Next Generation Sequencing to Identify Viruses Associated with Bovine Respiratory Disease - Dr. Ben Hause, Clinical Assistant Professor, Kansas State University, from the 2016 NIAA Annual Conference: From Farm to Table - Food System Biosecurity for Animal Agriculture, April 4-7, 2016, Kansas City, MO, USA. More presentations at http://www.trufflemedia.com/agmedia/conference/2016_niaa_farm_table_food_system_biosecurity

1. Next Generation Sequencing to Identify Viruses Associated
with Bovine Respiratory Disease
Kansas State Veterinary Diagnostic Laboratory
Ben Hause, MS, PhD

2. Next Generation Sequencing
• NGS generates massive amounts of sequence data
– Sanger= 1 read, ~800bp
– NGS=1M reads, 300bp
• NGS can be sequence dependent or independent
– Don’t need to know what your sequencing
– Unique to this technology
• Amount of data for $
– PRRS ORF5
• Sanger $182/600bp=$0.30/bp
• NGS: $300/15400bp=$0.02/bp
• More comprehensive picture of virus: whole genome vs. 1 gene
• Ability to detect multiple viruses or quasispecies
• Metagenomic sequencing: sequencing material directly recovered from the environment
– Nasal swab

3. Metagenomic viral
RNA and DNA
(sample pretreated
with DNase/RNase
cocktail
Random hexamer with
5’-20bp barcode
Reverse Transcription and
Second Strand Synthesis
(RNA -> cDNA->dsDNA)
PCR Amplification
using primer identical
to 20bp barcode
Amplicon pools
generated from
randomly amplified
virus nucleic acid

4. Data analysis
• De novo assembled sequences into contigs
• Analyze contigs by BLASTN
• Reassemble with best BLAST hit as a reference

5. Full genome sequence of porcine parainfluenza 1 (PPIV1)
virus from a nasal swab
• 11 M reads
• 52,111 mapped to
PPIV1
– 0.45% reads
• 361x average
coverage

6. Next Generation Sequencing for
Characterization of the Viral Ecology of Swine
at Slaughter
• Slaughter facilities in SE US often close in proximity to commercial
production facilities
• Common transport
• Sites of co-mingling

7. • Metagenomic sequencing of fecal/nasal swabs from 5 swine markets
in NC
– 2 slaughterhouses for healthy pigs (primary market)
– 2 cull slaughterhouses (secondary market)
– 1 buying station
• 5 pigs (nasal swab and fecal swab) per producer per site
– 5 producers per site
– 250 total swabs, analyzed in 50 pools (25 nasal, 25 rectal swab pools)
– 2 samplings, June/August, 2015
– Samples collected by veterinarian; animals fit for slaughter
Emerging Infectious Diseases, accepted

8. 0
10
20
30
40
50
60
70
80
90
100
Percentageofswabspositive
Site 1
Site 2
Virus detection in primary markets

9. Virus detection in secondary markets
0
10
20
30
40
50
60
70
80
90
100
Percentageofswabspositive
Site 3
Site 4

10. Virus detection in buying station
0
10
20
30
40
50
60
70
80
90
100
ssDNA virus bocavirus torovirus posavirus torque teno
sus virus
IAS virus picobirnavirus teschovirus enterovirus parvovirus pasivirus influenza A
virus
sapleovirus hokovirus
Percentageofswabspositive

11. • Large numbers of viruses identified=Virus Soup!
• Seneca valley virus identified from both samplings from 4/5
markets by sequencing
• qRT-PCR for SVV
– 26/50 (52%) June sampling
– 18/50 (36%) August sampling
• Primary market=1/40 (2.5%)
• Secondary market=43/60 (72%)
• Virus isolation, second sampling
– Positive for 5 samples (Ct values ~15-20)
• SVV much more common in lower health status pigs?
– Oral fluid testing for SVV, ~1% positive (ISU/UMN/SHIC)
– Cause versus effect???

12. 1
10
100
1000
10000
100000
0 5 10 15 20 25 30 35 40 45
NGSReadCount
SVV PCR Ct
pcr vs ngs
NGS sensitivity on par with
qRT-PCR

13. Metagenomic characterization of the virome associated with bovine
respiratory disease in feedlot cattle identified novel viruses
• BRD is the most costly disease of the cattle industry
• Widespread use of MLV vaccines for 20+ years; incidence is increasing
– Combos of live/inactivated
• Bovine viral diarrhea virus
• Bovine herpesvirus 1
• Parainfluenza virus 3
• Bovine respiratory syncytial virus
• Mannheimia haemolytica
• Histophilus somni
• Pasteurella multocida
• Mycoplasma bovis
• BRD has complex pathogenesis
– Host factors
– Environmental factors
– Pathogen factors

14. Can we use metagenomic sequencing to identify
pathogens associated with BRD?
• Experimental design
– 5 feedlots in Mexico and 5 feedlots in the U.S. (500-800# cattle)
– Nasal swabs collected from 5 animals with acute respiratory disease and 5 healthy pen mates at
each feedlot (n=100)
– Viral metagenomic sequencing

15. OR=2.9
P=0.134
OR=4.2
P=0.212
Vx Vx VxVx
OR=1.7
P=0.259
OR=2.62
P=0.265

16. • IDV only virus with even moderately correlated with BRD
• qRT-PCR to verify sequencing result
– 8 sick animals positive (Ct=20.3-36.6)
– 3 health animals positive (Ct=34.6-37.0)
• Difference in IDV titer (Ct values) in nasal swabs between sick and healthy animals?
– Wilcoxson test: P=0.04
• IAV titers higher in humans with influenza-associated pneumoniae vs. upper respiratory tract infection (Li et
al., 2010, Emerg Infect Dis 16:1265-1272)

17. IDV HEF Phylogeny

18. BRAV USII/02 (KU159364)
BRAV USII/09
BRAV USII/19
BRAV USII/18-2
BRAV USII/15
BRAV USII/12
BRAV BS02
BRAV USII/06
BRAV BS06
BRAV BS12
BRAV BSRI4 (KP264974)
BRAV BS07
BRAV USII/10
BRAV Sd-1 (KP236128)
BRAV BS16-2 (KU159362)
BRAV BS19
BRAV BS17
BRAV BS16-1
BRAV USII/05
BRAV USII/18-1
BRAV USII/20
BRAV 140032-1 (KP236129)
BRAV H-1 (JN936206)
BRAV MexB15 (KU159363)
6
62
3
23
31
37
100
100
82
62
0.02
BRAV1
BRAV2
BRBV BSRI1 (KP264980)
BRBV USII/03
BRBV BS10
BRBV USII/17
BRBV NIH (NC 010354)
BRBV BRV2 (EU236594)
BRBV MexB49
BRBV USII/13
BRBV USII/12
BRBV 140032-2 (KP236130)
BRBV MexB48 (KU159361)
BRBV BSRI3 (KP264975)
BRBV MexB09 (KU159360)
BRBV MexB29 (KU159358)
BRBV USII/19 (KU159359)
BRBV USII/05
BRBV MexB10 (KU159357)
BRBV MexB39
100
100
100
100
83
96
100
100
90
95
98
41
17
100
71
0.05
BRBV1
BRBV2
Fig.2(a)
Fig.(2b)
Proposed new
genotype/serotype

19. Enterovirus E K2577 (AF123432)
Enterovirus E LC-R4 (DQ092769)
Enterovirus E Vir404/03 (DQ092771)
Enterovirus E PS83 (DQ092793)
Enterovirus E PS42 (DQ092792)
Enterovirus E MexKSU/5 (KU172420)
Enterovirus E VG5-27 (D00214)
Enterovirus F BEV261 (DQ092770)
Enterovirus F IL/alpaca (KC748420)
Enterovirus F PS87 (AY508696)
Enterovirus F PS87/Belfast (DQ092794)
Enterovirus G (KF985175)
Enterovirus A (NC 001612)
Enterovirus B (AY843298)
Enterovirus J N125 (AF414372)
Enterovirus H (NC 003988)
Enterovirus D (NC 001430)
Enterovirus C/Poliovirus (NC 002058)
Enterovirus C/coxsackievirus A13 (DQ995644)
Rhinovirus B R93 (KF958309)
Rhinovirus A hrv-A101-v1(GQ415052)
Rhinovirus C JAL-1 (JX291115)
100
99
67
100
78
49
50
36
89
98
100
99
11
66
100
32
29
0.1
Enterovirus E PS83 (DQ092793)
Enterovirus E PS42 (DQ092792)
Enterovirus E K2577 (AF123432)
Enterovirus E D14/3/96 (DQ092786)
Enterovirus E Vir404/03 (DQ092771)
Enterovirus E VG5-27 (D00214)
Enterovirus E LC-R4 (DQ092769)
Enterovirus E MexKSU/5 (KU172420)
Enterovirus F PS87/Belfast (DQ092794)
Enterovirus F PS87 (AY508696)
Enterovirus F BEV261 (DQ092770)
Enterovirus F IL/alpaca (KC748420)
Enterovirus G (KF985175)
Enterovirus A (NC 001612)
Enterovirus J N125 (AF414372)
Enterovirus D (NC 001430)
Enterovirus H (NC 003988)
Enterovirus C/Poliovirus (NC 002058)
Enterovirus C/coxsackievirus A13 (DQ995644)
Enterovirus B (AY843298)
Rhinovirus C JAL-1 (JX291115)
Rhinovirus A hrv-A101-v1(GQ415052)
Rhinovirus B R93 (KF958309)
100
100
100
63
100
100
55
100
100
100
100
44
99
100
99
46
98
99
62
48
0.2
Fig.3(a) Fig.3(b)
New
genotype?
Polymerase
phylogeny
Capsid phylogeny

20. New species
First detection in North America
Parvoviridae Phylogeny

22. NGS/Metagenomic Sequencing
• Powerful method for veterinary diagnostics
• Complete viral genome sequencing
– Isolated virus
– Directly from clinical samples (with sufficient viral titer)
• Cases with unknown etiology
– Unusual clinical presentation
– Clinical symptoms with absence of usual suspects
• Profiling animal/herds
– Live exposure (rotavirus, PEDV, PDCoV, PRRSV)
• Screening for foreign animal diseases
• Affordable
– $300/sample
– Alternative: multiple PCRS, histopathology, culture, VI, etc., can easily
reach $300
• Need more widespread use!

23. • Ampliseq:
– Highly multiplexed PCR (1<primers<6,144)
– Amplicon sequencing with Ion Torrent PGM
– Widely used for cancer/inherited disease panels in humans
– Can we adapt to use for pathogen detection/characterization?
• Custom primer panel for BRD viruses/bacteria
• Conserved/variable regions
– Issues:
• Targets will be highly variable
– Primer redundancy
• Targets will often be present in low amounts
AmpliSeq: a hybrid between metagenomic
sequencing and PCR panels?

24. • AmpliSeq benefits
– Cost: sequence with
AmpliSeq pool for ~$50
– Turnaround time: ~1-2 days
– Data analysis: simple
templated assembly (a
couple minutes per sample)
• Ampliseq drawbacks
– Targeted
• Miss divergent pathogens
• Miss pathogens not
targeted
• Miss novel agents
• MiSeq drawbacks
• Time to results: 1-2 weeks
• Cost
• $300 metagenomic sequencing vs. $80
BRD panel
• Data analysis:
• Map reads to host
• De novo assembly
• BLAST

25. Goal: Highly multiplexed assay for BRD
detection and genetic characterization

26. • Proof of concept successful
• More development/characterization required:
– Ability to detect all viruses
– Ability to detect variant viruses
– Sensitivity
– Specificity
– Efficiency/user-friendliness
• automation

27. Acknowledgements
• Kansas State University
• Dr. Dick Hesse
• Dr. Lalitha Peddireddi
• Dr. Jianfa Bai
• Dr. Emily Collin
• Rachel Palinski
• Dr. Namita Mitra
• Aiswaria Padmanabhan
• Veterinarians
• Dr. Josh Duff
• Dr. Chad Smith
• Dr. Bob Smith
• National Pork Board grant #14-204
• Zoetis
• Boehringer Ingelheim
• Merck Animal Health
• Lapisa