Metagenomic sequencing for virus discovery and characterization

Kansas State Veterinary Diagnostic Laboratory

Ben Hause, MS, PhD

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

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

De novo assembly

BLAST to identify

PRRSVMycoplasma

pig

Templated assembly

PRRSV

Random PCR amplicons

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

Metagenomic sequencing uses:

- cases with unusual clinical presentations

-cases where the usual suspects arent identified

-herd profiling

-feedback/exposure inoculum screening

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

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)

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

Recent Findings and future studies

ISU was able to demonstrate APPV-like virus induced congenital shaker pigs after in uteroinoculation 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

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

Replicase=54% identical to bat circovirus Capsid=36% identical to PCV2 and duck circovirusORF3=39% identical to Murid herpes protein (unknown function)

REP

ORF 3

CAP

PCV3, 2000bp

Kidney

Skin

Lymph node

MAb14 (PCV3) 2 antibody only PCV3 negative tissues

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%)

PCV2 history First identified in mid-1990s Sporadic PMWS in Canada Severe systemic disease in Europe and Asia followed by U.S. in early/mid 2000s 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?

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.

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.

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)

Herd profiling: slaughter houses as areas of comingling and concentration

Primary Market HogsHause et al, 2016, JSHAP, accepted

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Secondary cull slaughterhouses

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.

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

Other new porcine viruses, clinical significance?

Porcine parvovirus 7

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

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

Metagenomic sequencing for virus discovery and characterizationMetagenomic sequencingSlide Number 3Slide Number 4Full genome sequence of porcine parainfluenza 1 (PPIV1) virus from a nasal swabMetagenomic sequencing uses:- cases with unusual clinical presentations-cases where the usual suspects arent identified-herd profiling-feedback/exposure inoculum screeningUnknown neurologic diseaseSlide Number 8Slide Number 9IHCRecent Findings and future studiesNew circovirus species, PCV3Slide Number 13Slide Number 14Slide Number 15Slide Number 16Novel viruses identified in unexplained respiratory disease casesPorcine parainfluenza virus 1Slide Number 19Herd profiling: slaughter houses as areas of comingling and concentrationSlide Number 21Slide Number 22Feral Swine ViromeOther new porcine viruses, clinical significance?Slide Number 25NGS/Metagenomic SequencingAcknowledgements