Metagenomic sequencing for virus discovery and characterization

Kansas State Veterinary Diagnostic Laboratory

Ben Hause, MS, PhD

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

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

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 PRRSV Epidemiology

• 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

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

RFLP174

Topological incongruences

Gene Combination Representative Strain Strains (n ) GP2 GP3 GP4 GP5 M1 14-79 (Inglevac ATP) 6 2.1 3.1 4.1 5.1 6.12 14-64 (Inglevac MLV) 4 2.2 3.2 4.2 5.1 6.23 ISU10 11 2.3 3.3 4.3 5.2 6.64 ISU49 1 2.3 3.3 4.3 5.5 6.65 ISU94 8 2.3 3.3 4.7 5.7 6.36 ISU81 5 2.4 3.4 4.4 5.6 6.47 ISU32 1 2.4 3.4 4.6 5.4 unassigned8 ISU28 6 2.4 3.4 4.6 5.2 6.69 ISU3 7 2.5 3.3 4.3 5.2 6.6

10 ISU23 5 2.5 3.3 4.3 5.5 6.411 ISU39 7 2.5 3.3 4.3 5.5 6.612 ISU90 4 2.5 3.4 4.5 5.4 6.513 104194 1 unassigned unassigned unassigned 5.1 unassigned

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

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

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

BLASTP analysis of APPV

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

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

ELISA results

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

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

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 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

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.

Hemagglutinin-Neuraminidase gene sequences

NE, IL, OK

HK

U.S. PPIV1 HN gene sequences 85-96% identical to PPIV1 from Hong Kong

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

In situ hybridization

Nasal turbinates trachea

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

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

• 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???

N.C. 2015

Brazil 2015

2015 U.S., >99% identical97% identical to Brazilian SVV, 201595% identical to Canadian SVV, 201187-92% identical to historical SVV

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

2,000bp

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

• 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!

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!

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