a metagenomics and case-control study to identify viruses

A metagenomics and case-control study to 1 identify viruses associated with bovine 2

respiratory disease 3 4 Terry Fei Fan Ng1,2,5 #, Nikola O. Kondov1,2 #, Xutao Deng1,2, Alison Van 5 Eenennaam3, Holly L. Neibergs4, Eric Delwart1,2* 6 7 1. Blood Systems Research Institute, San Francisco, California 8 2. Department of Laboratory Medicine, University of California at San 9 Francisco, San Francisco, California 10 3. Department of Animal Science, University of California, Davis, 11 California 12 4. Department of Animal Sciences, Washington State University, Pullman, 13 Washington 14 5. Current address: NCIRD, Centers for Disease Control and Prevention, 15 Atlanta, Georgia 16 17 # contributed equally to this work. 18 * Corresponding author: [email protected] 19 20

JVI Accepted Manuscript Posted Online 4 March 2015J. Virol. doi:10.1128/JVI.00064-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract: 21 Bovine respiratory disease (BRD) is a common health problem for both dairy and 22 beef cattle resulting in significant economic loses. In order to identify viruses 23 associated with BRD we used a metagenomics approach to enrich and sequence 24 viral nucleic acids in the nasal swab of fifty young dairy cattle with symptoms of 25 BRD. Following deep sequencing, de novo assembly, and translated protein 26 sequences similarity searches, numerous known and previously uncharacterized 27 viruses were identified. Bovine adenovirus-3, bovine adeno-associated virus, 28 bovine influenza D, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A and 29 multiple genotypes of bovine rhinitis B, were identified. The genomes of a 30 previously uncharacterized astrovirus and picobirnaviruses were also partially or 31 fully sequenced. Using real time PCR the detection rate of the eight viruses that 32 generated the most reads were compared in the nasal secretions of 50 animals with 33 BRD versus 50 location-matched healthy control animals. Viruses were detected in 34 68% of BRD-affected versus 16% of healthy control animals. Thirty eight % of sick 35 animals versus 8% of controls were infected with multiple respiratory viruses. 36 Significantly associated with BRD were bovine adenovirus 3 (P<0.0001), Bovine 37 rhinitis A (p=0.005), and the recently described bovine influenza D (P=0.006) which 38 were detected either alone or in combination in 62% of animals with BRD. A 39 metagenomics and real time PCR detection approach in carefully matched cases and 40 controls, can provide a rapid means to identify viruses associated with a complex 41 disease opening the way to effective intervention strategies. 42


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Importance 43 Bovine respiratory disease is the most economically important disease affecting the 44 cattle industry whose complex root causes includes environmental, genetics, and 45 infectious factors. Using an unbiased metagenomics approach we characterized the 46 viruses in respiratory secretions from BRD cases and identified known and 47 previously uncharacterized viruses belonging to seven viral families. Using a cases-48 control format with location-matched animals we compared the rate of viral 49 detection and identified 3 viruses associated with severe BRD signs. Combining a 50 metagenomics and case-control format can provide candidate pathogens associated 51 with complex infectious diseases and inform further studies aimed at reducing their 52 impact. 53 54 Introduction 55 Bovine respiratory disease (BRD) is the most common and costliest problem 56 in the cattle industry accounting for 70-80% of morbidity and 40-50% of 57 mortality in US feedlots (3, 25). The cattle industry is one of the largest 58 agricultural sectors of the United States economy with approximately ¾ of a 59 million farms raising cattle. The annual costs of BRD have been estimated at 60 over 1 billion dollars per year (17, 46). 61 In BRD, bacterial infections are thought to be opportunistic infections 62 precipitated by viral infections causing damage to the respiratory epithelium 63 (18, 47). The use of prophylactic antibiotic treatment has been shown to be of 64 limited utility in reducing BRD and concerns exists about decreasing efficacy 65 and the spread of antibiotic resistance to bovine or human pathogens (56). A 66 review of the efficacy of bacterial vaccination in feedlot cattle for Histophilus 67 somni, Mannheimia haemolytica, and Pasteurella multocida indicated little to 68 no benefit (18, 34). Overall, neither immunization nor antimicrobial 69 therapies have noticeably reduced the prevalence or severity of BRD (46). 70 Prior studies have implicated several viruses in BRD (14, 23, 25, 47). 71 Studies testing for one or a few viruses or measuring sero-conversions have 72


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reported the following as associated with BRD: bovine herpesvirus-1 (BHV-1) 73 (14, 48) (Herpesviridae), bovine viral diarrhea virus (BVDV in the Flaviviridae 74 family) (50, 71), bovine parainfluenza type 3 (PI3V) and bovine respiratory 75 syncytial virus (BRSV)(both in the Paramyxoviridae family)(10, 14, 33), 76 bovine adenovirus 3 (BAdV3)(11, 23) and bovine coronavirus (BoCV)(24, 33, 77 60). 78 Applications of viral metagenomics has recently allowed the rapid 79 genetic characterization of viral genomes and has found wide spread 80 applications in animal viral discovery (6, 12, 13, 59). Studies of farm animals 81 have revealed the presence of both known and new viruses occurring at high 82 frequency in healthy and sick animals (12, 31, 36, 39, 63, 72). Using deep 83 sequencing the Schmallenberg bunyavirus was recently identified in the 84 plasma of cows with fever, decreased milk production and diarrhea and 85 shown to induce symptoms following inoculations (26). A novel astrovirus 86 was recently identified in brain tissues as the likely cause of a common 87 neurological condition in cattle (8, 37). 88 In order to characterize the respiratory virome of cattle with BRD we 89 analyzed nasopharyngeal and pharyngeal recess swabs from 50 young dairy 90 calves with symptoms of BRD and 50 healthy controls collected by 91 researchers associated with the Bovine Respiratory Disease Complex 92 Coordinated Agricultural Project (BRDC-CAP)(51). Using partial and complete 93 viral genomes we then designed real time PCR assays to measure the 94 frequency of infection and viral loads in animals with BRD and matched 95 healthy controls to identify which respiratory viruses were associated with 96 this disease. 97 98 Materials and Methods 99 California dairy calves between the ages of 27 and 60 days of age housed in 100 hutches were closely monitored and considered for enrollment as either BRD 101 cases or controls, based on the assignment of calf health scores using a 102


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respiratory screening tool developed by McGuirk (45). Calves were observed 103 and assigned a numeric health score (0 for normal, 1 for slightly abnormal, 2 104 for abnormal, and 3 for severely abnormal) for each of the five health criteria: 105 Rectal temperature, cough, nasal discharge, and the greater of the two scores 106 for eye discharge, and ear tilt. Calves with a cumulative score ≥ 5 were 107 enrolled in the BRDC-CAP study as a BRD case. BRD calves studied here were 108 selected among those with the most severe signs with cumulative scores of 8-109 12. Immediately thereafter, a calf adjacent to the BRD case that possessed a 110 cumulative score ≤ 3 was enrolled as a matched control for this study. 111 All case and control calves had nasopharyngeal and pharyngeal recess 112 swabs collected. Samples collected from the nasopharyngeal region utilized a 113 six-inch sterile unguarded polyester swab that was inserted five inches into a 114 clean naris and rotated against the surface for 15 seconds. Thereafter, the 115 swab was removed and placed into 3 ml of viral transport media (minimum 116 essential media, HCO3, HEPES, Gentamycin, Amphotericin B). The pharyngeal 117 recess was swabbed using a 27-inch sterile guarded swab with a polyester 118 fiber tip (Kalajian Industries, Signal Hill CA 90755). The swab was rotated 119 against the pharyngeal recess surface for 15 seconds, retracted back into the 120 guarding sheath, and placed into the same tube of viral transport media 121 containing the nasopharyngeal swab. 122 123 Viral metagenomics 124 Viral transport media supernatants from nasopharyngeal and pharyngeal 125 recess swabs were collected on 50 BRD case animals with cumulative health 126 scores ranging from 8-12. Ten pools of five randomly selected BRD samples 127 were assembled using 32ul from each sample. The resulting 160µl pools were 128 passed through a 200 nm-filter and the resulting filtrates were incubated for 129 1.5 hours in a cocktail of DNAse and RNAse enzymes. Nucleic acids were then 130 extracted using QiaQuick Viral RNA column purification system according to 131 manufacturer’s instructions. Reverse transcription was performed using a 28 132 bases oligonucleotide whose 3’ end consisted of eight N (all four nucleotides 133


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at each of the eight 3’ positions) while the 5’ end 20-bases consisted of an 134 arbitrarily designed sequence (Primer N1, 135 CCTTGAAGGCGGACTGTGAGNNNNNNNN). Following denaturation of the first-136 strand cDNA product, a complimentary strand was synthesized using Klenow 137 fragment DNA polymerase extension (New England Biolabs). The resulting 138 double-stranded cDNA and DNA was then PCR amplified using AmpliTaq Gold 139 DNA Polymerase and a 20 bases primer (same as above minus the 8N) (Ng et 140 al., 2012). The randomly amplified nucleic acid was then subjected to 141 NexteraXT library preparation protocol (Illumina) according to 142 manufacturer’s instructions and sequenced using Illumina’s HiSeq Platform. A 143 total of 23.7 milions reads were generated, fewer than the 100-150 millions 144 expected due to sub-optimal cluster formation. 145 146 Bioinformatic analysis 147 An in-house analysis pipeline running on a 32-nodes Linux cluster was used 148 to process the data. One hundred bases reads from one lane of a HiSeq run were 149 debarcoded using vendor software from Illumina. Human reads and bacterial 150 reads were subtracted by mapping these reads to human reference genome 151 hg19 and bacterial RefSeq genomes release 66 using bowtie2 (32). Reads 152 were considered duplicates if bases 5 to 55 from 5’ end were identical. One 153 random copy of duplicates was kept. Low sequencing quality tails were 154 trimmed using Phred quality score 10 as the threshold. Adaptor and primer 155 sequences were trimmed using the default parameters of VecScreen (44). The 156 cleaned reads were de-novo assembled using an in-house de novo sequence 157 assembler consists of SOAPdenovo2 (40), ABySS (65) , meta-Velvet (49) and 158 CAP3 (28). The assembled contigs, along with singlets were aligned to an in-159 house viral proteome database using BLASTx. 160 Sequences belonging to different viral families are re-analyzed manually to 161 identify sequence diversity and viral strains (Geneious version R6). Viral 162 contigs from each pool and across pools were re-assembled with high 163 seqeuence identity criteria. Reads representing the same viral genome with 164


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high sequence identities (>95%) are re-assembled into larger contig or 165 genome. Reads representing different viral strains (sequence identities 166 <95%) were individually identified and analyzed (such as the different BRBV 167 strains), which were then confirmed by specific PCR and Sanger sequencing. 168 Finally, cleaned reads were mapped to the final viral sequence to account for 169 the number of reads to that sequence (Geneious version R6). 170 171 172 173 Genome Extension and RACE 174 Gaps in the viral genomes were filled using Superscript III-Platinum Taq One-175 Step RT-PCR kit for RNA sequences shorter than 2000 base-pairs, and Takara 176 Ex Taq PCR kit for DNA sequences shorter than 2000 base-pairs. Longer gaps 177 were obtained by bridging PCR amplification with Takara LA Taq. In the case 178 of RNA genomes, random cDNA templates were generated prior to PCR 179 amplification with Prime Script Reverse Transcriptase (Takara) using a 1:1 180 combination of randomly generated hexa- and octamer sequences. 181 Extremities of RNA genomes were obtained using Clontech SMARTerII RACE 182 cDNA kits followed by amplification with LA Taq (Takara). 183 184 Next-generation sequencing of individual samples 185 When deemed necessary due to a large number of gaps and/or an incomplete 186 genome, individual samples were prepared for next-generation sequencing in 187 a manner analogous to the one described above. Individual samples were re-188 sequenced using an Illumina MiSeq Platform. 189 190 Case-Control Real-Time PCR 191 Viral transport media supernatants from nasopharyngeal and pharyngeal 192 recess swabs from asymptomatic control calves (cumulative health scores 193 ranging from 0-2) were obtained in a manner analogous to that used for 194 symptomatic animals. Total nucleic acid was extracted from 140 µl using 195


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QiaQuick Viral RNA extraction kit (QiaGen). Random cDNA was generated 196 using Protoscript II Reverse Transcriptase (New England Biolabs). The 197 reaction mix for the reverse transcription consisted of 2 µl of a 1.0M KCl, 198 0.10M Tris Base, 0.0025M MgCl26H2O (pH8.3) and 100mM Tris, 25mM 199 MgCl26H2O, 10%Tween-20, 10%NP40 solutions mixed at a 1:1 ratio, 0.2 µl 25 200 mM dNTP, 0.075 µl 100N Random Hexamer mix, 0.5 µl RiboLock RNAse 201 Inhibitor (ThermoFisher Scientific) and 0.5 µl Protoscript II Reverse 202 Transcriptase (New England Biolabs). 3.28 µl of the above-descriped pre-mix 203 was mixed with 16.72 µl of total extracted nucleic acid, incubated at 25⁰C for 204 10 minutes, 42⁰C for 30 minutes and 95⁰C for 10 minutes to produced 205 randomized cDNA mix. For DNA virus screening, the cDNA synthesis step was 206 omitted, and total nucleic acid was used as a template. Real-Time PCR 207 amplification was performed in a Roche 480 Thermocycler using a Fam-Zen 208 TaqMan probe specific to the virus of interest. The Primer-probe set for each 209 virus investigated by real-time PCR, and the amplicon lengths are summarized 210 in Supplementary Table 1. The reaction mix for each sample consists of 15mM 211 KCl, 40mM Tris HCL, 25ug BSA, 5mM Mg++, 20mM dNTP, 1uM of each primer, 212 0.2uM TaqMan probe and 0.75 units Fastart Taq Polymerase (Roche). 213 Amplification followed the following cycling profile: 1 minute initial 214 dentaturation step at 95⁰C followed by 45 cycles of 30s 95⁰C denaturation 215 and 1 minute 60⁰C extension step. Ct values were thus used as a relative 216 measure of viral load. Ct values of 45 indicate viral nucleic acid below level of 217 detection. Lower Ct value indicates higher viral loads. Because the real time 218 PCRs were not calibrated with known concentrations of target nucleic acids 219 the Ct values only reflect relative viral loads for the same virus in different 220 samples and cannot be compared across different viruses. 221 222 Sequence and Phylogenetic analysis 223 The open reading frame of the viral nucleotide sequences were analyzed with 224 Geneious version R6. Complete or partial viral genomes sequences are 225 available in GenBank (Table 1). Sequences were aligned using MAFFT with the 226


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E-INS-I alignment strategy and previously described parameters (53). 227 Maximum likelihood (ML) trees were generated from translated protein 228 sequences using RAxML using Dayhoff similarity matrix parameters (66) ML trees 229 were run with 100 bootstrap replications with a gamma distribution for rates over 230 sites. Mid-point rooting was conducted using MEGA (67). 231 232 Results 233 Viral metagenomics 234 Deep nasal swabs collected from 50 animals with extreme symptoms of BRD 235 were randomly distributed in 10 pools of five samples that were then 236 processed for viral metagenomics using the Illumina Hiseq platform. After 237 quality control and removal of duplicates 11.5 million reads were used. 238 Following de novo assembly with the sequence reads from each pool both the 239 contigs and singlets were compared using BLASTx to the viral RefSeq 240 database in GenBank for translated protein similarity to the proteins of all 241 known eukaryotic viruses. The greatest number of hits using BLASTx to 242 eukaryotic viruses were to bovine rhinitis A virus > bovine adenovirus 3 > 243 adeno-associated virus > bovine rhinitis B virus > astrovirus-BSRI1 > bovine 244 influenza D> picobirnaviruses > bovine parvovirus 2 > bovine herpes virus 6. 245 We then generated complete or near complete genome sequences for a 246 subset of these viruses (Table 1). 247 248 Bovine Adenovirus 3 249 Deep sequencing revealed 4,279 sequences related to bovine adenovirus-3, 250 providing coverage of 82.6% of the total genome length. A complete DNA 251 polymerase sequence was obtained, which shared 98.4 % amino-acid identity 252 to bovine adenovirus-3 (AF030154)(Table 1). 253 254 Bovine Adeno-Associated Virus 255


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Bovine adeno-associated virus was detected with 3481 sequences. The 256 assembled genome contains replicase and capsid-encoding genes, and shared 257 98.5 % amino-acid identity in the replicase with previously described bovine 258 adeno-associated virus (BAAV) in the species dependoparvovirus B and 99% 259 identity in the capsid protein. Reads assembled into a complete genome contig 260 that shared 97 % nucleotide identities to BAAV reference genome 261 (AY388617) (Table 1). Phylogenetic analysis confirmed it to be closely related 262 to another bovine dependovirus BAAV originally found as a contaminant of 263 two isolates of bovine adenoviruses (62) (Fig 1A). 264 265 Bovine Rhinitis A and B Viruses 266 11,109 sequences were detected with sequence identities to bovine 267 rhinitis A virus (BRAV, Table 1). A near-complete genome (GenBank 268 KP264974) was assembled resulting in a 7,207 bases sequence. The complete 269 polyprotein showed 94% identity to that of the reference BRAV genome (and 270 single currently available genome). 512 bases of the 5’ UTR were generated 271 that were 91% identical to that of the reference genome. The 3D protein 272 sequence shared (98.3%) nucleotide identity to its closest homolog 273 (JN936206) (Table 1). 274 Another 2,639 sequence reads were more closely related to bovine 275 rhinitis B viruses (BRBV), and sequence analysis reveals several BRBV strains 276 existed in the data. With the use of de novo sequence assembly combined 277 with RT-PCR amplification of gapped regions and 3’RACE we obtained two 278 nearly complete (strain BSRI1 and 3) and one partial genome sequences 279 (strain BSRI2). The near complete genome of BRBV –BSRI1 was comprised of 280 7359 bases including the complete polyprotein, 420-base 5’UTR and 68-base 281 3’UTR excluding polyA end. The polyprotein of BRBV-BSRI shared 88% 282 identity to that of BRBV reference genome (NC_010354). The entire 283 polyprotein of BRBV-BSRI3 shared 81% identity to the BRBV reference. The 284 third strain, BRBV-BSRI2 was incomplete with only partial L-VP4-VP2-VP3-285


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VP1 (with 71% to BRBV reference), partial 2C-3B (77%) and complete 3D 286 (81%). 287 Overall these three distinct BRBV genotypes differ only slightly from 288 one another and the reference BRBV genome (NC_010354). The BRBV 289 reference shared 97.4%, 91.6% and 97% identity with the BRBV-BSRI1, -290 BSRI2 and -BSRI3 3D protein respectively while the three BRDV-BSRI strain 291 differed among themselves by 91.4 to 97.2%. The phylogenetic analysis 292 revealed multiple, closely related bovine rhinitis viruses from the genus 293 Aphthovirus, present in bovine respiratory disease sample set (Fig. 1B). 294 295 Bovine Influenza D Virus 296 Viral sequence closely related to Swine Influenza D/OK (JQ922308), including 297 the segment 4 that encodes the Hemagglutin-Esterase-Fusion (HEF) 298 glycoprotein were identified (92-98%). PCR amplification and Sanger 299 sequencing was used to identify an individual sample within a pool containing 300 influenza. Further deep sequencing of that sample generated 304 reads with 301 genome coverage of 72% of genome. These sequences shared 92-98 % 302 similarity to the seven segments of Swine Influenza D/OK (Suppl. Fig 1). Our 303 finding is consistent with the similar genomes that were recently described in 304 bovine respiratory disease samples (21, 22, 64)(Hause et al., 2014). 305 Maximum-likelihood phylogenetic analysis of the nucleotide sequence of the 306 HEF fragment is depicted in Figure 1C. 307 308 Bovine Astrovirus –BSRI1 309 Using 1666 sequence reads in combination with gap filling RT-PCR, RACE and 310 Sanger dideoxy sequencing, we obtained a complete genome of a divergent 311 bovine astrovirus (BoAstV-BSRI1). BoAstV-BSRI1 genome is 6,099 nt long, 312 with a GC content of 50.9 % (GenBank KP264970, Table 1). Consistent with 313 other members of the Astroviridae family, BoAstV-BSRI1 contains two open 314 reading frames (ORF1 and ORF2). ORF1 is segregated into two coding regions, 315 ORF1a and ORF1b, which encodes non-structure and RdRp protein 316


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respectively. The highly conserved AAAAAAC sequence, which constitutes the 317 ORF1a/b ribosomal frameshift site in astroviruses, was identified at residues 318 2,246-2,252. Additionally, the conserved promoter sequence 319 UUUGGAGNGGNGGACCNAAN14 AUGNC, was detected at the start of the ORF2. 320 ORF1b shared 83% nucleotide identities with a bovine astrovirus recently 321 identified in Korea (Table 1). 322 BoAstV-BSRI1belongs to astrovirus genogroup III, as shown by 323 phylogenetic analysis of the of the ORF2 capsid sequence (Fig 1D). ORF2 of 324 BoAstV-BSRI1 is most closely related to that of Bovine astrovirus B34/HK 325 (Genbank HQ916315), although with only 40.7 % protein identity. 326 327 Bovine Picobirnavirus 328 Two divergent picobirnaviruses were identified. Reverse transcription, PCR 329 amplification and Sanger sequencing confirmed the presence of these 330 sequences in two samples, both of which underwent further individual deep 331 sequencing. We obtained a total of 1, 286 and 1,366 base-pair long contigs for 332 these two picobirnaviruses. BLASTx analysis of the two partial RdRp sequence 333 revealed that they shared 33-34 % protein identity to Human PBV VS6600008 334 (KJ206569)(Table 1). Maximum-likelihood phylogenetic analysis of the 335 partial RdRP reveals a clear divergence between both viral sequences and 336 members of Genogroup I and II (Fig 1E), potentially placing these two bovine 337 picobirnaviruses in a distinct clade. 338 Unexpectedly both segments contain additional UGA stop codons. 339 One picobirnavirus (BPV-BSRI1) has six and BPV-BSRI2 had three premature 340 RdRp stop codons (GenBank accession numbers KP264972 and KP264973). 341 The presence of these stop codons was confirmed in both pooled, and 342 individual deep-sequencing runs using separate RNA extracts, as well as by 343 RT-PCR and Sanger sequencing. Premature stop codons have been reported in 344 other picobirnavirus RdRp genes (16, 52). Active RdRp may be provided by 345 minority helper picobirnavirus in the same cell or may be translated by 346 readthrough induced by an RNA stem loop structure. 347


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348 Bovine Parvovirus 2 349 79 sequences was identified with an average of 82.36 % nucleotide identity to 350 Bovine Parvovirus 2 (AF406966)(Table 1). An individual sample positive for 351 the presence of the genome was identified using PCR and Sanger sequencing, 352 and individually deep sequenced. We obtained 4,833 additional sequences 353 that assembled together and allowed for extension of the viral genome of 354 5,331 bases. Amino-acid comparison of the translated NS1 coding sequence 355 reveals 96 % identity to that of ungulate copiparvovirus 1 species 356 (AF406966) (2), placing it within the same species group as confirmed by 357 phylogenetic analysis (Fig 1A). 358 359 Bovine Herpesvirus 6 360 Deep sequencing revealed 19 sequences with an average nucleotide identity 361 of 98.16 % to Bovine Herpesvirus 6 (Table 1). 362 363 VIRUS DETECTION RATES IN BRD CASES VERSUS CONTROLS 364 To determine which of these viruses were associated with BRD real time PCR 365 assays were designed and Ct values were measured from nucleic acids in 366 nasal secretions measured in 50 BRD cases and 50 healthy controls. The PCR 367 primers and TaqMan probes used are listed in Supp Table 1. Fisher’s exact 368 test was used to determine whether the detection rate of tested viruses were 369 significantly higher in BRD than matched controls. The results from the real-370 time PCR case-control study are shown in Figure 2 and p values summarized 371 in Table 2. 372 373 Bovine Adenovirus -3 374 Twenty-four samples (48%) obtained from symptomatic cows were positive 375 for Bovine Adenovirus-3. In the asymptomatic control group, five cows (10%) 376 were positive for the virus. A significantly different rate of detection was 377 calculated using Fisher’s exact test p<0.0001. 378


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379 Bovine Rhinitis A Virus – BSRI4 380 Twelve animals (24%) from the symptomatic group had detectable amounts 381 of Bovine rhinitis A RNA. In the asymptomatic group, four animals (8%) 382 tested positive for the presence of the BSRI4 strain. A significantly different 383 rate of detection was calculated using Fisher’s exact test p=0.009 384 385 Bovine Rhinitis B Virus– Strains BSRI 1 and 2 386 We design real time PCR assay that targeted the conserved RdRp sequence of 387 BRBV-BSRI1+3 and 2. Among the symptomatic animals, four (8%) were 388 positive for strain BSRI1+3, while strain BSRI2 was detected in five (10%) 389 animals. The asymptomatic group had one animal each positive for BSRI1+3 390 and BSRI2. The resulting p-values were non-significant at 0.36 and 0.20 for 391 strains BSRI1+3 and BSRI2, respectively. 392 393 Bovine Influenza D Virus 394 Seven animals (14%) within the symptomatic group were positive for 395 Influenza D. None of the animals from the symptomatic group was found to be 396 positive for the virus. A significantly different rate of detection was calculated 397 using Fisher’s exact test p=0.012. 398 399 Bovine Parvovirus 2 strain BSRI 400 Among the symptomatic cows, four (8%) tested positive for Parvovirus-2 401 BSRI, while one animal in the asymptomatic control group also had detectable 402 levels of the virus. The resulting p-value was non-significant at 0.36. 403 404 Bovine Astrovirus BSRI 405 Four animals (8%) within the symptomatic group were positive for Bovine 406 Astrovirus BSRI1. No detectable amounts of the virus were detected in all fifty 407 asymptomatic animals. The resulting p-value were non-significant at 0.117. 408 409


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Bovine Picobirnavirus 410 Only a single animal with BRD and none of the healthy animals tested positive 411 for picobirnavirus-BSRI1. The resulting p-value were non-significant at 1.0. 412 413 Other viruses 414 Based on the small numbers of reads generated in the Illumina dataset we did 415 not design real time PCR assays for the bovine picobirnaviruses-BSRI2, bovine 416 rhinitis B virus strain BSRI3, and herpes 6 genomes. Because adeno-417 associated viruses have not been associated with disease it was not analyzed 418 further. 419 420 DISCUSSION 421 Our study revealed a large numbers of different viruses present in the nasal 422 secretions of animals in bovine feedlot. These viruses included both close 423 relatives of known viruses and “new” viruses (highly divergent to those 424 currently reported in GenBank). When the distribution of these viruses in 425 nasal secretions was compared between animals with BRD and healthy 426 controls, bovine adenovirus 3 (p<0.0001), bovine rhinitis A (p=0.005), and 427 bovine influenza D (p=0.006), were each associated with BRD. The detection 428 rate of these tentative viral pathogens in animals with BRD was 48 % for 429 BAdV3, 30% for BRAV and 14% for Bovine influenza D. Bovine astrovirus-430 BSRI infection was only found in BRD cases (4/50) but did not reach 431 statistical significance with a p value of 0.117. The set of symptoms defining 432 BRD was therefore associated with 3 viruses. A high level of co-infections was 433 also detected with more than one virus in 38% of BRD cases versus 8% in 434 controls. Whether co-infections or specific combinations of viruses are more 435 likely to result in symptomatic infections will require analyzing larger number 436 of cases and controls. A recent study of calves from Ireland with BRD, testing 437 for BVDV, BoCV, BoHV-1, BRSV and PI3V, reported a virus detection rate of 438


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35% with 40% of these infected animals co-infected with two or more viruses 439 (54). 440 In 32% of BRD cases none of the tested viruses were detected. Viruses 441 present below levels of detections, deeper in the respiratory track, or earlier 442 in disease progression, may account for such apparently virus negative cases. 443 Infections with bacterial respiratory pathogens alone may also account for 444 these virus negative cases. Infections with viruses with no homologue among 445 all the viral genomes in GenBank, and therefore unrecognizable using BLASTx, 446 may also conceivably account for such cases. 447 Bovine adenovirus is highly sero-prevalent in feedlot calves with 448 ~50% of animals sero-converting during the first month in feedlots and has 449 been associated with increased fever although not reduced weight gain (43). 450 Intratracheal inoculation of 4 months old colostrum-deprived calves also 451 induced fever and other symptoms (35). Here BAdV3 was the virus most 452 commonly detected by PCR in nasal secretions (48%) and most strongly 453 associated with BRD (p<0.0001). Because replication competent BAdV3 have 454 been used as vaccine vectors for other infections (5, 9) candidate BAdsV3 455 vaccines may fortuitously already be available for testing. 456 In 2013 a highly divergent influenza virus was described in a pig with 457 influenza-like illness that was unable to productively re-assort with human 458 influenza C virus and was also phylogenetically and antigenically distinct 459 enough to be classified as new genus named influenzavirus D in the 460 Orthomyxoviridae family (21, 22, 64). Related viruses were reported in 461 Chinese cattle (29). The cellular tropism of this virus was wider than that of a 462 human influenza C and was not inhibited at elevated temperatures. In 2014 463 RT-PCR testing of 208 nasal swab samples from BRD cases revealed that 5% 464 were positive with either of two antigenically distinct strains (D/OK and 465 D/660). No healthy controls were tested. Here we report a influenza D RNA 466 detection rate of 14% with none of 50 healthy controls positive using real 467 time PCR. Of the seven influenza D RNA positive animals all but one were co-468 infected, most frequently with adenovirus 3 (n=4) or BPV2 (n=3). 469


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Bovine rhinitis A virus or BRAV, together with foot and mouth disease 470 (FMDV), equine rhinitis A (ERAV), and bovine rhinitis B viruses (BRBV), is one 471 of four picornavirus species in the Apthovirus genus 472 (http://www.picornaviridae.com/aphthovirus/brv/brv.htm). BRAV consists 473 of up to 3 serotypes (70) but a single BRAV genome is currently available in 474 GenBank (JN936206). A single genome of BRBV is currently in GenBank 475 (NC_010354) (27) from a viral isolate derived from a specific pathogen free 476 calf that developed respiratory disease (58). Here one nearly complete 477 genomes of bovine rhinitis A and two of bovine rhinitis B viruses were 478 sequenced. Real time PCR for all three viruses showed only BRVA-BSRI4 to be 479 significantly associated with BRD. 480 The bovine astrovirus-BSRI1 genetically characterized here was found in 481 4/50 BRD cases and in none of the 50 healthy controls (p value of 0.117). 482 While these results are suggestive of a possible involvement in a fraction of 483 BRD cases, the study of a larger number of cases and controls will be needed 484 to confirm significant enrichment in BRD. Astroviruses are commonly 485 associated with gastrointestinal symptoms, particularly in very young or 486 immunodeficient human (1, 20, 69) and are commonly found in animal feces 487 (38, 63, 73). Astroviruses have also been found in the brain of human (57) 488 and animals (7), including cows, with neurological symptoms (8, 37) 489 indicating that its host range can extend beyond enteric tissues. Because all 4 490 BRD cases with this astrovirus also contained bovine adenovirus 3 and/or 491 bovine rhinitis A it remains possible that its presence aggravate symptoms 492 induced by other viral infections. 493 Picobirnaviruses are a group of genetically diverse viruses highly 494 prevalent in the feces of both healthy and diarrhetic human and animals (30, 495 42, 55, 68). In human, PBVs are associated with diarrhea mainly in 496 immunosuppressed individuals (19). Bovine PBV have been described (15, 497 41). Here a single infection was detected in a BRD animal co-infected with 498 both bovine adenovirus 3 and astrovirus-BSRI1. 499


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A viral metagenomics approach combined with real time PCR testing of 500 biological samples from well-matched BRD cases and healthy controls 501 provided a direct approach to the study of a complex infectious disease. 502 Multiple factors may influence the outcome of viral infections including pre-503 existing immunity, stressors, changes in diet, and antibiotics treatment 504 (3, 17, 25, 46). Mingling of large number of animals of different ages is also 505 likely to increase the amount and diversity of exposures to viral and bacterial 506 pathogens and affect the outcome of infections. Despite the multi-factorial 507 aspect of BRD the identification of viruses associated with this complex 508 disease open the way for further studies including replication of our findings 509 using animals from different herds, animal challenges to test viral 510 pathogenicity, and ultimately vaccination to measure the impact of reducing 511 viral infections. The impact of multivalent vaccination against viruses (BHV-1, 512 PI3V, BVDV, and BRSV) has been shown to reduce respiratory symptoms in 513 subsequently virally challenged animals (61) and could be extended to the 514 potential viral pathogens reported here. 515 Except for bovine adenovirus 3, the viruses traditionally tested for in 516 BRD, namely BHV-1, PI3V, BVDV, BCoV, and BRSV (14, 23, 25, 47) were not 517 detected here. Five DNA viruses recently reported in a viral metagenomics 518 study of beef (muscle tissue) (72) were also not detected in nasal secretions of 519 BRD animals. Because of the non-specific nature of the viral metagenomics 520 method used their non-detection reflects either their absence from the 521 samples analyzed or presence below the viral loads of the viruses that were 522 successfully detected. The mix of respiratory viruses infecting cattle may also 523 vary widely between herds, in different regions, or when sampled during 524 different years or even seasons (4). Whether the respiratory viruses detected 525 here reflect those infecting cattle with BRD throughout the US and other 526 countries will therefore require deep sequencing of respiratory samples 527 collected at different times from different locations. 528 529 530


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531 Table 1. Viruses identified by metagenomics and GenBank accession numbers. 532 533 Table 2. Virus detection rates in BRD cases versus controls. Significant p-534 values are noted with an asterisk. 535 536 Fig 1. Phylogenetic analysis of different viral protein sequences. Viruses 537 characterized in this study are labeled with triangle. The phylogenetic 538 analysis was performed based on: A) Parvoviruses and dependoparvoviruses 539 non-structural protein. B) Picornavirus RNA dependent RNA polymerase 540 protein of the Aphtovirus genus including BRAV-BSRI and BRBV-BSRI1/2/3, 541 other available members of these two species, and ERAV and FMDV species. 542 C) Influenza D hemagglutin esterase region including representative of other 543 genera in the Orthomyxoviridae family. D) Astrovirus ORF2 capsid proteins 544 including representative from various host species. E) Partial picobirnavirus 545 RdRp protein from major genogroups including representative from other 546 species. 547 548 Fig 2. Real time PCR Ct values for eight bovine viruses in respiratory samples 549 from 50 BRD and 50 healthy controls. Bovine rhinitis B virus – strain BSRI1 550 and 3 were screened using the same real time PCR assay targeting their 551 conserved RdRp region. 552 553 554


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Acknowledgment: We thank Steven Head at the Scripps Research Institute for DNA 555 sequencing, Jana Sachsenröder for help making the HiSeq library, and Lani Montalvo 556 for help and advice with real time PCR. Support was provided by BSRI and NIH R01 557 HL083254 to Dr. Delwart. The samples used were provided by the Bovine 558 Respiratory Disease Consortium Coordinated Agricultural Project, J. Womack (PD), 559 USDA Agriculture and Food Research Initiative Competitive Grant no. 2011-68004-560 30367 from the USDA National Institute of Food and Agriculture 561 562 563 564


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1. Afrad M. H., P. C. Karmakar, S. K. Das, J. Matthijnssens, F. Ahmed, S. 565 Nahar, A. S. Faruque, M. Z. Rahman, M. Rahman, and T. Azim. 2013. 566 Epidemiology and genetic diversity of human astrovirus infection among 567 hospitalized patients with acute diarrhea in Bangladesh from 2010 to 2012. J 568 Clin Virol 58:612-618. 569 2. Allander T., S. U. Emerson, R. E. Engle, R. H. Purcell, and J. Bukh. 2001. 570 A virus discovery method incorporating DNase treatment and its application 571 to the identification of two bovine parvovirus species. Proc Natl Acad Sci U S A 572 98:11609-11614. 573 3. Apley M. 2014. The clinical syndrome of BRD: what it is and what it is not. 574 Anim Health Res Rev :1-3. 575 4. Autio T., T. Pohjanvirta, R. Holopainen, U. Rikula, J. Pentikäinen, A. 576 Huovilainen, H. Rusanen, T. Soveri, L. Sihvonen, and S. Pelkonen. 2007. 577 Etiology of respiratory disease in non-vaccinated, non-medicated calves in 578 rearing herds. Vet Microbiol 119:256-265. 579 5. Baxi M. K., D. Deregt, J. Robertson, L. A. Babiuk, T. Schlapp, and S. K. 580 Tikoo. 2000. Recombinant bovine adenovirus type 3 expressing bovine viral 581 diarrhea virus glycoprotein E2 induces an immune response in cotton rats. 582 Virology 278:234-243. 583 6. Belák S., O. E. Karlsson, A. L. Blomström, M. Berg, and F. Granberg. 584 2013. New viruses in veterinary medicine, detected by metagenomic 585 approaches. Vet Microbiol 165:95-101. 586 7. Blomström A. L., F. Widén, A. S. Hammer, S. Belák, and M. Berg. 2010. 587 Detection of a novel astrovirus in brain tissue of mink suffering from shaking 588 mink syndrome by use of viral metagenomics. J Clin Microbiol 48:4392-4396. 589 8. Bouzalas I. G., D. Wüthrich, J. Walland, C. Drögemüller, A. Zurbriggen, 590 M. Vandevelde, A. Oevermann, R. Bruggmann, and T. Seuberlich. 2014. 591 Neurotropic astrovirus in cattle with nonsuppurative encephalitis in europe. J 592 Clin Microbiol 52:3318-3324. 593 9. Brownlie R., P. Kumar, L. A. Babiuk, and S. K. Tikoo. 2014. Recombinant 594 Bovine Adenovirus-3 Co-Expressing Bovine Respiratory Syncytial Virus 595


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Glycoprotein G and Truncated Glycoprotein gD of Bovine Herpesvirus-1 596 Induce Immune Responses in Cotton Rats. Mol Biotechnol . 597 10. Castleman W. L., J. C. Lay, E. J. Dubovi, and D. O. Slauson. 1985. 598 Experimental bovine respiratory syncytial virus infection in conventional 599 calves: light microscopic lesions, microbiology, and studies on lavaged lung 600 cells. Am J Vet Res 46:547-553. 601 11. Darbyshire J. H., A. R. Jennings, A. R. Omar, P. S. Dawson, and P. H. 602 Lamont. 1965. Association of adenoviruses with bovine respiratory diseases. 603 Nature 208:307-308. 604 12. Delwart E. 2012. Animal virus discovery: improving animal health, 605 understanding zoonoses, and opportunities for vaccine development. Curr 606 Opin Virol 2:344-352. 607 13. Delwart E. 2013. A roadmap to the human virome. PLoS Pathog 608 9:e1003146. 609 14. Ellis J. A. 2009. Update on viral pathogenesis in BRD. Anim Health Res 610 Rev 10:149-153. 611 15. Ghosh S., N. Kobayashi, S. Nagashima, and T. N. Naik. 2009. Molecular 612 characterization of full-length genomic segment 2 of a bovine picobirnavirus 613 (PBV) strain: evidence for high genetic diversity with genogroup I PBVs. J Gen 614 Virol 90:2519-2524. 615 16. Green J., C. I. Gallimore, J. P. Clewley, and D. W. Brown. 1999. Genomic 616 characterisation of the large segment of a rabbit picobirnavirus and 617 comparison with the atypical picobirnavirus of Cryptosporidium parvum. 618 Arch Virol 144:2457-2465. 619 17. Griffin D. 1997. Economic impact associated with respiratory disease in 620 beef cattle. Vet Clin North Am Food Anim Pract 13:367-377. 621 18. Griffin D., M. M. Chengappa, J. Kuszak, and D. S. McVey. 2010. Bacterial 622 pathogens of the bovine respiratory disease complex. Vet Clin North Am Food 623 Anim Pract 26:381-394. 624 19. Grohmann G. S., R. I. Glass, H. G. Pereira, S. S. Monroe, A. W. 625 Hightower, R. Weber, and R. T. Bryan. 1993. Enteric viruses and diarrhea in 626


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JQ922306 Influenza D virus (C/swine/Oklahoma/1334/2011)

Influenza D virus (C/bovine/California/2014)

JX963123 Influenza C virus (C/Singapore/DSO-070170/2006)

FR671421 Influenza C virus (C/JJ/1950)

JX963125 Influenza C virus (C/Singapore/DSO-070203/2006)

Influenza C

CY095673 Influenza A virus (A/swine/Texas/4199-2/1998(H3N2))

FJ966080 Influenza A virus (A/California/04/2009(H1N1))

CY034138 Influenza A virus (A/WSN/1933(H1N1))

Influenza A

Influenza B AF102005 Influenza B virus (B/Victoria/2/87)

46

98

100

100

100

70

NC_004004 FMDV

NC_010354 Ap

hth

oviru

s

X96871 Equine Rhinitis B virus

100

71

79

100

99

86

0

NC_004004 FMDV

BRBV

BRBV BSRI2

BRBV BSRI1

BRBV BSRI3

JN936206 BRAV H1

BRAV BSRI4

B) Picornavirus 3D

C) Influenza virus HEF

HM756259 Astrovirus swine/PoAstV12-4/Canada

HQ647383 Porcine astrovirus isolate JWH-1

JF713712 Porcine astrovirus 2 strain 51/USA


HQ916313 Bovine astrovirus B18/HK

HQ916317 Bovine astrovirus B76-2/HK

HM447046 Astrovirus deer/CcAstV-2

HM447045 Astrovirus deer/CcAstV-1



HM756260 Astrovirus swine/PoAstv 14-4/Canada

Bovine astrovirus BSRI


HM450382 Astrovirus rat/RS126/HKG

NC_016896 Astrovirus wild boar/WBAstV-1/HUN

GU562296 Porcine astrovirus PAstV-2/HUN


KC609001 Murine astrovirus strain BSRI1

ATVPOLY6A Human astrovirus type 1 strain Oxford

100

68

74

100

40

100

100

64

100

100

100

88

96

88

100

99

D) Astrovirus ORF2

KJ206569 Human PBV VS66000008 KJ663814 Human PBV CDC23

KF792838 Feline PBV Viseu GQ915028 Human PBV PAK-HPBV-1

Bovine PBV BSRI1 Bovine PBV BSRI2

98

100

83

83

77

77

100

E) Picobirnavirus Partial RdRp

Genogroup I

Genogroup II

NC006259 Bovine parvovirus 2

Bovine Parvovirus 2 BSRI

NC023860 Porcine parvovirus 6

KF937794 AAV Red junglefowl

JN420371 AAV California sea lion

AAV BSRI Bovine

NC005889 AAV Bovine

DQ335246 AAV Goat

NC006152 AAV5

DQ335247 Bovine parvovirus 1

100

100

100

100

100

100

100

100

100

96

100

69

97

0

Porcine parvoviruses 5

Porcine parvoviruses 4

A) Parvovirus NS1

Copiparvovirus

Dependoparvovirus

AAVs Human

0.2

0.2

0.5

0.05

0.5

Influenza D

Protoparvovirus

Bocaparvovirus


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Sample ID Status Bovine Adenovirus 3 Bovine Rhini�s A Bovine Rhini�s B - BSRI 1&3 Bovine Rhini�s B - BSRI 2 Bovine Influenza D Bovine Parvovirus 2 Bovine Astrovirus BSRI 1 Bovine Picobirnavirus BSRI1

H1 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H2 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H3 Asymptoma!c 33.94 40.59 >45 >45 >45 >45 >45 >45

H4 Asymptoma!c 41.98 >45 43.48 >45 >45 >45 >45 >45

H5 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H6 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H7 Asymptoma!c >45 >45 >45 37.88 >45 >45 >45 >45

H8 Asymptoma!c >45 29.96 >45 >45 >45 >45 >45 >45

H9 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H10 Asymptoma!c 39.21 42.9 >45 >45 >45 >45 >45 >45

H11 Asymptoma!c >45 31.37 >45 >45 >45 27.89 >45 >45

H12 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H13 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H14 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H15 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H16 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H17 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H18 Asymptoma!c 41.24 >45 >45 >45 >45 >45 >45 >45

H19 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H20 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H21 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H22 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H23 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H24 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H25 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H26 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H27 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H28 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H29 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H30 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H31 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H32 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H33 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H34 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H35 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H36 Asymptoma!c 29.4 >45 >45 >45 >45 >45 >45 >45

H37 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H38 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H39 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H40 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H41 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H42 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H43 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H44 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H45 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H46 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H47 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H48 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H49 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

H50 Asymptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 1 2676 Symptoma!c >45 >45 38.84 >45 >45 >45 >45 >45

pool 1 2745 Symptoma!c 38.78 >45 >45 31.99 >45 >45 >45 >45

pool 1 2778 Symptoma!c 39.89 >45 >45 >45 >45 >45 >45 >45

pool 1 2820 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 1 2870 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 2 2930 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 2 2944 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 2 2984 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 2 3058 Symptoma!c >45 37.98 31.53 >45 >45 >45 >45 >45

pool 2 3078 Symptoma!c 24.82 >45 >45 36.15 >45 >45 >45 >45

pool 3 3130 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 3 3172 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 3 3228 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 3 3282 Symptoma!c 22.77 >45 >45 >45 >45 >45 >45 >45

pool 3 3288 Symptoma!c 37.02 >45 >45 >45 >45 >45 >45 >45

pool 4 3368 Symptoma!c 35.31 >45 >45 >45 >45 >45 >45 >45

pool 4 3380 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 4 3416 Symptoma!c 28 >45 >45 >45 >45 >45 >45 >45

pool 4 3430 Symptoma!c 41.51 23.96 >45 >45 >45 >45 >45 >45

pool 4 3460 Symptoma!c 38.45 >45 >45 >45 >45 >45 20.50 27.56

pool 5 3486 Symptoma!c 37.7 28.93 >45 >45 27.84 >45 >45 >45

pool 5 3520 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 5 3528 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 5 3574 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 5 3582 Symptoma!c 39.37 >45 >45 >45 >45 >45 >45 >45

pool 6 3588 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 6 3616 Symptoma!c 31.54 31.18 >45 >45 >45 30.32 >45 >45

pool 6 3628 Symptoma!c 36.97 >45 >45 >45 >45 >45 >45 >45

pool 6 3718 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 6 3738 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 7 3758 Symptoma!c 28.29 23.38 >45 >45 40.39 23.85 >45 >45

pool 7 3772 Symptoma!c 35.82 37.3 >45 >45 38.56 >45 >45 >45

pool 7 3828 Symptoma!c 31.67 37.87 >45 >45 >45 >45 >45 >45

pool 7 3874 Symptoma!c 39.76 24.38 >45 >45 >45 >45 >45 >45

pool 7 3900 Symptoma!c >45 33.28 >45 >45 >45 >45 >45 >45

pool 8 3924 Symptoma!c 31.97 38.31 40.41 >45 >45 >45 >45 >45

pool 8 3936 Symptoma!c >45 24.93 >45 >45 >45 >45 40.51 >45

pool 8 3938 Symptoma!c >45 >45 >45 31.75 >45 >45 >45 >45

pool 8 3974 Symptoma!c 33.09 >45 >45 >45 >45 >45 >45 >45

pool 8 3982 Symptoma!c >45 >45 >45 >45 28.16 >45 >45 >45

pool 9 4014 Symptoma!c >45 >45 >45 35.83 35.92 26.14 >45 >45

pool 9 4088 Symptoma!c >45 24.4 >45 >45 32.08 24.99 >45 >45

pool 9 4090 Symptoma!c 41.31 >45 >45 >45 >45 >45 >45 >45

pool 9 4112 Symptoma!c >45 >45 >45 35.91 >45 >45 >45 >45

pool 9 4414 Symptoma!c >45 >45 >45 >45 >45 >45 >45 >45

pool 10 4116 Symptoma!c >45 >45 36.46 >45 33.85 >45 >45 >45

pool 10 4152 Symptoma!c 25.71 36.62 >45 >45 34.23 >45

pool 10 4228 Symptoma!c 34.69 >45 >45 >45 >45 >45 >45 >45

pool 10 4410 Symptoma!c 37.76 24.7 >45 >45 >45 >45 23.94 >45

pool 10 4496 Symptoma!c 40.78 34.51 >45 >45 >45 >45>45 >45

>45 >45


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Virus Family GenBank Accession Number

Number of sequence

reads Closest relative Amino acid identity

Bovine adenovirus 3 Adenoviridae KP264982 4,279 Bovine adenovirus 3 98% Bovine adeno-associated virus Parvoviridae KP264981 3,481 Bovine adeno-associated virus 97% Bovine rhinitis A virus - BSRI4 Picornaviridae KP264974 11,109 Bovine rhinitis A virus 92% (3D) Bovine rhinitis B virus - BSRI1 Picornaviridae KP264980 1,224 Bovine rhinitis B virus 89% (3D) Bovine rhinitis B virus - BSRI2 Picornaviridae KP264976-9 985 Bovine rhinitis B virus 89% (3D) Bovine rhinitis B virus - BSRI3 Picornaviridae KP264975 430 Bovine rhinitis B virus 81% (3D)

Bovine Influenza D virus - BSRI5 Orthomyxoviridae - 304 Swine Influenza D/OK 92-98%

Bovine astrovirus - BSRI1 Astroviridae KP264970 1,666 Bovine astrovirus BAstK08/51 83% (ORF1b) Picobirnavirus - BSRI1 Picobirnaviridae KP264972 21 Human picobirnavirus 33% Picobirnavirus - BSRI2 Picobirnaviridae KP264973 123 Human picobirnavirus 34%

Bovine Parvovirus 2 - BSRI Parvoviridae KP264971 79 Bovine parvovirus 2 82% Bovine Herpesvirus 6 Herpesviridae - 19 Bovine Herpesvirus 6 98%


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Virus Viral family Asymptomatic BRD p value

Bovine adenovirus 3 Adenoviridae 5/50 (10%) 24/50 (48%) <0.0001* Bovine rhinitis A virus - BSRI4 Picornaviridae 4/50 (8%) 12/50 (24%) 0.009*

Bovine rhinitis B virus - BSRI1 & 3 Picornaviridae 1/50 (2%) 4/50 (8%) 0.36 Bovine rhinitis B virus - BSRI2 Picornaviridae 1/50 (2%) 5/50 (10%) 0.2

Bovine influenza D virus-BSRI1 Orthomyxoviridae 0/50 (0%) 7/50 (14%) 0.012* Bovine parvovirus 2 - BSRI Parvoviridae 1/50 (2%) 4/50 (8%) 0.36 Bovine astrovirus - BSRI1 Astroviridae 0/50 (0%) 4/50 (8%) 0.117

Bovine picobirnavirus-BSRI1 Picobirnaviridae 0/50 (0%) 1/50 92%) 1


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a metagenomics and case-control study to identify viruses

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