Dr. Ben Hause - Metagenomic Sequencing for Virus Discovery and Characterization

Information about Dr. Ben Hause - Metagenomic Sequencing for Virus Discovery and...

Published on January 17, 2016

Author: trufflemedia

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

2. Why NGS vs. Sanger? More Data! • 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. 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

5. Next Generation Sequencing for PRRSV Epidemiology

6. • Attempted sequencing from sera NPB grant 14-204, Development of next generation sequencing methodology for full genome characterization of PRRSV • NGS on sera from 182 samples that were qRT-PCR positive – 66 complete genomes – Others had PRRSV sequence but not complete • Parvoviruses

7. -phylogenetic analysis of glycoproteins identified 4-7 well supported clades -<10% diversity within clade; >10% diversity between clades RFLP174

8. Topological incongruences Gene Combination Representative Strain Strains (n) GP2 GP3 GP4 GP5 M 1 14-79 (Inglevac ATP) 6 2.1 3.1 4.1 5.1 6.1 2 14-64 (Inglevac MLV) 4 2.2 3.2 4.2 5.1 6.2 3 ISU10 11 2.3 3.3 4.3 5.2 6.6 4 ISU49 1 2.3 3.3 4.3 5.5 6.6 5 ISU94 8 2.3 3.3 4.7 5.7 6.3 6 ISU81 5 2.4 3.4 4.4 5.6 6.4 7 ISU32 1 2.4 3.4 4.6 5.4 unassigned 8 ISU28 6 2.4 3.4 4.6 5.2 6.6 9 ISU3 7 2.5 3.3 4.3 5.2 6.6 10 ISU23 5 2.5 3.3 4.3 5.5 6.4 11 ISU39 7 2.5 3.3 4.3 5.5 6.6 12 ISU90 4 2.5 3.4 4.5 5.4 6.5 13 104194 1 unassigned unassigned unassigned 5.1 unassigned

9. Nsp2 phylogeny and deletions VR2332-like MN184-like Novel • 11/66 genomes (17%) identified as PRRSV RFLP174 • >95% identity in GP2a, GP3, GP4, GP5, M, N • 74-100% identity in nsp2

10. Why perform PRRS full genome sequencing? • GP5 is the major surface glycoprotein, is highly variable and contains a neutralizing epitope, however… – Delayed GP5 aby response after clearance of viremia • Equivalent diversity in minor glycoproteins (GP2a/GP3/GP4) – GP2a/GP4 are receptor binding proteins – Known B-cell and neutralization epitopes • Phylogenetic incongruence= which tree do you believe for epi studies? – Viruses with same clade of GP5 but different clades for GP2a-4 • Nsp2 related to pathogenicity • Can detect other viruses in serum (new pestivirus APPV, porcine parvovirus 6) • Not significantly more money that Sanger

11. Atypical porcine pestivirus (APPV) • Highly divergent pestivirus identified in pig sera sequenced for NPB PRRSV genome sequencing project • Very distantly related to BVDV, CSFV (~25% identity) • More closely related to recently partially described bat pestivirus in China (~68% identity) • 5/182 positive serum samples (2.7%) Hause et al, 2015, Journal of General Virology

12. BLASTP analysis of APPV

13. • MEGA6 phylogenetic trees: a) polyprotein b) Npro c) Erns d) NS3

14. qPCR results • Two qRT-PCR assays were developed targeting either E2 or the Erns region of the genome • 3 of 5 were positive on both assays, one positive only on Erns assay and another sample on tested on E2 due to qns – Sample only positive for Erns showed only 88% aa identity for the partial E2 sequence • Samples originated from NE, AZ, NC, MN, and KS

15. ELISA results • ELISA to detect antibodies cross-reactive to Erns peptides produced in E. coli

16. APPV and shaker pigs • Starting in 2014, 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 • Metagenomic sequencing of brain homogenate mapped 195 reads to APPV at 87% identity • qRT-PCR from pig on brain, lymph nodes, heart tissue and serum were positive for APPV • August 2015 new group of pigs 10-16 weeks of age from same system showed similar symptoms, lymph nodes from one pig was APPV qRT-PCR positive Transboundary and Emerging Diseases, submitted

17. 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

18. 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

19. 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 Journal of General Virology, under revision U.S. H.K.

20. Hemagglutinin-Neuraminidase gene sequences NE, IL, OK HK U.S. PPIV1 HN gene sequences 85-96% identical to PPIV1 from Hong Kong

21. PPIV1 incidence in U.S. • 6.1% (17/279) lung/oral fluid/nasal swabs PCR+ for PPIV1 • Serology – ELISA (recombinant F protein)=63% positive – Immunocapture PCR= 55% positive

22. In situ hybridization Nasal turbinates trachea

23. PPIV1 • Common in U.S. pigs • Acute respiratory disease in pigs on sow farms/early nursery • Include in differential diagnostics for influenza/PRRS • KState has qRT-PCR ($30/sample) • No vaccines available – Virus has not been isolated

24. Seneca Valley Virus • 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 Emerging Infectious Diseases, submitted

25. • Large numbers of viruses identified=Virus Soup! – Olivia Myers, NC State CVM at AASV, 2016 • 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???

26. N.C. 2015 Brazil 2015 2015 U.S., >99% identical 97% identical to Brazilian SVV, 2015 95% identical to Canadian SVV, 2011 87-92% identical to historical SVV

27. 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 Emerging Infectious Diseases, submitted

28. 2,000bp Replicase=54% identical to bat circovirus Capsid=36% identical to PCV2 and duck circovirus ORF3=39% identical to Murid herpes protein (unknown function)

29. • Incidence determined by qPCR: 34/271 samples positive (12.5%) • Quantified virus in symptomatic cases – Sows with PDNS, Ct=28-30 – Mummies, Ct=17-21 • 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!

30. 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!

31. Acknowledgements • Kansas State University • Dr. Dick Hesse • Dr. Lalitha Peddireddi • Dr. Jianfa Bai • Dr. Erin Schirtzinger • Dr. Emily Collin • Rachel Palinski • Dr. Namita Mitra • South Dakota State University • Travis Clement • Iowa State University • Dr. Phillip Gauger • Swine Veterinarians • Dr. Josh Duff • Dr. Chad Smith • Dr. Emily Byers • Dr. John Prickett • Those unnamed • National Pork Board grant #14-204 • Zoetis • Boehringer Ingelheim

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