain stem, 160

cerebellum and spinal cord. 161

162

Liver from an adult, male rainbow lorikeet with histological evidence of moderate hepatocellular 163

single cell necrosis, karyomegaly, and unusual amphophilic and variably shaped (stellate, 164

discrete and dense, and ground-glass) intranuclear inclusions (Figure 1a) was included in the 165

meta-transcriptomic investigation based on suspicion of one or more underlying viral infections. 166

This bird presented recumbent, with extensive subcutaneous epithelial lined cysts encapsulating 167

clusters of mites distributed along the wings and cranium, and loss of the distal primary feathers 168

and central tail feathers in a pattern suggestive of psittacine circovirus infection (Registry 169

4771.1). 170

171

Clinical and histological description of black and white bird disease 172

A total of 2781 birds in the order Passeriformes were included in the analysis. This identified 51 173

birds with myocardial degeneration or non-suppurative myocarditis. Two cases were excluded 174

from the analysis, due to incongruity of histological lesions compared with those of all other 175

birds (n = 49) examined. The signalments, clinical signs and histological lesions from these birds 176

are summarized chronologically in Table S2. 177

178

Although BWBD was first recognized during an epizootic extending across Victoria, NSW and 179

into Queensland (QLD) in 2006, records from the Australian Registry of Wildlife Health 180


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identified the index cases as magpies and currawongs found in Sydney and the NSW central 181

coast in August 2003. The majority of birds examined were magpies (n = 26), currawongs (n = 182

15), and ravens (n = 6), with adults, juveniles, males and females evenly represented. A solitary 183

adult female figbird and one adult female magpie lark were also among the affected birds. 184

Concurrent mortality was observed in crested pigeons (Ocyphaps olphotes), common koels 185

(Eudynamys scolopacea) silver gulls (Larus novaehollandiae) and indian mynahs (Acridotheres 186

tristis). Presentation of affected birds varied, occurring as nine mass morbidity and mortality 187

events, and 25 sporadic cases. The birds examined from outbreaks in 2003, 2006, 2013 and 2015 188

represent a small fraction of affected birds, and accumulatively total 105 birds reported formally 189

and more than 250 reported informally. Suspected intoxication was a common history in those 190

birds presenting en masse. Although seven birds were found dead or died prior to veterinary 191

examination, clinical signs among the remaining birds included paresis (Figure 2a), recumbency 192

or profound weakness (27), loss of righting reflex or difficulty righting (12), and less commonly 193

dyspnea (4), watery or bloody diarrhea (4), clenched claws (1) and a moribund state (1). 194

Although most birds were recumbent, they were not paralyzed as they had good cloacal tone, 195

withdrawal reflexes, and could stand and shuffle, flap or walk across the room when stimulated. 196

Most ravens had additional clinical signs indicative of central nervous system dysfunction, 197

including ataxia, head tilt, circling, and unusual head posture (4/6). Other species were alert and 198

could accurately grasp items with their beaks, despite being recumbent. The body condition of 199

affected birds varied with 21 noted to be in good body condition, while 27 were classified as 200

thin, very thin or emaciated. 201

202

Gross lesions in affected birds were rare, but included hydropericardium, epicardial hemorrhage, 203

pulmonary congestion to hemorrhage, hemorrhage into the gastrointestinal lumen and fibrinous 204


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serositis. Microscopic lesions characteristic of BWBD include the unifying feature of 205

degeneration of cardiac myocytes with or without non-suppurative perivascular and interstitial 206

cardiac inflammation (Figure 2 b-e). Myocyte degeneration or non-suppurative inflammation 207

within skeletal muscle was observed in 24 of the 45 birds examined (four animals were 208

unavailable for examination). Non-suppurative inflammation of the central nervous system was 209

observed in 25 birds. Lesions were most severe in the central cerebellar white matter and 210

included one to four cell deep perivascular cuffs, spongiotic change of the neuropil, degeneration 211

to necrosis of nerve cell bodies and multifocal gliosis. Other common histological features of the 212

syndrome included acute hepatic necrosis or inflammation (25/49), necrosis or non-suppurative 213

gastro-intestinal inflammation (21/49), non-suppurative interstitial nephritis (8/49), mild 214

vasculopathy to marked fibrino-necrotising vasculitis (19/49), non-suppurative perivascular 215

infiltrates throughout serosal surfaces (20/49) and non-suppurative interstitial pancreatitis 216

(11/49). Less common lesions included perivascular pulmonary, cardiac and neural hemorrhage, 217

meningitis and peripheral neuritis. Ravens generally had more severe and widespread central 218

nervous system lesions, and lacked skeletal muscle, renal and serosal inflammation that was 219

evident in the other species. In birds other than ravens, paresis was most likely associated with 220

lesions in striated muscle and the peripheral nervous system. Although Leucocytozoon and 221

microfilaria are common hemoparasites of the bird species examined, and were found in our 222

metagenomic data (32), these organisms were not relevant within disease-related lesions in our 223

cases. Ancillary diagnostic testing for known bacterial and viral pathogens, Chlamydophyla 224

species and intoxication revealed no significant findings. 225

226

Meta-transcriptomic virus identification 227


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Archived samples including brain, liver, heart, and kidneys from representative cases of diseased 228

birds were pooled and used to generate nine RNA-sequencing libraries that resulted in 7,725,034 229

to 26,555,569 paired reads per pool, for meta-transcriptomic analysis. From these data we 230

discovered eight viruses from the RNA virus families, Astroviridae, Paramyxoviridae, and 231

Picornaviridae, the DNA virus families Adenoviridae, Circoviridae, Parvoviridae and 232

Polyomaviridae. Two additional viruses - a hepacivirus and a narnavirus - were also identified 233

but unlikely associated with any clinical syndrome. 234

235

Avian avulavirus in brain samples of lorikeets with clenched claw syndrome 236

The Paramyxoviridae are a large group of enveloped linear negative-sense RNA viruses that 237

range from 15.2 to 15.9 kb in length. We identified paramyxovirus-like contigs in the brain 238

libraries of rainbow lorikeets. A RT-PCR designed to amplify the paramyxovirus L protein (301 239

nt) identified matching RNA in 4/5 brain tissues, corresponding to the four birds presenting with 240

neurological symptoms. Ten overlapping RT-PCRs were then performed to confirm the genomic 241

sequence (Table S4), revealing a typical 3’-N(455 aa)-P(446 aa)-M(366 aa)-F(619 aa)-HN(528 242

aa)-L(2263)-5’ organization. Based on the phylogenetic analysis of the L and M protein, the 243

novel paramyxovirus identified here - termed Avian paramyxovirus 5/rainbow 244

lorikeet/Australia/CCS - fell into the clade comprising the genus Avulavirus, exhibiting 86.7% 245

and 89.1% amino acid pairwise identity to its closest relative - APMV-5/budgerigar/Japan/TI/75 246

(GenBank accession number: LC168759). 247

248

To provide a provisional assessment of the virulence of this novel virus, we compared F protein 249

cleavage sites between the lorikeet avulavirus and known pathotypes of NDV (Avulavirus 1) that 250

commonly causes disease in avian species, including the velogenic, lentogenic, mesogenic and 251


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asymptomatic vaccine types. Specifically, the precursor F glycoprotein (F0) is cleaved into F1 252

and F2 subunits for the progeny virus to be infectious and to enable replication. The F protein 253

cleavage position for virulent or mesogenic strains contain a furin recognition site comprising 254

multiple basic amino acids. Notably, the lorikeet avulavirus had an F cleavage ‘RRRKKRF’ 255

motif identical to pathogenic NDV strains, suggesting its potential virulence, although this 256

remains to be confirmed (Figure 3). 257

258

DNA viruses identified in sporadic cases or mortality events rainbow lorikeets 259

In addition to RNA viruses, we identified three DNA viruses in the context of sporadic mortality 260

events of rainbow lorikeets: avian chapparvovirus, beak and feather disease virus and lorikeet 261

adenovirus. In addition to the RNA-sequencing results, we extracted DNA from corresponding 262

tissues for DNA virus validation through PCR and rolling circle amplification (RCA) assays. 263

264

Lorikeet chapparvovirus 265

The Parvoviridae are a family of small, non-enveloped, dsDNA animal viruses with linear 266

genomes of ~5 kb in length. We identified parvovirus-like transcripts in both liver RNA-seq 267

libraries from diseased lorikeets. Using RCA to enrich for circular DNAs, we recovered a 268

complete genome of a novel lorikeet chapparvovirus, comprising 4,271 nt with two distinct 269

ORFs that encoded the non-structural protein NS1 (670 aa) and the structural protein VP (542 270

aa). We further designed specific primers to amplify the targeted VP region (~248 bp) for 271

screening both the RNA (positive in two liver samples) and DNA (all positive) products. In 272

addition, we inferred two separate phylogenetic trees based on the complete NS1 and VP 273

proteins to determine the evolutionary relationships between the lorikeet virus and other 274

parvoviruses. This revealed that the closest relative to the lorikeet chapparvovirus identified here 275


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was an avian-associated red-crown crane parvovirus, yc-9 (GenBank accession number: 276

KY312548.1), although this shares only 48.2% amino acid identity in NS1 and 46.9% identity in 277

VP (Figure 4). 278

279

Beak and feather disease virus 280

Beak and feather disease virus (BFDV), a member of Circoviridae, is highly prevalent in 281

Australian wild birds, particularly psittacine species (33). We identified BFDV-like contigs in all 282

lorikeet RNA libraries, sharing 95% nucleotide identity to known strains of BFDV. Remapping 283

the raw reads to the closest BFDV strain (GenBank accession number: KM887928) gave 284

coverage of the whole virus genome, comprising 2,014 nt, and encoding a replication-associated 285

protein (290 amino acids; aa) and a capsid protein (247 aa) (Figure 5A). We then screened all the 286

RNA samples from rainbow lorikeets using PCR primers targeting the capsid protein (596 nt) 287

(GenBank accession number: KM887928), with the PCR products then Sanger sequenced. The 288

BFDVs identified from individual birds carried distinctive single nucleotide variants, and 289

sequences from each case formed strong phylogenetic clusters suggesting that these results are 290

not due to cross-sample contamination (Figure 5B). 291

292

Lorikeet adenovirus 293

Adenovirus-like contigs were identified from both the brain and liver libraries of rainbow 294

lorikeets. Phylogenetic analyses were performed based on the products of PCR primers designed 295

based on the obtained sequences of polymerase (pol, 843 aa) and hexon (hexon, 580 aa) proteins. 296

The virus identified, termed lorikeet adenovirus (LoAdv), exhibited 76% amino acid identity to 297

the most closely related skua adenovirus (GenBank accession number: YP.0049359313) (Figure 298

6). 299


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300

A novel lorikeet hepatovirus in clenched clawed lorikeets 301

A complete hepatovirus-like (Picornavirales) contig was identified in one of the liver libraries of 302

diseased lorikeets, exhibiting relatively high read abundance (~10,000x coverage depth). The 303

genome of this novel lorikeet hepatovirus, termed lorikeet hepatovirus (LoHepaV), is 7,339 nt in 304

length, and encodes a polyprotein of 2070 amino acids, excluding poly-A tails, that is similar in 305

structure to most Avihepatoviruses (Figure 6). Interestingly, this virus was most closely related 306

to Hepatovirus sp. isolate HepV-bat3206/Hipposideros_armiger/2011 identified from a round-307

leaf bat, although these two viruses only exhibit 46.3% amino acid identity across the 308

polyprotein. Recently, a lorikeet picornavirus-LoPV-1 (GenBank accession number: MK443503) 309

was identified in fecal samples from rainbow lorikeets in China (34), although this only 310

exhibited 17.4% amino acid identity with the lorikeet hepatovirus identified here (Figure 7). 311

312

Novel picornavirus identified in black and white bird disease cases 313

The Picornaviridae are a large family of single-strand positive-sense RNA viruses, with 314

genomes ranging from 6.7-10.1 kb in length. We identified a highly abundant complete 315

picornavirus-like contig in the library of archived black and white diseases cases. Remapping 316

raw reads to the picornavirus-like transcript showed complete genome coverage (~300x coverage 317

depth). We named this novel virus black and white bird picornavirus (BWBDpicoV). A RT-PCR 318

targeting this picornaviral contig (~220 bp) was positive in 2/6 of the brain tissues tested from an 319

Australian magpie (Cracticus tibicen) and an Australian raven (Corvus coronoides): notably, the 320

two positive birds were those presenting with non-suppurative encephalomyelitis. 321

322


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BWBDpicoV exhibits classical picornavirus features, with a genome length of 7,011 nt 323

excluding the poly A tails. This genome encodes a polyprotein of 2070 amino acid residues, as 324

well as a 5’ untranslated region (UTR) of 508 nt that includes the putative internal ribosome 325

entry site (IRES). The genome organisation of BWBDpicoV is similar to that of other 326

picornaviruses, with the characteristic gene order: 5′-VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 327

3Cpro, 3Dpol-3′ (Figure 7). BWBDpicoV also exhibited such typical picornavirus features as 328

rhv-like domains (111-222,307-424 aa), an RNA-helicase (1172-1271 aa), and an RNA-329

dependent RNA polymerase (RdRp; 2903-2154 aa). Phylogenetic analysis revealed that 330

BWBDpicoV was most closely related to seal picornavirus (GenBank accession number: 331

NC.009891.1), a member of the genus Aquamavirus (Figure 7). However, BWBD picornavirus 332

exhibited low amino acid similarity to the polyproteins of any of these viruses: only 29.5% with 333

Kunsagivirus A (GenBank accession number: YP_009505615.1) and 30.8% with Seal 334

picornavirus type 1 (GenBank accession number: YP_001487152.1). According to the 335

International Committee on Taxonomy of Viruses (ICTV), members of a genus within the 336

Picornaviridae should share at least 40% amino acid sequence identity in the polyprotein region. 337

As such, our BWBD may represent a new virus genus. 338

339

A divergent black and white bird astrovirus in a BWBD outbreak from Nowra, NSW 340

Several astrovirus-like reads were found in the RNA-seq library of the suspected BWBD (i.e. 341

non-suppurative encephalitis of undetermined etiology) outbreak from Nowra, NSW (Table 1). 342

This novel virus, termed black and white bird astrovirus (BWBDastV), was highly divergent in 343

sequence, and contained only 25 reads that covered the partial RdRp of Astrovirus 344

Pygoscelis/DT/2012 (GenBank accession number: KM587711.1) (see below). 345

346


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Reads from BWBDastV were used to develop PCR assays to bridge the gap in the RdRp. Using 347

RT-PCR amplification of a 195 nt region targeting the RdRp, we obtained positive hits for 348

BWBDastV in 4 of 5 brain samples. Based on RdRp protein identity and phylogenetic analysis, 349

there was 69.8% amino acid sequence similarity to the closest astrovirus, Astrovirus 350

Pygoscelis/DT/2012 (GenBank accession number: KM587711) sampled from Adelie penguins 351

(Pygoscelis adeliae) in Antarctica (Figure 8). Combining the PCR results with clinical and 352

histopathology patterns of diseased birds from the Nowra outbreak, we suggest that BWBDastV 353

might be the cause of the BWBD outbreak (Figure 10B). 354

355

Novel magpie polyomavirus in the Nowra outbreak 356

In addition to the novel astrovirus, our RNA-seq analysis identified a novel circular avian 357

polyomavirus in brain and heart tissue from one non-diseased Australian magpie from the Nowra 358

outbreak. Of note, the histopathology of BWBD was not consistent with polyomavirus infection 359

which is not known to be associated with myositis or encephalitis in birds. The genome of the 360

novel magpie polyomavirus - termed magpolyV - comprised 5115 bp with an overall GC content 361

of 46.5%, and we were able to recover full length genome of this virus using RCA and PCR 362

assays. Based on sequence similarity with existing reference polyomavirus proteins we predict 363

that the genome of the novel virus encodes capsid proteins VP1, VP2, VP3 and open reading 364

frame-X (ORF-X) from the late region, as well as two alternative transcripts, large T antigen 365

(LT) and small T antigen (ST) (Figure 9). Consistent with most known polyomaviruses, 366

magpolyV retains the typical conserved motifs and splicing sites in LT, including HPDKGG 367

(DnaJ domain), LRELL and LLGLL (LXXLL- CR1 motif), LFCDE (LXCXE,a pRB1-binding 368

motif) and GAVPEVNLE (ATPase motif). However, the consensus sequence CXCXXC for 369

protein phosphatase 2A binding, mostly found in mammalian polyomaviruses, was not present. 370


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Phylogenetic analysis revealed that this magpie polyomavirus consistently clustered within avian 371

lineages, exhibiting 90.3% amino acid its closest relative - butcherbird polyomavirus isolate 372

AWH19840 (accession number: KF360862) - in the LT protein, and 92% amino acid similarity 373

in VP1. 374

375

Discussion 376

We characterize the clinical and histological features of CCS and BWBD and apply a meta-377

transcriptomic approach to identify potential viral etiological agents in wild Australian birds 378

presenting with hepatic inclusion bodies, paresis and non-suppurative myocarditis or 379

encephalomyelitis, elucidating the complex nature of viral disease investigation amid multiple 380

infections. Next-generation metagenomic sequencing, in combination with traditional gross and 381

histological examination, identified several candidate pathogens for disease syndromes that had 382

been reported for decades as undiagnosed, but likely of viral origin. Application of this 383

methodology in the face of emergent disease syndromes or mass mortality events will facilitate 384

the early detection of viruses infecting wildlife, greatly contributing to disease control, 385

conservation of wild populations and the more rapid identification of novel pathogens. 386

387

Clenched claw syndrome 388

A key element of our paper was the investigation of CCS, a paretic disease of unknown etiology 389

in rainbow lorikeets (Trichoglossus haematodus) and a single red-collared lorikeet 390

(Trichoglossus rubritorquis) in Australia. The diseased lorikeets present with paresis and were 391

initially often found recumbent or sitting on their hocks with feet clenched, the passive position 392

for the avian foot. Affected birds kept in care progressively developed head tilt, ataxia, and 393

paralysis. The disease syndrome has primarily occurred along coastal New South Wales and 394


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South-East Queensland since the early 1980s. Although previous studies report 5-10% of free-395

living rainbow lorikeets rescued annually presenting with this syndrome, it seems likely that the 396

clinical syndrome in fact encompasses two or more disease etiologies (35). For example, lead 397

poisoning and plant based intoxication have been proposed as contributing to syndromes 398

described as clenched claw or drunken lorikeet syndrome (36). We focused on the investigation 399

of 55 cases of CCS characterized by non-suppurative encephalomyelitis and often 400

ganglioneuritis in which a viral etiology has been suspected. The meta-transcriptomic results, in 401

conjunction with the clinical signs, histopathologic data and confirmation through PCR assays, 402

suggest that the Avulavirus (APMV-5) played a potential leading role in the pathogenesis of 403

CCS in these diseased birds (Figure 10a). An increasing incidence of clinical AMPV infection in 404

wild birds is reported worldwide (37). APMVs have been categorized into 12 serotypes, and 405

APMV1 (NDV) causes significant economic impact on the poultry industry as well as population 406

level impacts on wild birds (37). Both NDV and APMV5 have been reportedly associated with 407

disease outbreaks where mortality rates approach 100%, although the pathogenicity of APMV5 408

varies substantially among aviary species. APMV5 was first isolated from a fatal outbreak of 409

caged budgerigars in Japan in 1974, followed by epizootic outbreaks in budgerigars in the UK 410

(38) and Australia (39). Interestingly, APMV5 will not replicate in chicken embryonated eggs, it 411

lacks a virion hemagglutinin, and it is not pathogenic to chickens (40). Phylogenetically, the 412

AMPV5 identified here is closely related to the Brisbane and Japanese strains identified from 413

budgerigars (41). Because of the possibility of cross-species transmission, the prevalence and 414

pathogenicity of avian paramyxoviruses circulating among wild birds in Australia clearly merits 415

further investigation. 416

417


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Detection of avian chapparvovirus, avian adenovirus and beak and feather disease viruses in 418

rainbow lorikeets 419

Chapparvoviruses are being increasingly identified in metagenomic studies, and seemingly have 420

a wide host range (42), including bats (43), and the feces of turkeys (44), red-crowned cranes 421

(45), wild rats (46) and pigs (47). Importantly, a murine kidney parvovirus was recently found in 422

immunodeficient laboratory mice strains, causing renal failure and kidney fibrosis (48), 423

indicating that some chapparvoviruses are associated with highly virulent infections. 424

425

Avian polyomavirus, beak and feather disease virus, and avian adenovirus appear to be prevalent 426

in psittacine species in Australia (49), compatible with the data presented here. As multi-viral 427

infection is common, our analysis of viral communities associated with neglected avian diseases 428

enabled a comprehensive understanding beyond the one-host, one-virus system (13). Many 429

known viral infections are relevant clinical issues in psittacine species due to their association 430

with acute death, disease, the capacity to induce immunocompromise, and difficulties in 431

treatment and control (50). These have been associated with a variety of RNA viruses including 432

avian paramyxovirus (37), avian influenza virus (51, 52), and avian bornavirus (53), and DNA 433

viruses such as psittacine beak and feather disease virus (PBFD) (54), avian polyomavirus 434

infection (APV) (55), psittacid herpesvirus (PsHV) (56), psittacine adenovirus (PsAdV) (57), 435

poxvirus infection (58), and papillomavirus infection (59, 60). 436

437

The role of astroviruses, polyomaviruses and picornaviruses in black and white bird disease 438

Viruses of the family Astroviridae comprise genomes of 6.8-7kb, infect multiple mammalian 439

species including humans, and also avian species. Astrovirus infection is most commonly 440


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21

associated with gastroenteritis, and rarely neurological symptoms. A number of novel divergent 441

novel astroviruses in wild birds have recently been discovered through metagenomic surveillance 442

(61-63). Many avian astroviruses, including avian nephritis virus, duck and turkey astroviruses 443

and duck hepatitis virus 2, have considerable economic impact on the poultry industry and these 444

viruses are widely distributed in wild birds (62). Despite this, the diversity and ecology of these 445

viruses, including their potential interspecies transmission events, are largely unknown. 446

Previously, astrovirus-associated central nervous system impairment had been reported in mink, 447

human, bovine, ovine, and swine (64-68). 448

449

In mammals such as dogs and cats, astroviruses are commonly isolated with enteric bacterial 450

pathogens in sporadic gastroenteritis outbreaks (69), while in birds, astrovirus infection 451

manifests as a broader disease spectrum including enteritis, hepatitis and nephritis, but rarely 452

neurological symptoms. The astrovirus identified here (BWBDastV) was present in each bird 453

with non-suppurative encephalomyelitis, concurrent with myositis, coelomitis and myocarditis 454

from a single outbreak. However, formal classification of the virus will require additional 455

sequence information. In addition, further surveillance and in situ diagnostic modalities are 456

required to reveal the association between BWBDastV, clinical illness of the birds, and the 457

histological lesions observed. 458

459

A novel polyomavirus - magpolyV - was identified from the brain and heart of one of the non-460

diseased Australian magpies (Cracticus tibicen) that died in the same BWBD outbreak. An avian 461

polyomavirus (APV) - budgerigar fledgling disease polyomavirus – has been documented in 462

young budgerigars and other psittacine species, causing feather abnormalities, abdominal 463

distension, head tremors and skin hemorrhages, reaching infection rates of up to 100% in aviaries 464


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22

worldwide (70). The clinical presentation and degree of susceptibility to APV infection varies 465

from skin diseases to acute death. In the case of non-psittacine species, goose polyomavirus has 466

been characterized as the etiologic agent of fatal hemorrhagic nephritis and enteritis of European 467

geese (HNEG) (71). Other species of polyomaviruses have been identified in finches (72), crows 468

(73) butcherbirds (74), and a novel APV was recently associated with a fatal outbreak in canaries 469

(75). Moreover, a recent study revealed an APV isolated from pigeon feces in China showing 470

almost 99% identity with previously identified psittacine strains, suggesting a much broader host 471

range of APVs and undermined cross-species transmission (76). 472

473

Picornaviruses are commonly identified infecting a variety of avian species in metagenomic 474

studies. In the context of overt disease, notable avian picornaviruses include avian 475

encephalomyelitis viruses in chickens, pheasants, turkeys (77), duck hepatitis A in ducklings 476

(78), and a novel poecivirus that has been identified strongly associated with Avian keratin 477

disorder (AKD) in Alaskan birds (79, 80). We identified two novel picornaviruses in this study - 478

black and white bird picornavirus and a lorikeet hepatovirus - the pathogenic potential of which 479

merits further investigation. Interestingly, a novel lorikeet picornavirus (LoPv-1, GenBank 480

accession number MK443503) was identified in the feces of healthy rainbow lorikeets 481

(Trichoglossus haematodus) in China (34), although the polyprotein only shared 12.6% 482

similarity with the lorikeet hepatovirus identified here. 483

484

The high frequency of microbial co-infections suggests that rather than being the primary cause 485

of the disease outbreak, some novel avian viruses might augment another pathogen, causing 486

immunosuppression in immuno-compromised hosts (81). Although we were successful in 487

elucidating seven candidate viral pathogens that eluded detection through viral culture, it is clear 488


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that additional experimental evidence such as in vivo inoculation experiments and in situ 489

methodologies to identify pathogens within lesions are critical for clarifying the relationship 490

between infection and the emergent disease syndromes we consider. Notably, the metagenomic 491

approach utilized here is not limited to viruses and can be useful in the detection of bacteria, 492

fungi, protozoan and metazoan parasites. For example, the same data set provided a meta-493

transcriptomic signal of Leucocytozoon manifestation in BWBD affected birds (32), although 494

our histological data suggests that this was not a primary or secondary pathogen. 495

496

The utility of meta-transcriptomics in the context of a thorough and multi-disciplinary diagnostic 497

approach is to rapidly identify viruses and other microbial species, including those that are 498

highly divergent, with greater sensitivity and capability than conventional diagnostic tests. The 499

sequencing data and PCR assays developed here enhance diagnostic capabilities for avian 500

disease and enable epidemiological surveillance to better understand the ecology and impacts of 501

the viruses described. More broadly, our study highlights the value of proactive viral discovery 502

in wildlife, and targeted surveillance in response to an emerging infectious disease event that 503

might be associated with veterinary and public health. 504

505

Materials and methods 506

Animal ethics 507

Wild birds were examined under the auspices of NSW Office of Environment and Heritage 508

Licenses to Rehabilitate Injured, Sick or Orphaned Protected Wildlife (#MWL000100542). 509

Diagnostic specimens from recently deceased birds were collected under the approval of Taronga 510

Animal Ethics Committee’s Opportunistic Sample Collection Program, pursuant to NSW Office 511

of Environment and Heritage issued scientific licenses #SL10469 and SL100104. 512


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24

513

Sample collection 514

Samples were collected between 2002 and 2013 from 18 birds, predominantly from within the 515

Sydney basin and the coastal center of NSW. Fresh portions of brain, liver, heart, and kidney 516

were collected aseptically and frozen at -80oC. Additionally, a range of tissues were fixed in 10% 517

neutral buffered formalin, processed in ethanol, embedded with paraffin, sectioned, stained with 518

hematoxylin and eosin, and mounted with a cover slip prior to examination by light microscopy. 519

Giemsa stains were applied to a subset of tissue samples to determine whether protozoa were 520

present within lesions. 521

522

Historical Case Review 523

The records of the Australian Registry of Wildlife Health dating back to 1981 were interrogated 524

to identify rainbow lorikeets with clinical signs relating to the nervous system, and those with 525

unexplained non-suppurative inflammation in the central nervous system. An additional search 526

was conducted to identify passerine birds with unexplained non-suppurative inflammation within 527

cardiac or skeletal muscle. Retrieved records were investigated to determine the signalment, 528

clinical signs and histological lesions of affected animals. 529

530

The severity of non-suppurative inflammation was graded on a scale of 0-4, where 0 indicated no 531

discernible lesions and 4 represented severe and extensive mononuclear cell inflammation. 532

Degeneration and necrosis were graded on a scale of 0-4 where 1 represented mild, multifocal 533

single cell degeneration or necrosis, and 4 characterized extensive coagulative necrosis or 534

malacia. Wallerian degeneration of the white matter tracts within the brain, spinal cord, central 535

and peripheral nerves was noted when present. In rainbow lorikeets the lesions were graded in 536


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25

the cerebrum, brain stem, cerebellum, spinal cord, ganglia, and peripheral nerves. Whereas in the 537

passerines, degeneration or necrosis, and non-suppurative inflammation were graded in skeletal 538

and cardiac muscle, brain, liver, gastrointestinal tract, pancreas, blood vessel walls, and renal 539

interstitium. 540

541

Avian influenza and NDV were excluded using real-time PCR on oropharyngeal, conjunctival 542

and cloacal swabs from 22 magpies and 12 other wild birds (including currawongs, magpies and 543

ravens) (Flu DETECT, Zoetis). Serological testing of plasma from the same birds comprised 544

hemagglutination inhibition to identify NDV antibodies, and competitive ELISA tests targeting 545

Avian influenza virus and flavivirus antibodies. Sixty-eight tissue samples from a sub-group of 546

13 magpies, currawongs and ravens identified as having acute histological lesions suggestive of 547

viral infection were subjected to further avian influenza, NDV and WNV specific rt-PCR and 548

virus isolation. Virus isolation was attempted in both mammalian and insect cell lines (Peter 549

Kirland pers comm.). Additional viral culture was attempted on the same samples inoculated into 550

chick embryos while assessing hemagglutinating agents, and in Vero cells assessing cytopathic 551

effect consistent with WNV infection. Clearview antigen capture ELISA (Unipath, Mountain 552

View, Calif.) targeting Chlamydophila species was conducted on splenic tissue from eight birds 553

with BWBD (magpies, currawongs and a raven and magpie lark). Liver and lung tissues from 554

eight magpies and currawongs were subjected to routine aerobic and anaerobic bacterial and 555

fungal culture. Finally, liver tissue from eight magpies and currawongs, representing the 2006 556

and 2015 BWBD epizootics, were subjected to toxicological testing to exclude the presence of a 557

variety of acaricides, fungicides, organophosphates, carbamates, synthetic pyrethroids, and 558

organochlorines and their metabolites. 559

560


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26

RNA extraction, library construction and sequencing 561

Viral RNA was extracted from the brain and liver, heart and kidney samples of animals using the 562

RNeasy Plus Mini Kit (Qiagen, Germany). RNA concentration and integrity were determined 563

using a NanoDrop spectrophotometer (Thermo) and TapeStation (Agilent). RNA samples were 564

then pooled in equal proportions based on animal tissue type and syndrome. Illumina RNA 565

libraries were prepared on the pooled samples following rRNA depletion using a RiboZero Gold 566

kit (Epidemiology) at the Australian Genome Research Facility (AGRF), Melbourne. The rRNA 567

depleted libraries were then sequenced on an Illumina HiSeq 2500 system (paired 100 nt reads) 568

569

Virome meta-transcriptomics 570

Unbiased sequencing of RNA aliquots extracted from diseased animals in each outbreak were 571

pooled based on host species, clinical syndrome and histological findings as shown in Table 1. 572

The RNA sequencing reads were trimmed of low quality bases and any adapter sequences before 573

de novo assembly using Trinity 2.1.1 (82). The assembled sequence contigs were annotated using 574

both nucleotide and protein BLAST searches against the NCBI non-redundant sequence 575

database. To identify low abundance organisms, the sequence reads were also annotated directly 576

using a BlastX search against the NCBI virus RefSeq viral protein database using Diamond (83) 577

with an e-value cutoff of <10−5. Open reading frames were then predicted from the viral contigs 578

in Geneious v11.1.2 (84) with gene annotation and functional predictions made against the 579

Conserved domain databases (CDD) (85). All sequences were aligned using the E-INS-i 580

algorithm in MAFFT version 7 (86). Virus abundance was assessed using a read mapping 581

approach, in which reads were mapped to the assembled viral contigs using the BBmap program 582

(87). 583

584


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27

Rolling-circle amplification assays for circular DNA viruses 585

To recover the complete genomes of the polyomaviruses and circoviruses identified through our 586

RNA-Seq analysis, we enriched for circular DNA using rolling-circle amplification (RCA) (88). 587

Briefly, genomic DNA extracted from animal tissue and combined with a reaction buffer 588

containing random primers, 1X phi29 Buffer, DTT, Bovine Serum Albumin, dNTPs, and phi29 589

DNA polymerase (Thermo Scientific, Australia) and then incubated at 30°C for 16h. The RCA 590

products were then purified using the Monarch PCR & DNA Cleanup Kit (NEB), then quantified 591

using the Qubit dsDNA broad range assay. Nextera XT DNA libraries were then prepared and 592

sequenced on an Illumina MiSeq to a depth of ~2M reads (2 X 150nt). 593

594

RT-PCR assays and Sanger sequencing 595

Total liver and brain RNA from individual birds was reverse transcribed with SuperScript IV 596

VILO Mastermix (Invitrogen). The cDNA generated from the sampled tissues was used for viral 597

specific PCRs targeting regions identified by RNA-Seq. RT-PCR primers were designed to 598

bridge any genomic gaps based on detected transcripts of the paramyxoviruses, polyomaviruses, 599

adenoviruses, and astroviruses identified in the RNA-seq data (Tables S3 and S4). Accordingly, 600

all PCRs were performed using Platinum SuperFi DNA polymerase (Invitrogen) with a final 601

concentration of 0.2 µM for both forward and reverse primers. All PCR products were visualized 602

by agarose gel electrophoresis and Sanger sequencing. Long RT-PCR products were also 603

sequenced using Nextera XT and MiSeq, as per RCA assays above. 604

605

Phylogenetic analysis 606

Conserved domains within the viral proteins produced were used in phylogenetic analyses to 607

determine the evolutionary relationships of the viruses identified here. Specifically, in the case of 608


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28

RNA viruses we used amino acid sequences of the RNA-dependent RNA polymerase (RdRp) 609

that is the most conserved protein among this group. After removing all ambiguously aligned 610

regions using TrimAl (89), phylogenetic trees were inferred using the maximum likelihood 611

method (ML) implemented in PhyML version 3.0 (90), employing a Subtree Pruning and 612

Regrafting topology searching algorithm and the LG model of amino acid substitution. Bootstrap 613

resampling with 1000 replications under the same substitution model was used to assess nodal 614

support. All phylogenetic trees were then visualized using FigTree v1.4.3 615

(http://tree.bio.ed.ac.uk/software/figtree). 616

617

Nucleotide Sequence Accession Numbers 618

The RNA sequencing data in this study has been deposited in the GenBank Sequence Reads 619

Archive under accession numbers PRJNAXXXX. All genome sequences of identified viruses 620

have been uploaded in GenBank under accession numbers XXXXX to XXXXXX. 621

622

Acknowledgements 623

This research was supported by the Taronga Conservation Society Australia, Taronga 624

Conservation Science Initiative, and New South Wales National Parks and Wildlife Service. 625

ECH is supported by an ARC Australian Laureate Fellowship (FL170100022). We thank Drs. 626

Bill Hartley, Rod Reece, Cheryl Sangster, Shannon Donahoe, Richard Montali, and Cathy 627

Shilton for their diagnostic contributions on individual cases. Virology personnel at the NSW 628

Department of Planning, Industry and Environment’s Elizabeth Macarthur Agricultural Institute, 629

and CSIRO’s Australian Animal Health Laboratories are acknowledged for their considerable 630

body of work pursuing viral culture in the BWBD investigation. 631

632


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Novel picornavirus associated with avian keratin disorder in Alaskan birds. MBio 7: 854

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novel viruses from birds. Curr Opin Virol 10:63-9. 859

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84. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper 865

A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious 866

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85. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He 869

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Yamashita RA, Zhang D, Zheng C, Bryant SH. 2015. CDD: NCBI's conserved domain 871

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88. Rockett R, Barraclough KA, Isbel NM, Dudley KJ, Nissen MD, Sloots TP, Bialasiewicz S. 877

2015. Specific rolling circle amplification of low-copy human polyomaviruses BKV, 878

HPyV6, HPyV7, TSPyV, and STLPyV. J Virol Methods 215-216:17-21. 879

80. Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. 2009. trimAl: a tool for automated 880

alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972-3. 881

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performance of PhyML 3.0. Syst Biol 59:307-21. 884

885


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Table 1. Details of the 9 RNA-seq libraries, the avian species, and associated disease syndrome 886

studied here. 887

Library Host Organ Reads (pair

reads)

Suspected Disease

RBL-L-2 Rainbow lorikeet Liver 26,555,569 Sporadic mortality events

RBL-L-3 Rainbow lorikeet Liver 25,509,669 CCS,sporadic mortality events

RBL-B-9 Rainbow lorikeet Brain 23,994,093 CCS, sporadic mortality events

RBL-B-46 Rainbow lorikeet Brain 24,868,929 CCS

Nowra-B-14 Australian magpie, Magpie

lark, Pied Currawong

Brain 25,598,183 Nowra BWBD outbreak

Nowra-L-15 Australian magpie, Magpie

lark, Pied Currawong

Liver 20,413,140 Nowra BWBD outbreak

BWBD-L-6 Australian magpie, Pied

currawong, Australian raven

Liver 7,725,034 Archived BWBD

BWBD-B-8 Australian magpie, Pied


Brain 25,779,043 Archived BWBD

BWBD-O-

13

Australian magpie, Pied


Heart,

Kidney

23,374,876 Archived BWBD

*CCS - Clenched Claw syndrome. BWBD - Black and white bird disease. 888


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41

Figure Legends 889

890

Figure 1. (a) Photomicrograph of rainbow lorikeet hepatocellular intranuclear inclusion bodies 891

(closed arrowheads) and karyomegaly with a stellate chromatin pattern (open arrowhead). (b) 892

Paretic rainbow lorikeet with clenched claws. (c-e) Photomicrographs of lesions characteristic of 893

clench claw syndrome in rainbow lorikeets. Encephalitis within the central cerebellar white 894

matter (c) with perivascular lymphoplasmacytic infiltrates (black arrow), vascular intramural 895

mononuclear cell infiltrates (black arrowhead), gliosis, dilation of axonal chambers (*) and 896

degenerating nerve cell bodies (open arrowheads). Spinal ganglion (d) with multifocal 897

mononuclear cell infiltrates (*), swollen axons and dilated axonal chambers within a white 898

matter tract (white bar). A peripheral nerve (e) with perivascular mononuclear cell infiltration 899

(black arrow), and Wallerian degeneration illustrated by dilated axonal chambers (black 900

arrowhead) and a macrophage within an axonal chamber signifying a digestion chamber (open 901

arrowhead). 902

903

Figure 2. (a) A paretic, but alert, Australian magpie with black and white bird disease. (b-e) 904

Photomicrographs of lesions characteristic of black and white bird disease. Severe perivascular 905

pulmonary hemorrhage (*). Myocardium (c) with a mononuclear cell infiltrate (*) and myocyte 906

degeneration (black arrowhead) signified by hypereosinophilic myofibrils and a pyknotic and 907

peripheralized nucleus. Proventricular mononuclear cell infiltrates (d) within the lamina propria 908

(white *) and surrounding serosal blood vessels (black *). Acute hepatic necrosis (e*). 909

910

Figure 3. Phylogeny and characterization of a novel avian avulavirus identified from a rainbow 911

lorikeet with clenched claw disease. (a) A maximum likelihood phylogeny of the M gene (366 912


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42

amino acids) of avian avulavirus plus representative members of the Paramyxoviridae, including 913

viruses from the genera Avulavirus, Rubulavirus, Ferlavirus, Henipavirus, Morbillivirus, 914

Aquaparamyxovirus and Respirovirus. The tree was midpoint rooted for clarity only. The scale 915

bar indicates the number of amino acid substitutions per site. Bootstrap values >70% are shown 916

for key nodes. (b) Characterization of the virulence determination site in the F gene by 917

comparison to representative NDV strains. The typical RRKKR cleavage site motif is 918

highlighted (red rectangle). 919

920

Figure 4. Characterization and phylogeny of the lorikeet chapparvovirus identified here. (b) 921

Schematic representation and read abundance of the genome of lorikeet chapparvovirus. 922

Maximum likelihood phylogenies of the (B) NS gene and (C) VP gene. The tree was midpoint 923

rooted for clarity only. The scale bar indicates the number of amino acid substitutions per site. 924

Bootstrap values >70% are shown for key nodes. 925

926

Figure 5. Genomic organization and phylogeny of the beak and feather disease virus (BFDV) 927

identified in this study. (a) BFDV circular genome, annotated with two genes - the capsid protein 928

(Cap) and the replicase-associated protein (replicase). (b) Sanger sequencing of each case 929

detected positive with BFDV. The ML phylogeny of the cap gene (596 nt) was inferred and the 930

BFDVs identified from the same case fell into the corresponding clade. Phylogenetic trees were 931

estimated based on (c) the Cap gene and (d) the Replicase protein. The tree was midpoint rooted 932

for clarity only. The scale bar indicates the number of amino acid substitutions per site. 933

Bootstrap values >70% are shown for key nodes. 934

935


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Figure 6. Phylogeny of the novel lorikeet adenovirus identified in this study. ML phylogenies of 936

the (a) Hexon and (b) polymerase protein were inferred with representative members of the 937

Adenoviridae. The tree was midpoint rooted for clarity only. The scale bar indicates the number 938

of amino acid substitutions per site. Bootstrap values >70% are shown for key nodes. 939

940

Figure 7. Black and white bird disease picornavirus and lorikeet hepatovirus identified in this 941

study. (a) Schematic representation and read abundance of the black and white picornavirus 942

(upper left) and lorikeet hepatovirus (upper right). (b) ML phylogenetic tree of the polyprotein 943

(containing the RdRp gene) of both viruses. Bootstrap resampling (1,000 replications) was used 944

to assess node support where >70%. GenBank accession numbers are given in the taxa labels. 945

Trees were midpoint rooted for clarify. The scale bars shows the number of amino acid distance 946

in substitution per site. 947

948

Figure 8. Phylogenetic analysis of magpie astrovirus. ML phylogeny of the partial RdRp gene 949

(267 amino acids) of astroviruses was inferred along with representative members of the 950

Avastrovirus and Mamastrovirus genera. GenBank accession numbers are given in the taxa 951

labels. Trees were midpoint rooted for clarity only, and branch support was estimated using 952

1,000 bootstrap replicates, with those >70% shown at the major nodes. 953

954

Figure 9. Genome characterization and phylogenetic analysis of the black and white bird 955

polyomavirus identified in this study. (a) Schematic depiction and read abundance of the 956

butcherbird polyomavirus/magpie. ML phylogenetic trees of the (b) large antigen (blue) and (c) 957

VP1 (yellow) protein of the butcherbird polyomavirus/magpie. Bootstrap support (1,000 958


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44

replicates) was used to assess node support where >70%. GenBank accession numbers are given 959

in the taxa labels. The scale bars show the numbers of substitutions per site. 960

961

Figure 10. Summary of cases with meta-transcriptomic pathogen identification and confirmation 962

through PCR assays. (a) Clenched claw syndrome. (b) Black and white bird disease. Blue: PCR 963

positive, yellow: negative, dark yellow: weak positive, NS: non-suppurative, CCS: Clenched 964

claw syndrome, BWBD: Black and white bird disease. 965

966

Supplementary Information 967

Table S1. Presentation and pathology of rainbow lorikeets with clench-claw syndrome. 968

Table S2. Presentation and pathology of passerines with myocardial degeneration and 969

myocarditis. 970

Table S3. PCR primers used to amplify viral sequences from bird tissues. 971

Table S4. Primers used to recover full length genome of the viruses identified. 972


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Figure 1 973

974


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Figure 2 975

976


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Figure 3 977

978

979


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48

Figure 4 980

981

982

983


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Figure 5 984

985

986

2,014 bp

Replicase

Circovirus

Cyclovirus

A B

Capsid

Beak and feather disease virus

0.3

C D

YP 009506308 Chimp Cyclovirus

YP 610962 Starling circovirus

AKO84210 Fox circovirus

YP 803548 Gull circovirusNP 059530 Columbid circovirus

YP 009051962 Human circovirus

NP 047277 Beak and feather disease virus

YP 004152330 Goat Cyclovirus

NP 150370 Goose circovirus

YP 004152334 Chicken Cyclovirus

YP 007697653 Canine circovirus

Beak and feather disease virus/lorikeet

NP 065679 Porcine circovirus 1NP 937957 Porcine circovirus 2

YP 009389457 Giant panda circovirus

YP 009506324 Cyclovirus PK5034

YP 764456 Raven circovirus

YP 009315911 Porcine circovirus 3

YP 004152332 Bat Cyclovirus

YP 009134741 Zebra finch circovirus

NP 573443 Canary circovirus

YP 009091699 Swan circovirus

YP_009506322 Cyclovirus TN25

YP 803551 Finch circovirus

ACV69956 Duck circovirus

100

97

98

98

100

98

71

92

100

100

83

100

100

70

0.5

Beak and feather disease virus/lorikeet

NP 065678 Porcine circovirus 1

YP 009389456 Giant panda circovirus 1

NP 150368 Goose circovirus

YP 009051960 Human circovirus

YP 004152329 Goat Cyclovirus YP 009506305 Chicken Cyclovirus

YP 009506323 Cyclovirus PK5034

YP 803546 Gull circovirus

YP 803549 Finch circovirus

NP 047275 Beak and feather disease virus

ACV69955 Duck circovirus

YP 009506307 Chimp Cyclovirus

AKO84209 Fox circovirus

YP 610960 Starling circovirus

YP 007697652 Canine circovirus

YP 009091698 Swan circovirus

YP 764455 Raven circovirus

YP 009134739 Zebra finch circovirus

YP 009506321 Cyclovirus TN25

NP 573442 Canary circovirus

NP 059527 Columbid circovirus

NP 937956 Porcine circovirus 2

YP 004152331 Bat Cyclovirus

100

9876

84

90

77

90

93

100

84

88

96

0.02

Lorikeet 5604.1 Liver

Lorikeet 2989.1 Brain






78

100

100

96

100


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Figure 6 987

988

989

PolA BHexon

0.3

YP 009047094.1 Pigeon adenovirus

YP 009051654.1 Lizard adenovirus

NP 046314.1 Bovine adenovirus

YP 009505686.1 Bottlenose dolphin adenovirus

YP 009508600.1 Psittacine aviadenovirus

YP 009252208.1 Penguin siadenovirus A

YP 009047155.1 Duck adenovirus

YP 004414800.1 Raptor adenovirus

YP 003933581.1 Turkey adenovirus

AP 000478.1 Turkey siadenovirus A


ADP30814.1 Skua adenovirus 1

YP 006383556.1 Goose adenovirus

YP 009389490.1 Squirrel adenovirus

NP 659515.1 Ovine adenovirus

YP 009373238.1 Deer mastadenovirus

NP 108658.1 Porcine adenovirus

YP 009414575.1 Odocoileus adenovirus

AAX51172.1 Avirulent turkey hemorrhagic enteritis virus

AP 000342.1 Murine mastadenovirus

ACW84422.1 Great tit adenovirus

YP 001552247.1 Snake adenovirus

AAL89791.1 Sturgeon chtadenovirus A

AAC64523.1 Turkey adenovirus

YP 009112716.1 Psittacine adenovirus 3

AAF86924.1 Frog adenovirus

92

99

100

100

100

100

88

100

93

100

95

99

100

98

77

100

75

98

100

100

100

82

100

94

81

82

100

98

100

Lorikeet adenovirus

0.3

YP 009505695.1 Bottlenose dolphin adenovirus

YP 009047163.1 Duck adenovirus 2


AP 000351.1 Murine mastadenovirus

ADP30822.1 Skua adenovirus

AAC64532.1 Turkey adenovirus 3

YP 004414808.1 Raptor adenovirus

YP 009508608.1 Psittacine aviadenovirus

YP 009389499.1 Squirrel adenovirus

YP 001552255.1 Snake adenovirus

YP 009373247.1 Deer mastadenovirus

AP 000486.1 Turkey siadenovirus

NP 046323.1 Bovine adenovirus

ALB78146.1 Penguin siadenovirus A

NP 659523.1 Ovine adenovirus

YP 009112724.1 Psittacine adenovirus

Lorikeet adenovirus

AAX51180.1 Avirulent turkey hemorrhagic enteritis virus

YP 009414582.1 Odocoileus adenovirus

YP 009051662.1 Lizard adenovirus

AAF86932.1 Frog adenovirus

YP 006383564.1 Goose adenovirus 4

CAD42235.1 Sturgeon ichtadenovirus

100

100

100

100

100

100

100

100

76

83100

100

10095

100

100

82

71

86

100

88

100

Fowl aviadenovirus

Fowl aviadenovirus

Bovine adenovirus

Bovine adenovirusDuck atadenovirus

Duck atadenovirus

Bat adenovirus Bat adenovirus

Primate adenovirus Primate adenovirus


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Figure 7 990

991

992

993


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Figure 8 994

995

996


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Figure 9 997

998


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Figure 10 999

1000

1001

+ Positive+ weak postive

Negative

A

B


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