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. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): BoNV MANUSCRIPT REVISION A.docx 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-