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Rapid detection of hepatitis virus and murine Interstate

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Rapid detection of hepatitis virus and murine Interstate Powered By Docstoc
					 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 Center
             Delaware State University
       Epidemiology of foodborne disease
            outbreaks- U.S. 2006

                                                                     • Norovirus- most common
                                                                       cause (54% of outbreaks
                                                                       and 11,879 cases)
                                                                     • Hepatitis A virus- second
                                                                       most common viral cause
                                                                       (<1% of outbreaks and 50
                              2009. MMR Weekly 58:609-615.             cases)

• 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).
                    Virus Overview




          Norovirus                     Hepatitis A virus
• Caliciviridae                   • Picornaviridae
• Non-enveloped, icosahedral,     • Non-enveloped, spherical, 30
  38-40 nm in diameter              nm in diameter
• 7-8kb ssRNA(+) genome           • 7.5kb ssRNA(+) genome
• Fecal-oral transmission         • Fecal-oral transmission
• Incubation- 12-48 hours         • Incubation- 28 days
• Symptoms- acute-onset           • Symptoms- nausea, anorexia,
  vomiting, diarrhea, abdominal     malaise, jaundice, abdominal
  cramps, and nausea                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



      Extract RNA with the Rneasy Mini Kit (Qiagen)




               Real-time qRT-PCR analysis
        HAV & MNV distribution in laboratory-
               contaminated oysters




Viral RNA is detected in oyster hemocytes as well as in tissues of HAV and
MNV laboratory-contaminated oysters.
Detection of HAV & MNV in laboratory-
         contaminated oysters
      Laboratory-contamination with HAV and MNV



       Prepare hemocyte and whole oyster samples



                      Extract RNA:
      Hemocytes- Dynabeads Oligo(dT)25 (Invitrogen)
                 Rneasy Mini Kit (Qiagen)
      Whole Oysters- GPTT



               Real-time qRT-PCR analysis
         Detection of HAV & MNV in laboratory-
                  contaminated oysters




Viral RNA extracted from hemocytes and tissues is detected at similar
levels in laboratory-contaminated oysters.
Detection of HAV & MNV in seeded oysters
        Prepare hemocyte and whole oyster samples



                  Seed with HAV and MNV



                       Extract RNA:
       Hemocytes- Dynabeads Oligo(dT)25 (Invitrogen)
                  Rneasy Mini Kit (Qiagen)
       Whole Oysters- GPTT



                Real-time qRT-PCR analysis
  Detection of HAV & MNV in seeded oysters




Viral RNA extracted from hemocytes has lower LODs than in tissues in
virus-seeded oysters.
      Detection of HAV & MNV in 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.
   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
 Dilute MNV in PBS or oyster homogenate



  Electron-beam inactivation (0-5.5 kGy)



            MNV plaque assay
             6.00
                           Inactivation of MNV in PBS - Combined


             5.00
                                                                                  y = -0.3985x + 5.3463
                                                                                        D10 = 2.51


             4.00
Log PFU/ml




             3.00




             2.00




             1.00




             0.00
                    0.00          1.00         2.00          3.00          4.00          5.00             6.00
                                                       E-beam Dose (kGy)
                              MNV Inactivation in Oyster Homogenate -Combined
                    5.8



                    5.6


                                                                           y = -0.1563x + 5.441
                    5.4                                                         R² = 0.9808
                                                                            D-10 = 6.3979 kGy

                    5.2
Log PFU/ ml of OH




                     5



                    4.8



                    4.6



                    4.4



                    4.2



                     4
                          0    1           2              3            4                   5      6
                                                   E-Beam Dose (kGy)
           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.
  Acknowledgements
      USDA, ARS, FSIT
Microbial Safety of Aquaculture
Products Center of Excellence
        David Kingsley, Ph.D.
        Gary Richards, Ph.D
          Michael Watson
           Gloria Meade

				
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posted:4/13/2011
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