Diagnostic investigation of

Senecavirus A-associated

diseases in Brazil and US

Fabio Vannucci, Daniel Linhares, Douglas Marthaler

Leman Conference 2015

Lessons learned from investigation of vesicular diseases in swine

Acknowledgements SHIC

Dr. Paul Sundberg

ISU-VDL Dr. Rodger Main Dr. Phil Gauger Dr. Chris Rademacher

NCFAD, Canada Dr. Ming Yang

Swine Vet Center Dr. Laura Bruner Dr. Steven Tousignant Dr. Tim Loula

UFRGS (Brazil) Dr. David Barcellos

PIC Dr. Eliana Paladino Dr. Jean Paul Cano

UMN-VDL Dr .Jim Collins Pathologist team Elizabeth Wiedenman Matthew Jarvis Dr. Han Soo Joo Wendy Wiese Jan Shivers

Outline Background

Brazil Diagnostic investigation

SVV: neonatal losses and vesicular disease

US Surveillance and retrospective cases

Recent cases and ongoing projects

Antigenic and genetic characterization

SVV: virus characterization

Senecavirus A: virus characterization

Cell culture medium (Hales et al, 2002)

Porcine trypsin or fetal bovine serum

Non-enveloped RNA (~7Kb)

Picornaviridae family (FMD and SVD)

PRRSv ~15Kb PEDv ~28 Kb PCV2 ~1.7 Kb

ncbi.nlm.nih.gov/genome/viruses/

Fecal sample SVV (Jul 27, 2015)

IL-USA Neonatal losses

Senecavirus A: distribution

MG

GO

SP

MT

PR

SC

RS

Canada, Australia, Italy and New Zealand

* *

* *

*

US: Sporadic cases reported since 1988 (NVSL ~30)

* Disease-associated cases (2012-2015)

Brazil: diagnostic investigation October 12th, 2014

Acute neonatal mortality (sudden death and diarrhea)

Off-feed sows but no fever

November 4th, 2014 (*vet visiting)

Mortality and sow consumption back to normal

Brazil: diagnostic investigation

Diagnostics work up Diarrhea: Bact, Rota(s), PED, TGE..

Reproductive: Parvo, PCV2, PRRS, Lepto

Respiratory: Mhyo, SIV

Findings Rota B (Ct 36.4) and C (Ct 38)

C. perfringens A

Brazil: diagnostic investigation Additional findings: subsequent cases

Dehydration/Anorexia

Myocarditis PCV2 + A. suis septicemia

Thromboembolic pneumonia

Necrotizing hepatitis Rota(s) A, B and C

Suppurative colitis C. difficile

Brazil: diagnostic investigation

Other speculations (non-infectious) Toxicity (residues, feed, mycotoxins)

Nov - Dec, 2014 Lameness in sows (along with neonatal losses)

Reportable diseases investigation FMD, VS, SVD and VES

Brazil: diagnostic investigation

January, 2015 Four major reportable diseases ruled out by official

laboratory (Ministry of Agriculture, Livestock and Food Supply)

January 29th, 2015 Samples submitted to MNVDL for unknown pathogen

discovery through NGS.

UMN BSL3 lab: inactivation and retest for FMD, VS, SVD and VES.

February 20th, 2015

Brazil: diagnostic investigation

Brazil: identification of SVV

0.05.0

10.015.020.025.030.035.0

Ct V

alue

Breeding herds

(gilts/sows)

Vesicular disease

Commercial herds

(neonatal piglets)

Acute death

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

Spleen Liver Lung Heart Kidney SmallIntestine

Colon Brain

Ct V

alue

US: retrospective cases and surveillance

ISU and UMN: PCR Screening 2,033 oral fluids samples 8/24 to 9/01/2015

441 cases: 5 positive cases (AR, IA, IL, IN, MN)

1 case: found vesicular lesions later

*

* *

* *

US: retrospective cases and surveillance

UMN-VDL: PCR Screening (2014-2015) as 9/16/15 4,079 samples: oral swabs, tonsils, serum, blood swabs, feces

and tissue homogenate (neonatal deaths).

4 positive cases with no information about vesicular disease

1 case: vesicular disease

5 cases: neonatal losses o no information about vesicular lesions in the sows

o 2001 and 1999: Picornavirus-like particle isolated

Fecal sample SVV (Jul 27, 2015)

IL89-USA Neonatal losses

Recent case: sow farm

US: recent cases and ongoing projects

SVV detection vs disease Koch postulate not fulfilled 2-3 attempts

Detection of SVV within the lesions IHC (Antibody): Dr. Ming Yang (NCFAD, Canada)

Hybridoma supernatant

Virus characterization in culture: OK

Virus identification in infected tissue: (?)

In situ hybridization (ISH): Primer-based approach (PCR specificity)

Hybridize with virus genome (VP1)

US: recent cases and ongoing projects

SVV detection vs disease Detection of SVV within the lesions

US: recent cases and ongoing projects

Vesicular lesions

Intraepidermal vesicles Nonspecific staining

IHC (pilot)

Replicating virus with tropism to stratum spinosum (similar to FMD)

ISH

No evident lesions Acute viremia Death ?????

Spleen

Neonatal losses

Focal necrosis but no presence of virus within the necrotic foci

Liver Virus diffusely distributed.

Acute death in neonatal piglet

Acute death in neonatal piglet

Heart

Jejunum

Preliminary antigenic comparison: IFA Monoclonal antibody produced using NVSL 1988 isolate (Yang et al 2011)

SVV: antigenic and genetic characterization

USA/15 BRA/15 USA/01 USA/99 USA/88

SVV serology: IFA (in development) Selecting the best cell line

Establishing the best titer dilutions

SVV: antigenic and genetic characterization

Genetic comparison: whole genome/partial sequences

SVV: antigenic and genetic characterization

- 2001

- 1997

99.5% 98% (140 nt)

99.5%

96%

94%

Genetic comparison: VP1 gene

SVV: antigenic and genetic characterization

- 1997

- 2001

99.5% 97%

(29 nt)

99.6%

94% 95-96%

Lessons (being) learned FAD investigation is priority!

Economic impact: packing plants

Continuing education: investigation of vesicular lesions

SVV + vesicular lesions: strong association

SVV + neonatal syndrome: virus but not lesions

Short lived viremia

Sequence interpretation (?)

Lack of information on basic questions

Incubation, shedding, transmission, potential sources

Diagnostic investigation of Senecavirus A-associated diseases in Brazil and USAcknowledgementsOutlineSVV: virus characterizationSenecavirus A: virus characterizationSenecavirus A: distributionBrazil: diagnostic investigationBrazil: diagnostic investigationBrazil: diagnostic investigationBrazil: diagnostic investigationBrazil: diagnostic investigationBrazil: diagnostic investigationBrazil: identification of SVVUS: retrospective cases and surveillanceUS: retrospective cases and surveillanceUS: recent cases and ongoing projectsUS: recent cases and ongoing projectsUS: recent cases and ongoing projectsSlide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23SVV: antigenic and genetic characterizationSVV: antigenic and genetic characterizationSVV: antigenic and genetic characterizationSVV: antigenic and genetic characterizationLessons (being) learnedSlide Number 29