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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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

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24

25 Abstract: 248 words

26 Importance: 109 words

27 Text: 6296 words

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

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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.

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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

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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

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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 in rainbow lorikeets in

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

122 Paramyxoviridae, and Parvoviridae in common urban wild birds.

123 Results

124

125 Clinical and histological description of rainbow lorikeets

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

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

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

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

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

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

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

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

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

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

136

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

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

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

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

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

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

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

144 was euthanasia (n = 37).

145

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

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

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

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

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

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

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

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

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

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

156

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

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

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

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

161 cerebellum and spinal cord.

162

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

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

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

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

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

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

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

170 4771.1).

171

172 Clinical and histological description of black and white bird disease

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

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

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

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

177 are summarized chronologically in Table S2.

178

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

201 thin, very thin or emaciated.

202

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

225 species and intoxication revealed no significant findings.

226

227 Meta-transcriptomic virus identification

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

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

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

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

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

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

234 but unlikely associated with any clinical syndrome.

235

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

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

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

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

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

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

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

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

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

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

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

247 (GenBank accession number: LC168759).

248

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

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

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

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

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

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

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

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

257 remains to be confirmed (Figure 3).

258

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

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

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

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

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

264

265 Lorikeet chapparvovirus

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

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

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

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

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

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

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

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

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

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

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

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

278 VP (Figure 4).

279

280 Beak and feather disease virus

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

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

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

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

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

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

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

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

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

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

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

292

293 Lorikeet adenovirus

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

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

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

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

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

299 6).

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300

301 A novel lorikeet hepatovirus in clenched clawed lorikeets

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

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

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

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

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

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

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

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

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

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

312

313 Novel picornavirus identified in black and white bird disease cases

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

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

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

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

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

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

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

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

322

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

339

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

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

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

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

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

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

346

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

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

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

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

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

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

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

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

355

356 Novel magpie polyomavirus in the Nowra outbreak

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

374 in VP1.

375

376 Discussion

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

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

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

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

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

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

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

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

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

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

387

388 Clenched claw syndrome

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

416 further investigation.

417

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

419 rainbow lorikeets

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

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

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

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

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

425

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

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

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

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

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

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

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

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

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

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

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

437

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

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

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

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

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

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

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

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

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

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

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

449

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

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

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

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

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

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

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

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

458 histological lesions observed.

459

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

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

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

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

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

21 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.

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

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

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

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

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

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

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

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

473

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

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

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

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

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

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

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

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

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

483 similarity with the lorikeet hepatovirus identified here.

484

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

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

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

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

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

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

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

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

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

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

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

496

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

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

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

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

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

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

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

504 might be associated with veterinary and public health.

505

506 Materials and methods

507 Animal ethics

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

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

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

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

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

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513

514 Sample collection

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

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

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

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

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

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

521 present within lesions.

522

523 Historical Case Review

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

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

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

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

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

529 clinical signs and histological lesions of affected animals.

530

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

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

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

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

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

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

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

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

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

540 interstitium.

541

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

559 organochlorines and their metabolites.

560

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561 RNA extraction, library construction and sequencing

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

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

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

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

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

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

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

569

570 Virome meta-transcriptomics

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

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

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

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

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

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

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

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

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

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

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

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

583 (87).

584

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585 Rolling-circle amplification assays for circular DNA viruses

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

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

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

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

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

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

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

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

594

595 RT-PCR assays and Sanger sequencing

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

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

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

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

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

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

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

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

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

605

606 Phylogenetic analysis

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

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

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

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

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

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

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

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

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

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

617

618 Nucleotide Sequence Accession Numbers

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

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

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

622

623 Acknowledgements

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

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

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

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

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

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

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

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

632

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

887 studied here.

Library Host Organ Reads (pair Suspected Disease

reads)

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 Brain 25,598,183 Nowra BWBD outbreak

lark, Pied Currawong

Nowra-L-15 Australian magpie, Magpie Liver 20,413,140 Nowra BWBD outbreak

lark, Pied Currawong

BWBD-L-6 Australian magpie, Pied Liver 7,725,034 Archived BWBD

currawong, Australian raven

BWBD-B-8 Australian magpie, Pied Brain 25,779,043 Archived BWBD

currawong, Australian raven

BWBD-O- Australian magpie, Pied Heart, 23,374,876 Archived BWBD

13 currawong, Australian raven Kidney

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

40 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.

889 Figure Legends

890

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

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

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

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

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

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

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

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

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

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

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

902 arrowhead).

903

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

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

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

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

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

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

910

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

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

41 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.

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

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

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

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

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

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

919 highlighted (red rectangle).

920

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

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

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

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

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

926

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

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

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

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

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

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

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

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

935

42 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.

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

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

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

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

940

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

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

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

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

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

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

947 in substitution per site.

948

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

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

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

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

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

954

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

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

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

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

43 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.

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

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

961

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

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

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

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

966

967 Supplementary Information

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

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

970 myocarditis.

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

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

44 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.

973 Figure 1

974

45 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.

975 Figure 2

976

46 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.

977 Figure 3

978

979

47 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.

980 Figure 4

981

982

983

48 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.

984 Figure 5

985

A B Lorikeet 8856.2 Brain

100 Lorikeet 4771.2 Liver 78

Lorikeet 5789.1 Brain Beak and feather Lorikeet 5604.1 Liver disease virus 100

2,014 bp Lorikeet 5604.1 Brain 100

Lorikeet 2989.1 Liver 96

Lorikeet 2989.1 Brain 0.02

C Capsid D Replicase

YP_009506322 Cyclovirus TN25 YP 009506321 Cyclovirus TN25 83 Cyclovirus 98 YP 004152334 Chicken Cyclovirus 98 84 YP 004152329 Goat Cyclovirus 76 71 YP 009506308 Chimp Cyclovirus YP 009506305 Chicken Cyclovirus YP 004152330 Goat Cyclovirus 90 YP 009506307 Chimp Cyclovirus YP 004152332 Bat Cyclovirus 84 YP 009506323 Cyclovirus PK5034 YP 009506324 Cyclovirus PK5034 YP 004152331 Bat Cyclovirus YP 803548 Gull circovirus YP 803546 Gull circovirus 70 97 NP 059530 Columbid circovirus Circovirus YP 610962 Starling circovirus NP 059527 Columbid circovirus YP 610960 Starling circovirus 100 NP 573443 Canary circovirus NP 573442 Canary circovirus 92 98 YP 764456 Raven circovirus 93 98 YP 764455 Raven circovirus YP 803551 Finch circovirus 77 YP 803549 Finch circovirus YP 009134741 Zebra finch circovirus YP 009134739 Zebra finch circovirus 100 NP 047277 Beak and feather disease virus 100 NP 047275 Beak and feather disease virus Beak and feather disease virus/lorikeet Beak and feather disease virus/lorikeet 100 NP 150370 Goose circovirus 90 100 NP 150368 Goose circovirus YP 009091699 Swan circovirus 100 YP 009091698 Swan circovirus ACV69956 Duck circovirus ACV69955 Duck circovirus 100 YP 007697653 Canine circovirus 88 AKO84209 Fox circovirus AKO84210 Fox circovirus YP 009315911 Porcine circovirus 3 YP 007697652 Canine circovirus 96 NP 065678 Porcine circovirus 1 100 NP 065679 Porcine circovirus 1 NP 937957 Porcine circovirus 2 NP 937956 Porcine circovirus 2 YP 009389456 Giant panda circovirus 1 YP 009389457 Giant panda circovirus YP 009051962 Human circovirus YP 009051960 Human circovirus 986 0.3 0.5

49 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.

987 Figure 6

988

A Hexon B Pol

100 AAX51180.1 Avirulent turkey hemorrhagic enteritis virus 100 YP 009310429.1 Pigeon adenovirus AP 000486.1 Turkey siadenovirus 94 YP 009047094.1 Pigeon adenovirus 88 AAC64532.1 Turkey adenovirus 3 93 YP 009508600.1 Psittacine aviadenovirus 71 ALB78146.1 Penguin siadenovirus A 100 82 YP 003933581.1 Turkey adenovirus YP 004414808.1 Raptor adenovirus Fowl aviadenovirus 100 ADP30822.1 Skua adenovirus 100 YP 009047155.1 Duck adenovirus 86 YP 006383556.1 Goose adenovirus 100 Lorikeet adenovirus 77 AAC64523.1 Turkey adenovirus AAF86932.1 Frog adenovirus 100 AP 000478.1 Turkey siadenovirus A CAD42235.1 Sturgeon ichtadenovirus 95 AAX51172.1 Avirulent turkey hemorrhagic enteritis virus YP 009310437.1 Pigeon adenovirus YP 009252208.1 Penguin siadenovirus A 100 YP 009508608.1 Psittacine aviadenovirus 98 YP 004414800.1 Raptor adenovirus 100 ADP30814.1 Skua adenovirus 1 82 100 Fowl aviadenovirus 75 82 Lorikeet adenovirus YP 009047163.1 Duck adenovirus 2 ACW84422.1 Great tit adenovirus 100 98 YP 006383564.1 Goose adenovirus 4 AAF86924.1 Frog adenovirus YP 009051662.1 Lizard adenovirus AAL89791.1 Sturgeon chtadenovirus A 100 YP 001552255.1 Snake adenovirus YP 009112716.1 Psittacine adenovirus 3 100 YP 009414582.1 Odocoileus adenovirus 100 100 Duck atadenovirus 100 YP 009051654.1 Lizard adenovirus 100 NP 659523.1 Ovine adenovirus 100 88 YP 001552247.1 Snake adenovirus 95 Bovine adenovirus NP 659515.1 Ovine adenovirus 98 100 100 Duck atadenovirus 99 YP 009414575.1 Odocoileus adenovirus 76 100 100 YP 009112724.1 Psittacine adenovirus Bovine adenovirus AP 000351.1 Murine mastadenovirus AP 000342.1 Murine mastadenovirus 100 100 NP 046314.1 Bovine adenovirus YP 009505695.1 Bottlenose dolphin adenovirus 99 YP 009373238.1 Deer mastadenovirus 100 NP 046323.1 Bovine adenovirus 92 81 83 YP 009505686.1 Bottlenose dolphin adenovirus YP 009373247.1 Deer mastadenovirus NP 108658.1 Porcine adenovirus 100 100 Bat adenovirus Bat adenovirus YP 009389499.1 Squirrel adenovirus YP 009389490.1 Squirrel adenovirus 100 Primate adenovirus Primate adenovirus 100

989 0.3 0.3

50 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.

990 Figure 7

991

992

993

51 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.

994 Figure 8

995

996

52 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.

997 Figure 9

998

53 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.

999 Figure 10

1000

A

B

+ Positive + weak postive 1001 Negative

54