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Discoveryofunconventionalviruses Associatedw Ithbovinerespiratory 2019-01-03 Introduction Discoveryofunconventionalviruses associatedw ithbovinerespiratory diseases– w iththehelpofhigh Non-culturab le ordifficult- Nove lorm utate d organism throughputsequencing to-culture organism Yanyun Huang Prairie Diagnostic Services (PDS) Inc. High Throughput Sequencing (HTS) h ttp s://www.google.ca/search ?q=ce ll+culture &rlz=1C1CHBF_e nCA758CA758&source 1 2 Introduction Introduction First Generation Sequencing Second Generation Sequencing Why High Throughput Sequencing Virusdiscove ry Mutation discove ry and p rofiling In 2011, Sch m allenb e rg virus(SBV) waside ntified and DNA Prote inInte ractions nam e d using High th rough p utseque ncing (HTS) from cattle and she e p inGe rm any and th e Ne th e rlands. Transcriptom e Analysis Ap p lication of HTS inve te rinary m e dicine islimite d inCanada. Advantage s: 1. Se que nce a large num b e rof DNA Limitation: m oleculesinp arallel; 1. Sm allam ountsof DNA 2. Do not ne e d p riorknowledge of wh at p roce ssed p e runittime ; y ou’re seque ncing. 2. h igh costp e rb ase: firsth um an 3. Ch e ap costp e rb ase: a single h um an ge nom e $100 m illion ge nom e $1000 h ttp s://www.b iote ch articles.com /Ge ne tics-Article/Sange r-Se que ncing-Ch ain-te rmination-m e th od-of-DNA-Se que ncing-3937.h tm l 3 4 Introduction Introduction $1 b illion, annually. Controlled with $30 p e rm orbidcalf. Th e re are sub stantialstudiesto supp ort th at viralinfe ction gre atly incre ased antimicrob ialsand susce p tibility of b e e f cattle to b acte rialinfe ction. 70-80% of th e m orbidity viralvaccination (USDA, 2013) First60 day s Th e re are alsosom e diagnosticcases,wh e re lesionssugge stvirale tiology, h owe ve r, conve ntionalvirusescannot b e de te cte d b y PCR assay s. Use of antimicrob ials prom ote sde ve lopm e nt of antimicrob ial- re sistance (AMR). Conve ntionalviruses= traditionally associate d with BRD, include d inBRD vaccine sand availab le diagnosticassay s h ttp ://th re e rive rsve tgroup.co.uk/ve te rinary-service s/farm-animals/info-for-farme rs/b ovine -re spiratory-disease-b rd-p ne umonia/ 5 6 1 2019-01-03 Hypothesis Sample information 240 sam p les(120 p aire d nasalswab sand trach e alwashe s)kindly p rovide d b y Dr.Tre vorW . Alex ande r (Le th b ridge Re search Ce nte r,Agriculture and Agri-Food Canada) Healthy control cattle BRD cattle Th e re are unconve ntional virusesassociate d with BRD 1. No visualBRD signs 1. One visualBRD sign, at least 2. Re ctalte m p e rature < 40 °C 2. Re ctal te m p e rature ≥ 40 °C 3. No tre atm e nt againstoth e rdisease 3. Ab norm allung sounds 4. No ab norm allung sounds 4. No tre atm e nt againstoth e rdiseases 5. If sick with in60 day s,re m ove d h ttp s://www.google.ca/search ?rlz=1C1CHBF_e nCA758CA758&tb m =isch &q=h ap p y +ste e r+cartoon&ch ips=q:h ap p y +ste e r+cartoon 7 8 Materials and Methods Materials and Methods NexteraXT library preparation and sequencing Sample preparation 5’ 3’ RNA Trach e aland Ex traction of viral 0.2µg Input DNA Nasalswab nucleicacids Re verse transcriptase + random p rime rs+ dNTPs First-strand synthesis 1 Bioanalyzer 5’ 3’ Ce ntrifuge DNA-RNA hybrid 6 3’ 5’ DNase/ RNase 2 5 Adde d RNase H and Se que nase Library Normalization and Pooling Tagmentation of Input DNA 7 PCR Amplification Hostce lls 3’ 5’ Second-strand synthesis with Index Primers 3’ 5’ Viruses 4 Bacte ria Polym e rase and p rime rs with out6 Ns Amplification h ttp s://www.ab m good.com /m arke ting/knowledge _b ase/ne x t_ge ne ra PCR Clean-up Sample loading and Data analysis tion_seque ncing_e x p e rime ntal_de sign.p h p 9 10 Materials and Methods Raw data Assem b led th e re st re ads Ob taine d h igh quality re ads Map p e d th e (Trimm om atic) assem b led seque nce s to Re fSe q datab ase Map p e d to cow ge nom e to re m ove h ost-de rive d seque nce s h ttp s://e n.wikipe dia.org/wiki/Illum ina_dy e _seque ncing 11 12 2 2019-01-03 Data Analysis Sample Total No. of Reads No. of Reads unmapped to cattle Percentage Nasal swab 33,813,931 9,618,962 28.45% Tracheal wash 48,936,263 24,026,779 49.10% Total 82,750,194 33,645,741 40.66% Virus 1,812,011 2.19% 13 14 Table 1. Viruses identified by metagenomics Largest contig Total No. of Largest contig Table 2. Virus identification rates and association between presence of viruses and the probability of an Virus Family Closest relative length (bp) sequence reads amino acid identity (%) animal showing symptoms of BRD IDV Orthomyxoviridae 1,587 17,297 99 (PB2) Influenza D virus No. of positive cattle/total BRD ICV Orthomyxoviridae 1,010 307 100 (PB1) Influenza C virus or control cattle (% positive) BRBV Picornaviridae 2,431 38,648 99 (polyprotein) Bovine rhinitis B virus Virus Odds ratio 95% CI for odds ratio P value BRAV Picornaviridae 1,296 1,022 100 (polyprotein) Bovine rhinitis A virus Symptomatic Asymptomatic EVE Picornaviridae 3,186 20,124 98 (polyprotein) Enterovirus E BRSV Paramyxoviridae 1,169 121,005 98 (RdRp) Bovine orthopneumovirus IDV 13 (22) 3 (5) 6.145 1.435 - 26.310 0.015* BPIV3 Paramyxoviridae 279 49 99 (M) Bovine respirovirus 3 BRBV 16 (28) 6 (10) 3.836 1.245 - 11.821 0.020* BCV Coronaviridae 7,513 197,921 99 (ORF1ab) Bovine coronavirus BRSV 10 (17) 1 (2) 13.422 1.454 - 123.885 0.022* BNV Coronaviridae 4,782 86,392 99 (PP1a/b) Bovine nidovirus TCH5 BPV2 Parvoviridae 1,149 1,427 93 (nonstructural protein) Bovine parvovirus-2 BCV 11 (19) 2 (3) 7.392 1.354 - 40.346 0.021* BAAV Parvoviridae 1,096 1,002 99 (Cap) Bovine adeno-associated virus BRAV 7 (12) 2 (3) 5.659 0.982 - 32.602 0.052 UTPV1 Parvoviridae 4,375 1,061,037 98 (NS1, VP1 and VP2) Bovine hokovirus 2 BPV2 7 (12) 3 (5) 3.289 0.682 - 15.865 0.137 UBPV6 Parvoviridae 4,518 263,902 99 (nonstructural protein) Ungulate bocaparvovirus 6 BNV 4 (7) 23 (40) 0.078 0.021 - 0.288 0.000ǂ BVDV1 Flaviviridae 602 12 97 (NS5b) Bovine viral diarrhea virus 1 ICV 0 (0) 6 (10) --- --- 0.967 HCV Flaviviridae 528 11 97 (core protein) Bovine hepacivirus BAdV3 Adenoviridae 366 14 99 (284R) Bovine adenovirus 3 BAV 5 (9) 1 (2) 4.485 0.459 - 43.798 0.195 BAV Astroviridae 1,220 884 98 (NSP1ab) Bovine astrovirus UTPV1 26 (45) 16 (28) 1.878 0.812 - 4.348 0.140 ssCDV Genomoviridae 676 158 91 (Rep) Gila monster-associated gemycircularvirus UBPV6 8 (14) 20 (34) 0.296 0.108 - 0.814 0.019ǂ WUPyV Polyomaviridae 731 337 77 (large T antigen) WU Polyomavirus WUPyV 3 (5) 6 (10) 0.421 0.081 - 2.185 0.300 PBCV Phycodnaviridae 351 288 96 (CVM1) Paramecium bursaria Chlorella virus EVE 6 (10) 2 (3) 4.258 0.704 - 25.740 0.113 HPV Papillomaviridae 763 174 100 (major capsid protein) Human papillomavirus type 40 BAdV3 1 (2) 2 (3) 0.339 0.025 - 4.619 0.414 15 16 Conclusionandimplications Table 3. Association between presence of at least one of the following four viruses and the probability of an animal showing symptoms of BRD •There are unconventional viruses in BRD samples Total No. of cattle positive •Several viruses are associated with BRD in the current data set Virus containing at least 1 of 4 viruses Odds ratio 95% CI for odds ratio PDOF value P value Symptomatic Asymptomatic •Further studies needed to understand the roles of these viruses in IDV BRD BRBV 36 10 7.484 2.167-25.849 0.001* 0.002* BRSV •HTS is a powerful technique for virus detection BCV 17 18 3.
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