Next Generation Sequencing to Identify Viruses Associated

with Bovine Respiratory Disease

Kansas State Veterinary Diagnostic Laboratory

Ben Hause, MS, PhD

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

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

Data analysis

• De novo assembled sequences into contigs

• Analyze contigs by BLASTN

• Reassemble with best BLAST hit as a reference

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

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

• 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

0

10

20

30

40

50

60

70

80

90

100

Per

cen

tage

of

swab

s p

osi

tive

Site 1

Site 2

Virus detection in primary markets

Virus detection in secondary markets

0

10

20

30

40

50

60

70

80

90

100

Per

cen

tage

of

swab

s p

osi

tive

Site 3

Site 4

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

Per

cen

tage

of

swab

s p

osi

tive

• 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???

1

10

100

1000

10000

100000

0 5 10 15 20 25 30 35 40 45

NG

S R

ead

Co

un

t

SVV PCR Ct

pcr vs ngs

NGS sensitivity on par with qRT-PCR

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

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

OR=2.9 P=0.134

OR=4.2 P=0.212

Vx Vx Vx Vx

OR=1.7 P=0.259

OR=2.62 P=0.265

• 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)

IDV HEF Phylogeny

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

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

New species

First detection in North America

Parvoviridae Phylogeny

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!

• 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?

• 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

Goal: Highly multiplexed assay for BRD

detection and genetic characterization

• Proof of concept successful

• More development/characterization required:

– Ability to detect all viruses

– Ability to detect variant viruses

– Sensitivity

– Specificity

– Efficiency/user-friendliness

• automation

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