Journal of General Virology

Discovery of a novel nidovirus in cattle with respiratory disease --Manuscript Draft--

Manuscript Number: VIR-D-14-00290R1 Full Title: Discovery of a novel nidovirus in cattle with respiratory disease Short Title: Genetic characterization of a novel nidovirus in cattle Article Type: Short Communication Section/Category: Animal - Positive-strand RNA Viruses Corresponding Author: Rafal Tokarz Columbia University New York, NY UNITED STATES First Author: Rafal Tokarz Order of Authors: Rafal Tokarz Stephen Sameroff Richard A. Hesse Ben M. Hause Aaloki Desai Komal Jain W. Ian Lipkin Abstract: The family Coronaviridae represents a diverse group of vertebrate RNA viruses, all with genomes greater than 26,000 nucleotides. Here, we report the discovery and genetic characterization of a novel virus present in cattle with respiratory disease. Phylogenetic characterization of this virus revealed that it clusters within Torovirinae, a subfamily of Coronaviridae. The complete genome consists of only 20,261 nucleotides, and represents the smallest reported Coronaviridae genome. We identified seven open reading frames, including the canonical nidovirus open reading frames 1a and b. Analysis of polyprotein 1ab revealed that this virus, tentatively named bovine nidovirus (BoNV), shares the highest homology with the recently described python-borne nidoviruses and contains several conserved nidovirus motifs, but does not encode the NendoU or O-MT domains that are present in other viruses within Coronaviridae. In concert with its reduced genome, the atypical domain architecture indicates that this virus represents a unique lineage within the order Nidovirales.

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1 Discovery of a novel nidovirus in cattle with respiratory disease

2

3 Rafal Tokarz1 *, Stephen Sameroff1, Richard A. Hesse2, Ben M. Hause2, Aaloki

4 Desai1, Komal Jain1, W. Ian Lipkin1

5

6 1Center for Infection and Immunity, Mailman School of Public Health, Columbia

7 University, NY, 10032 USA

8 2 Kansas State Veterinary Diagnostic Laboratory and Department of Diagnostic

9 Medicine and Pathobiology, Kansas State University, Manhattan, KS 66508,

10 USA

11 * Corresponding author

12 Rafal Tokarz

13 Center for Infection and Immunity

14 Mailman School of Public Health, Columbia University

15 722 West 168th Street, Room 1701, New York, NY 10032

16 phone: (212) 631 335 5021

17 Email: [email protected]

18

19 The GenBank accession number for the bovine nidovirus sequence is KM589359

20

21 Running title: Genetic characterization of a novel nidovirus in cattle

22 Contents: Animal-Positive strand viruses

23 Word count Abstract: 150 Results: 2293 24 Abstract

25 The family Coronaviridae represents a diverse group of vertebrate RNA viruses,

26 all with genomes greater than 26,000 nucleotides. Here, we report the discovery

27 and genetic characterization of a novel virus present in cattle with respiratory

28 disease. Phylogenetic characterization of this virus revealed that it clusters within

29 the Torovirinae, a subfamily of Coronaviridae. The complete genome consists of

30 only 20,261 nucleotides, and represents the smallest reported Coronaviridae

31 genome. We identified seven open reading frames, including the canonical

32 nidovirus open reading frames 1a and b. Analysis of polyprotein 1ab revealed

33 that this virus, tentatively named bovine nidovirus (BoNV), shares the highest

34 homology with the recently described python-borne nidoviruses and contains

35 several conserved nidovirus motifs, but does not encode the NendoU or O-MT

36 domains that are present in other viruses within Coronaviridae. In concert with its

37 reduced genome, the atypical domain architecture indicates that this virus

38 represents a unique lineage within the order Nidovirales.

39

40

41

42

43

44

45

46 47 Summary

48 The order Nidovirales is comprised of enveloped, positive-sense, single-

49 stranded RNA viruses that share similar genome organization, homology in the

50 replicase polyprotein, and a unique replication strategy (deGroot et al., 2011a;

51 Gorbalenya et al., 2006). The genomic architecture of nidoviruses is

52 characterized by two large partially overlapping open reading frames (ORFs),

53 ORF1a and ORF1b, that encode components of the viral replicase and occupy

54 approximately two-thirds of the genome. ORFs encoding the structural

55 components are located downstream of ORF1a/b and are expressed from 3’ co-

56 terminal subgenomic mRNAs (Brian & Baric, 2005; Sawicki et al., 2007).

57 Nidoviruses are taxonomically divided into four families, Arteriviridae,

58 Coronaviridae, Mesoniviridae, and Roniviridae (deGroot et al., 2011a; Lauber et

59 al., 2012). In addition to phylogenetic classification, genome size is a distinctive

60 feature of these viral families. Arteriviruses (genome size of 12.7 kb to 15.7 kb)

61 have been referred to as “small nidoviruses”, mesoniviruses (~20 kb) as

62 “intermediate”, and both roniviruses (~26 kb) and coronaviruses (26-33 kb) as

63 “large” (deGroot et al., 2011a; Gorbalenya et al., 2006; Nga et al., 2011). Based

64 on genetic and phenotypic differences, the family Coronaviridae is further divided

65 into subfamilies Coronavirinae and Torovirinae (deGroot et al., 2011b).

66 Torovirinae consists of two genera, Torovirus and Bafinivirus. The Torovirus

67 genus includes four viruses originating from mammals (human torovirus, bovine

68 torovirus, equine torovirus, and porcine torovirus) (Beards et al., 1984; Draker et

69 al., 2006; Sun et al., 2014; Weiss et al., 1983). The Bafinivirus genus includes 70 two viruses isolated from fish (White Bream virus and White minnow nidovirus)

71 (Batts et al., 2012; Schutze et al., 2006). Within the past year, nidoviruses have

72 been described in pythons with pneumonia (Python nidovirus (PVN) and Ball

73 python nidovirus (BPVN)) that are proposed to represent a novel genus within

74 Torovirinae (Bodewes et al., 2014; Stenglein et al., 2014; Uccellini et al., 2014).

75 Here, we report the discovery and genomic characterization of a novel virus that

76 represents a distinctive lineage within this subfamily.

77 In 2013, an outbreak of severe respiratory disease occurred in a cattle

78 feedlot located in Southwestern United States. The animals suffered from severe

79 tracheitis and pneumonia, with a high mortality rate. Although a viral agent was

80 suspected, initial culture efforts were unsuccessful. In an effort to examine the

81 breadth of viral agents present, post-mortem tissue samples were obtained from

82 four animals and analyzed by high throughput sequencing (HTS) using the

83 Illumina Hiseq 2500 platform (Table 1).

84 Analysis of HTS data identified the presence of a virus with <35% amino

85 acid (aa) homology to viruses within Torovirinae in samples from three animals

86 (Table 1). The complete genome of this virus, provisionally called bovine

87 nidovirus (BoNV), was assembled from the lung sample of one of the animals

88 (GenBank under accession number KM589359). The authenticity of the draft

89 genome was confirmed by PCR using specific primers designed to generate

90 overlapping fragments along the length of genome and was followed by dideoxy

91 sequencing of the resulting amplification products. The genome termini were

92 obtained by 5’ and 3’ rapid amplification of cDNA ends. Using this genome 93 sequence as a reference, we assembled <80% of the BoNV genomes present in

94 the other two virus-positive animals. These sequences were 99% identical to the

95 reference sequence, suggesting that all three animals were infected with the

96 same strain of BoNV.

97 The complete genome of BoNV is comprised of 20,261 nucleotides (nt)

98 and contains seven ORFs (Fig.1(a)). Flanking the ORFs are 486 nt 5’ and 453 nt

99 3’ untranslated regions. The initial two ORFs are homologous to the canonical

100 ORFs 1a and 1b of Nidovirales. During viral replication, the expression of ORF1a

101 yields polyprotein 1a (pp1a) while translation of ORF1b is initiated by a -1

102 ribosomal frameshift sequence upstream of the ORF1a stop codon and results in

103 polyprotein 1ab. In BoNV, the putative frameshift sequence of UUUAAAC was

104 identical to the sequence found in Toroviruses and Banifiviruses but differs from

105 the sequence identified in the python-borne viruses (AAAAAAC). BlastP

106 homology searches using the amino acid sequences of both pp1a and pp1b

107 revealed that BoNV is most similar to viruses within Torovirinae, although limited

108 overall sequence identity was observed throughout either polyprotein (<30%).

109 The highest identity to BoNV within pp1a or pp1b was observed within the

110 python-borne nidoviruses PNV and BPVN (Table S1). Notably, the combined

111 size of BoNV pp1ab was the smallest among all Coronaviridae. Of all

112 nidoviruses, only arteriviruses were found to contain a smaller polyprotein.

113 The pp1a and pp1ab contain several conserved domains encoding

114 enzymes that are processed into their individual components following post-

115 translational cleavage of the polyprotein (deGroot et al., 2011a). We used protein 116 prediction tools including INTERPRO, HMMER3, and BLASTp to identify such

117 signature motifs within the polyproteins of BoNV. Analysis of pp1a revealed the

118 presence of a serine/threonine protein kinase, three hydrophobic regions (TM1-

119 TM3) and the main protease (Mpro) involved in posttranslational processing of

120 pp1a/b (Fig.1(b)). The Mpro domain was encoded between TM2 and TM3, a

121 location conserved in all nidoviruses. We also identified a homolog of

122 Coronavirus non-structural protein 10 (nsp10), a zinc-binding protein that is

123 involved in RNA synthesis (Bouvet et al., 2014).

124 ORF1b encodes the more highly conserved portion of pp1ab and is

125 characterized by a set of domains encoding replicative enzymes. The order of

126 these domains is preserved and in coronaviruses encompasses the RNA-

127 dependent RNA polymerase (RdRp), superfamily 1 helicase (Hel1), 3’ to 5’

128 exoribonuclease (ExoN), N7-methyltransferase (NMT), uridylate-specific

129 endonuclease (NendoU), and 2’-O-methyltransferase (O-MT). Although all

130 nidoviruses contain the RdRp and Hel, the presence of the remainder of these

131 domains varies across nidoviruses. Analysis of the pp1b of BoNV revealed that it

132 encodes domains homologous to RdRp, Hel1 and ExoN in the canonical order.

133 We did not identify an NMT domain, consistent with its absence in all viruses

134 within Torovirinae. Surprisingly, we also did not identify a homolog of NendoU

135 and O-MT domains. All nidoviruses reported to date encode at least one of these

136 enzymes.

137 For phylogeny inference, the aa sequences of the Hel and RdRp domains

138 of viruses within Coronaviridae were aligned and compared in Mega 6.0 (Tamura 139 et al., 2013). For both domains, BoNV forms a monophyletic branch within

140 Torovirinae, indicative of a novel taxonomic group within this subfamily (Fig. 2

141 and Fig. S1).

142 BoNV encodes five structural ORFs downstream of ORF1ab (Fig.1(a)).

143 ORF2 is located within the region of the genome typically occupied by the gene

144 encoding the S (“spike”) protein, a type I glycoprotein involved in receptor binding

145 and membrane fusion. This ORF is 1689 nt in length, and encodes a putative

146 562 aa protein containing a signal peptidase cleavage sequence between aa 18

147 and 19, a transmembrane domain between aa 537 and 559 and multiple

148 potential glycosylation sites. Homology searches revealed similarity only to the S

149 of the python-borne viruses, although with only 17% aa sequence identity within

150 the complete protein. In addition, although the BPNV and PNV S proteins are

151 among the shortest within Coronaviridae, the BoNV S is only 58% of their size

152 and represents the shortest Coronaviridae spike protein reported to date. ORF3

153 of BoNV encodes a putative 231 aa protein that contains multiple trans-

154 membrane (TM) spanning regions similar to Coronaviridae membrane proteins

155 (M). ORF4 encodes a 178 aa protein predicted by genomic location as a

156 nucleocapsid (N) protein. Similar to other Torovirinae N proteins it is short

157 (approximately half the size of typical Coronavirus N) and highly basic with a

158 predicted isoelectric point of 12.0. ORF 5 encodes a 455 aa protein that contains

159 multiple glycosylation sites and a predicted TM region between aa 429-448 and

160 likely represents another glycoprotein (designated G2). We also identified a very

161 short, putative 267 nt ORF that would encode an 88 aa protein near the 3’ end of 162 the genome. Unlike ORFs 1a/b and ORF2, ORFs 3-6 do not demonstrate

163 significant similarity to any viral or non-viral proteins.

164 To examine the seroprevalance of BoVN, we generated a Luciferase

165 Immunoprecipitation assay (LIPS) to measure antibody levels against BoVN

166 (Burbelo et al., 2009). We implemented this assay to examine 127 bovine sera

167 originating from central and western US (Kansas, Idaho, Missouri and Texas). All

168 samples were previously submitted for diagnostic testing for other bovine

169 infectious agents. Eleven sera (9%) positive for BoVN (Fig. S2). The specificity of

170 the BoNV LIPS assay was demonstrated by the lack of reactivity with bovine

171 coronavirus-positive sera.

172 The classification of Nidovirales is strictly based on sequence similarity.

173 Based on this criteria, the phylogeny of BoNV suggests that it is a member of a

174 novel genus within subfamily Torovirinae. However, BoNV has several genetic

175 characteristics that are atypical of viruses in this subfamily. First, at

176 approximately 20,000 nt, the BoNV genome is much shorter that the eight

177 viruses currently classified as Torovirinae that have genomes that range from

178 26,000 to 33,000 nt. The “intermediate” size of BoNV genome is more

179 characteristic of mesoniviruses though it shares little sequence homology with

180 viruses in this family. Second, BoNV would currently be the only virus within

181 Torovirinae lacking an O-MT and NendoU homolog in its genome. Interestingly,

182 BoNV shares the highest aa similarity with PNV and BPNV, viruses with the

183 largest RNA genomes discovered to date. For contrast, the genome of BoNV

184 measures only 60% of the size of these python-borne viruses. 185 We speculate that this virus has undergone an atypical route of genome

186 expansion. The expansion of the nidovirus genome has been correlated with an

187 evolutionary procurement of enzymes controlling RNA replication fidelity. The

188 acquisition of 3’-to 5’ exoribonuclease (ExoN), a homolog of a DNA proofreading

189 enzyme, has been proposed to be of particular importance in the transition from

190 “small” genomes (arteriviruses) to “intermediate” and “large” (genomes of <20 kb)

191 (Gorbalenya et al., 2006; Lauber et al., 2013; Nga et al., 2011). Consistent with

192 this hypothesis, we identified an ExoN homolog within the “intermediate” sized

193 genome of BoNV. The gain of additional enzymes such as O-MT or NendoU, and

194 their role in expansion of nidovirus genome size is less understood. If, as

195 proposed in Lauber et al, NendoU was present in an ancestral lineage that

196 culminated in the four known families of nidoviruses, it would suggest that this

197 domain was lost at some point in the BoNV evolutionary history. Such domain

198 loss would not be unprecedented as NendoU has been lost in both

199 mesoniviruses and roniviruses (Lauber et al., 2013; Nga et al., 2011). In contrast,

200 the O-MT domain is present in all nidoviruses with “intermediate” and “large”

201 genomes. It was suggested that this enzyme may have been acquired following

202 the split of the larger nidoviruses from arteriviruses and if so, its absence in

203 BOVN suggests it too would have been lost similar to NendoU. Alternatively, it is

204 also possible that the ancestral line that gave rise to BoNV never acquired it.

205 The clinical significance of BoNV is unknown. Although coronaviruses are

206 frequently implicated in disease in mammals, we do not have conclusive data to

207 associate BoNV with illness. BoNV was not the only viral agent detected by HTS 208 (Table 1). Furthermore, samples from healthy cows from the same feedlot were

209 not available for analysis.

210 In summary, we propose that BoNV may be a descendant of the ancestral

211 nidovirus lineage, one that acquired ExoN, facilitating the expansion of its

212 genome beyond the size of “small” nidoviruses. Based on their sequence

213 similarity, BoNV and the python-associated viruses may have shared a common

214 ancestor and likely split prior to a major expansion of the Torvirinae genomes.

215

216 Acknowledgments

217 This work was supported by grants from the National Institutes of Health

218 1U19AI109761 (Center for Research in Diagnostics and Discovery) and by

219 Zoetis Inc.

220

221

222

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299

300

301

302 Figure Legends

303 Figure 1. (A) Schematic representation of the bovine nidovirus genome. Each

304 ORF is designated by an arrow corresponding to its approximate size. The

305 ribosomal frameshift sequence UUUAAAC that results in translation of ORF1ab

306 is indicated by a black arrow. The ORF designations S, M, and N correspond to

307 the glycoprotein spike, membrane and nucleocapsid proteins, respectively. The

308 nucleotide coordinates are displayed on top. Genome assemblies were done in

309 Geneious v6.1.5. (B) Comparison of the length and domain organization of

310 polyprotein 1ab of bovine nidovirus and its closest homolog, the Ball python

311 nidovirus. The location and approximate length of each domain is shown. The

312 ORF1a portion of the polyprotein is indicated in gray, ORF1b in white. The amino

313 acid coordiates are shown on top. The schematic of the Ball python nidovirus

314 was modified from Stenglein et al.

315

316 Figure 2. Maximum likelihood phylogeny of Coronaviridae based on the amino

317 acid sequence of the RdRp (A) and the helicase (B). Trees were generated using

318 the JTT matrix-based model. Each genus within the Coronavirinae and 319 Torovirinae subfamilies is highlighted by a gray box. For Coronavirinae, only

320 representative ICTV-approved species with available complete sequence were

321 included. For Torovirinae, newly reported viruses were also included. The

322 accession numbers for sequences used in the tree were: Feline infectious

323 peritonitis virus (NC002306), Human coronavirus 229E (NC002645), Human

324 coronavirus NL63 (NC005831), Porcine epidemic diarrhea virus (NC003436), Bat

325 coronavirus HKU2 (NC009988), Scotophilus bat coronavirus 512 (NC009657),

326 bovine coronavirus (NC003045), Human coronavirus HKU1 (NC006577), murine

327 hepatitis virus (NC001846), Pipistrellus bat coronavirus HKU5 (NC009020),

328 Rousettus bat coronavirus HKU9 (NC009021), Severe acute respiratory

329 syndrome coronavirus (NC004718), Tylonycteris bat coronavirus HKU4

330 (NC009019), Munia coronavirus HKU13 (NC011550), Thrush coronavirus

331 HKU12 (NC011549), Bulbul coronavirus HKU11 (FJ376619), avian coronavirus

332 (NC001451), Beluga whale coronavirus SW1 (NC010646), Fathead minnow

333 nidovirus (GU002364), Porcine torovirus (NC022787), Breda virus (NC007447),

334 Berne virus (X52374), python nidovirus (KJ935003) Ball python nidovirus

335 (NC024709)

336

337

338

339

340

341 342

343

344 Table1. List of samples analyzed by high-throughput sequencing.

Other viruses identified Animal Sample type Bovine nidovirus* by HTS lung homogenate - bovine coronavirus

1 parvovirus, bovine lymph node homogenate + herpesvirus

2 trachea homogenate - bovine herpesvirus

lung homogenate + bovine rhinovirus B

abomasum homogenate - 6 > 3 trachea homogenate - bovine rhinovirus B, 4 trachea homogenate + polyomavirus 345

346

347 * nidovirus sequences were identified by HTS and were confirmed by PCR

348 Figure 1 Click here to download Figure: FIGURE 1.pdf

A

1 4000 8000 12000 16000 ORF 3 ORF 5 20261 ORF1A ORF1B M G2 S ORF1A/B ORF 2 N ORF 6 ORF 4

B 1 1000 2000 3000 4000 5000 6000 7000 8108

Pkinase Mpro RdRp Hel NendoU BPNV

ExoN O-MT ExoN BONV

Pkinase Mpro nsp10 RdRp Hel Figure 2 Click here to download Figure: FIGURE 2 PHYLOGENY RDRP AND HELICASE.pdf

Figure 2A Figure 2B

Feline infectious peritonitis virus 88 Porcine epidemic diarrhea virus 97 Rhinolophus bat coronavirus HKU2 Scotophilus bat coronavirus 97 92 Porcine epidemic diarrhea virus Miniopterus bat coronavirus HKU8 76 Scotophilus bat coronavirus 512 Alphacoronavirus 50 Human coronavirus 229E Alphacoronavirus Miniopterus bat coronavirus HKU8 31 Human coronavirus NL63 75 Human coronavirus 229E 11 Rhinolophus bat coronavirus HKU2 67 73 93 86 Human coronavirus NL63 26 Feline infectious peritonitis virus 79 Murine hepatitis virus 1 59 SARS coronavirus Coronavirinae 100 Human coronavirus HKU1 Coronavirinae 42 Rousettus bat coronavirus HKU9 54 Bovine coronavirus Tylonycteris bat coronavirus HKU4 40 85 SARS coronavirus 49 100 Pipistrellus bat coronavirus HKU5 Betacoronavirus Betacoronavirus 78 Rousettus bat coronavirus HKU9 Bovine coronavirus 94 Murine hepatitis virus 1 Tylonycteris bat coronavirus HKU4 100 100 100 88 Human coronavirus HKU1 99 Pipistrellus bat coronavirus HKU5 Infectious bronchitis virus Infectious bronchitis virus Gammacoronavirus Gammacoronavirus 74 Beluga Whale coronavirus 98 Beluga Whale coronavirus SW1 Munia coronavirus Munia coronavirus HKU13 Bulbul coronavirus HKU11 Deltacoronavirus 99 Bulbul coronavirus Deltacoronavirus 85 80 Thrush coronavirus 50 Thrush coronavirus HKU12 84 Breda virus 100 Berne virus 99 Breda virus Torovirus Berne virus Torovirus 44 43 Porcine torovirus Porcine torovirus 100 White bream virus White bream virus Bafinivirus Bafinivirus 99 Fathead minnow nidovirus Torovirinae 67 Fathead minnow nidovirus Torovirinae 100 Python nidovirus Python nidovirus Ball python nidovirus 60 100 Ball python nidovirus 68 Bovine nidovirus Bovine nidovirus 0.2 * * 0.2 Supplementary Material Files Click here to download Supplementary Material Files: SUPPLEMENTAL MATERIAL.pdf

Additional Material for Reviewer Click here to download Additional Material for Reviewer: MARKED UP FILE BoNV MANUSCRIPT REVISION .docx