Rapid detection of hepatitis a virus and murine norovirus in hemocytes of contaminated

oysters.

B.A. Dancho and D.H. Kingsley USDA, ARS, FSIT

Eastern Regional Research CenterDelaware State University

Epidemiology of foodborne disease outbreaks- U.S. 2006

• The economic burden of foodborne illness in the U.S. is estimated at $152 billion in health-related costs each year (2010. Sharff, R.L. Produce Safety Project at Georgetown University).

• Norovirus- most common cause (54% of outbreaks and 11,879 cases)

• Hepatitis A virus- second most common viral cause (<1% of outbreaks and 50 cases)

2009. MMR Weekly 58:609-615.

Virus Overview

Norovirus• Caliciviridae • Non-enveloped,

icosahedral, 38-40 nm in diameter

• 7-8kb ssRNA(+) genome• Fecal-oral transmission• Incubation- 12-48 hours• Symptoms- acute-onset

vomiting, diarrhea, abdominal cramps, and nausea

Hepatitis A virus• Picornaviridae• Non-enveloped, spherical,

30 nm in diameter• 7.5kb ssRNA(+) genome• Fecal-oral transmission• Incubation- 28 days• Symptoms- nausea,

anorexia, malaise, jaundice, abdominal cramps

Human norovirus and hepatitis A virus contaminate shellfish

2008. Bosh, A., et. al. Curr Opin Biotechnol 19(3): 295-301.

Human norovirus and hepatitis A virus contaminate shellfish

• Recent oyster-associated outbreaks– 2010. Westrell, T., et. al. Norovirus outbreaks linked to oyster consumption in

the United Kingdom, Norway, France, Sweden and Denmark, 2010. Euro Surveill 15.

– 2009. Guillois-Becel, Y., et al. An oyster-associated hepatitis A outbreak in France in 2007. Euro Surveill 14.

– 2008. Le Guyader, F. S., et. al. Aichi virus, norovirus, astrovirus, enterovirus, and rotavirus involved in clinical cases from a French oyster-related gastroenteritis outbreak. J Clin Microbiol 46:4011-7.

– 2007. Bialek, S. R., et. al. Use of molecular epidemiology to confirm a multistate outbreak of hepatitis A caused by consumption of oysters. Clin Infect Dis 44:838-40.

• Viral presence in 5-55% of random market samples of oysters perfomed in the U.S. and Europe– 2010. Depaola, A., et. al. Bacterial and Viral Pathogens in Live Oysters: U.S.

Market Survey 2007. Appl Environ Microbiol. 76:2754-68. – 2010. Terio, V., et. al. Norovirus in retail shellfish. Food Microbiol 27:29-32.

Goals

Part 1• To determine whether oyster hemocytes

could be utilized in cell-based viral extraction procedures with commercially available RNA extraction methods.

Part 2• To determine the D10 value (the radiation

dose required for one log reduction of viral load) of MNV in PBS and oyster homogenate.

Need for rapid viral detection in shellfish

• FDA and USDA diagnostics for viral contamination of shellfish are time-consuming (1-2 days)• 2010. Depaola, A., et. al. Appl Environ Microbiol. 76:2754-68. • 2001. Kingsley, D. H., and G. P. Richards. Appl Environ Microbiol

67:4152-7. – Glycine, polyethylene glycol, TRI-reagent, poly dT bead (GPTT) viral RNA extraction

protocol

• Methods using commercial RNA extraction kits are more rapid– 2009. Gentry, J., et. al. J Virol Methods 156:59-65. – 2007. de Roda Husman, A. M., et. al. J Food Prot 70:967-74.

Oyster hemocytes are a site of viral persistence

• Enteric viruses persist in oysters for extended periods– Hepatitis A virus persists for 6 weeks

(Kingsley, D. H., and G. P. Richards. 2003. J Food Prot 66:331-4.)

– Hemocytes are a site of viral persistence

• Oysters have an open circulatory system– Circulate hemolymph

– Hemocytes -oyster multifunctional blood cells (immunity & digestion)

– Hemolymph can easily be drawn out with a pipette, and the hemocytes can be separated by centrifugation.

HAV & MNV distribution in laboratory-contaminated oysters

Laboratory-contamination with HAV and MNV (NoV surrogate)

Separate hemocytes from oyster tissues

Real-time qRT-PCR analysis

Extract RNA with the Rneasy Mini Kit (Qiagen)

Viral RNA is detected in oyster hemocytes as well as in tissues of HAV and MNV laboratory-contaminated oysters.

HAV & MNV distribution in laboratory-contaminated oysters

Detection of HAV & MNV in laboratory-contaminated oysters

Laboratory-contamination with HAV and MNV

Prepare hemocyte and whole oyster samples

Real-time qRT-PCR analysis

Extract RNA:Hemocytes- Dynabeads Oligo(dT)25 (Invitrogen)

Rneasy Mini Kit (Qiagen) Whole Oysters- GPTT

Viral RNA extracted from hemocytes and tissues is detected at similar levels in laboratory-contaminated oysters.

Detection of HAV & MNV in laboratory-contaminated oysters

Detection of HAV & MNV in seeded oysters

Seed with HAV and MNV

Prepare hemocyte and whole oyster samples

Real-time qRT-PCR analysis

Extract RNA:Hemocytes- Dynabeads Oligo(dT)25 (Invitrogen)

Rneasy Mini Kit (Qiagen) Whole Oysters- GPTT

Detection of HAV & MNV in seeded oysters

Viral RNA extracted from hemocytes has lower LODs than in tissues in virus-seeded oysters.

Viral RNA extracted from seeded oyster hemocytes had a higher recovery for both HAV and MNV than in RNA extracted from seeded whole oyster homogenates.

Detection of HAV & MNV in seeded oysters

Summary & Conclusions-Part 1• Viral RNA is detected in oyster hemocytes as

well as in tissues of HAV and MNV laboratory-contaminated oysters.

• Viral RNA extracted from hemocytes had a lower LOD than in whole oyster homogenates in laboratory-contaminated oysters.

• Viral RNA extracted from seeded oyster hemocytes had a higher recovery than in RNA extracted from whole oyster homogenates.

• Oyster hemocytes can successfully be used instead of whole oyster tissues to expedite viral extraction from oysters.

Goals

Part 1• To determine whether oyster hemocytes

could be utilized in cell-based viral extraction procedures with commercially available RNA extraction methods.

Part 2• To determine the D10 value (the radiation

dose required for one log reduction of viral load) of MNV in PBS and oyster homogenate.

Electron Beam

• The National Center for Electron Beam Research, Texas A&M University System

• The current maximum FDA-approved irradiation dose for shellfish is 5.5 kGy

D10 value of MNV in PBS

Electron-beam inactivation (0-5.5 kGy)

Dilute MNV in PBS or oyster homogenate

MNV plaque assay

Summary-Part 2

• D10 value of MNV in PBS is 2.5 kGy

• D10 value of MNV in oyster homogenate is 6.4 kGy

• Studies need to be performed in live oysters to determine whether doses below 5.5 kGy will be effective in reducing the viral load.

AcknowledgementsUSDA, ARS, FSIT

Microbial Safety of Aquaculture Products Center

of ExcellenceDavid Kingsley, Ph.D.Gary Richards, Ph.D

Michael WatsonGloria Meade