Pathogen Discovery Using Metagenomic Sequencing - Dr. Ben Hause, College of Veterinary Medicine, Kansas State University, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA. More presentations at http://www.swinecast.com/2016-leman-swine-conference-material

1. Metagenomic sequencing for virus discovery and
characterization
Kansas State Veterinary Diagnostic Laboratory
Ben Hause, MS, PhD

2. • Metagenomic sequencing: sequencing material directly recovered from the environment
– Nasal swab, sera, oral fluids, tissue, etc.
– Next generation sequencing (Illumina Miseq)
Metagenomic sequencing
Sanger Sequencing
• 1 read
• ~800 bp
• $80/gene
• Targeted
• Quasispecies: no
• Single gene/region
Metagenomic/NGS
• ~1 million reads
• ~300 bp
• $300/sample
• Targeted or non-targeted
• Quasispecies: yes
• Complete genomes/multiple
organisms

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. De novo assembly
BLAST to identify
PRRSV
Mycoplasma
pig
Templated assembly
PRRSV
Random PCR amplicons

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. Metagenomic sequencing uses:
- cases with unusual clinical presentations
-cases where the usual suspects aren’t identified
-herd profiling
-feedback/exposure inoculum screening

7. Unknown neurologic disease
• Starting in spring 2015, approx. 700 5-14 week old pigs
exhibited intention tremors, increased respiratory rates,
and as disease progressed, inability to swallow
– Mortality was 100% within 4 days of onset
• Conventional diagnostics failed to find pathogens
• August 2015 new group of pigs 10-16 weeks of age from
same system showed similar symptoms

9. • Metagenomic sequencing of brain homogenate mapped 195 reads to atypical porcine
pestivirus (APPV, 87% identity)
• Only virus identified
• qRT-PCR from pig on brain, lymph nodes, heart tissue and serum were positive for APPV
• APPV:
– Highly divergent pestivirus identified in pig sera as part of NPB PRRSV genome sequencing project
(Hause et al., 2015, J Gen Virol. 96:2994-8)
– Very distantly related to BVDV, CSFV (~25% identity)
– More closely related to recently partially described bat pestivirus in China (~68% identity)

10. IHC
• Anti-APPV Erns antibodies
were generated in mice
• IHC detected virus in the
liver, spleen and lymph
nodes
– Brain was negative
– Large number positive cells
in LN, localization in
cytoplasm

11. Recent Findings and future studies
• ISU was able to demonstrate APPV-like virus induced congenital shaker pigs after in utero
inoculation of fetuses
• How serious is this disease in the swine industry?
– Congenital tremors have been sporadically observed with no definitive etiologic agent
– Intention tremors and resulting mortality in older pigs new?
• Is there potential for persistent infection of piglets?
• Multiple subtypes resulting in different disease presentation?
– Observed ~15% E2 nt diversity
• Newly emerged virus or circulating unnoticed for some time?
– Virus X or pestivirus-like viruses isolated from pigs reported in the past

12. New circovirus species, PCV3
• NC sow farm, chronic poor reproductive performance
• Acutely dead sows with lesions consistent with PDNS
• Sows with PDNS-like skin lesions aborting
• Samples sent to ISU
– Lesions consistent with PCV2, however…
– PCV2 negative by PCR and IHC
• Samples sent to KState for metagenomic sequencing

13. Replicase=54% identical to bat circovirus
Capsid=36% identical to PCV2 and duck circovirus
ORF3=39% identical to Murid herpes protein (unknown function)
REP
ORF 3
CAP
PCV3, 2000bp

14. Kidney
Skin
Lymph
node
MAb14 (PCV3) 2° antibody only PCV3 negative
tissues

15. • Quantified PCV3 in symptomatic cases
– Sows with PDNS, Ct=28-30
– Mummies, Ct=17-21
• Incidence determined by qPCR: 34/271 samples positive (12.5%)
– Samples submitted to ISU for respiratory disease testing
• Screened 48 PDNS cases from ISU that were negative for PCV2 by IHC
– 45 positive by PCR for PCV3 (94%)
– Confirmed subset of 5 positive samples by PCR/sequencing
– PCV3 IHC on 5 PCR positive slides, 3 positive (60%)

16. • PCV2 history
– First identified in mid-1990’s
– Sporadic PMWS in Canada
– Severe systemic disease in Europe and Asia followed by U.S. in early/mid 2000’s
– Retrospective studies found evidence of PCV2 circulation for decades prior to
widespread disease
– Uncontrolled until commercial vaccines developed
• PCV3 on a similar trajectory???
– More research needed, including vaccine development!
– role in PDNS?
– Subclinical infection?

17. Novel viruses identified in unexplained
respiratory disease cases
New genotype of porcine astrovirus 4
identified in numerous cases of acute
respiratory disease in swine
Padmanabhan and Hause. 2016. Detection and characterization of a novel
genotype of porcine astrovirus 4 from nasal swabs from pigs with acute
respiratory disease. Archives of Virology 161:2575-9.

18. Porcine parainfluenza virus 1
• 10-21 day old pigs with acute respiratory disease
– Negative for influenza A virus and PRRSV
• Metagenomic sequencing of nasal swabs from IL and NE
– Complete PPIV1 genomes
U.S. PPIV1 genomes 90-95% identical to
Hong Kong PPIV1
Palinski et al., J Gen Virol. 2016, 97(2):281-6. doi: 10.1099/jgv.0.000343.
U.S.
H.K.

19. • 6.1% (17/279) lung/oral fluid/nasal swabs PCR+ for PPIV1
• Serology
– ELISA (recombinant F protein)=63% positive
– Immunocapture PCR= 55% positive
• Replication largely limited to upper respiratory tract (ISH)

20. Herd profiling: slaughter houses as areas of
comingling and concentration
Primary Market HogsHause et al, 2016, JSHAP, accepted

21. 0
20
40
60
80
100
120
Percentageofswabspositive
Site 3
Site 4
Secondary cull
slaughterhouses

22. • 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???
Hause et al., 2016. Emerg Infect Dis 22:1323-1325.

23. Feral Swine Virome
• Novel swine orthopneumovirus
• 30% seropositivity both in
domestic and feral swine
• 5% domestic swine respiratory
samples positive
• Role in disease?
Hause et al., 2016, J Gen Virol, doi: 10.1099/jgv.0.000554

24. Other new porcine viruses, clinical significance?
Porcine parvovirus 7

26. NGS/Metagenomic Sequencing
• Powerful new 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)
• Affordable
– $300/sample
– Alternative: multiple PCRS, histopathology, culture, VI, etc., can easily reach
$300
• Need more widespread use!
• Need follow up studies to establish clinical relevance

27. Acknowledgements
• Kansas State University
• Dr. Dick Hesse
• Dr. Lalitha Peddireddi
• Dr. Jianfa Bai
• Dr. Erin Schirtzinger
• Dr. Emily Collin
• Rachel Palinski
• Dr. Namita Mitra
• Aiswaria Padmanabhan
• South Dakota State University
• Travis Clement
• Iowa State University
• Dr. Phillip Gauger
• Dr. Pablo Pineyro
• Dr. Paulo Arruda
• Dr. Kent Schwartz
• Swine Veterinarians
• Dr. Josh Duff
• Dr. Chad Smith
• Dr. Emily Byers
• Dr. John Prickett
• National Pork Board grant #14-204
• Zoetis
• Boehringer Ingelheim
• Swine Health Information Center
• Kansas Bioscience Authority/Center for
Excellence Emerging Zoonotic Animal
Diseases
New Contact Info:
bhause@cambridgetechnologies.net