178
G.P. Richards et al.
General Principles of Virus Extraction
Oysters, clams, mussels and cockles are filter-feeding bivalve molluscs which
can concentrate viruses to high levels within their tissues. The first step in virus
extraction is the collection and transport of samples from the environment, food
distribution center, or from the consumer. Since enteric viruses are incapable
of growth within shellfish, no increases in numbers will occur during transport;
however, samples should be refrigerated or frozen to prevent product degradation.
Viruses may be extracted from whole shellfish or from digestive tissues dissected
from the shellfish. The latter has the benefit of reducing the volume of sample.
Tissues may be diluted in a high salt and high pH buffer before homogenization
for 2–3 min in a blender. The homogenized sample is centrifuged and the viruscontaining supernatant is retained, while the pelleted tissues are discarded. Viruses
may be precipitated using polyethylene glycol followed by centrifugation. Additional chemical treatments may be applied to remove potential assay inhibitors
from the shellfish extracts, like the addition of cetyltrimethyl-ammonium bromide
(CTAB), which can be used to reduce polysaccharides from the sample, and Freon
TF (DuPont, Wilmington, DE), Vertrel XF (DuPont), or chloroform-butanol to
remove lipids, as previously described (Richards et al. in press). Protein may also be
removed using Pro-Cipitate (Biotech Support Group LLC, North Brunswick, NJ).
At this point in the purification, intact viruses may be present. Intact viruses may
also be separated from the mix by immunomagnetic capture of whole viruses on
antibody-conjugated beads. A simpler method involves homogenization of shellfish
digestive tissues followed by enzyme (protease K) digestion, centrifugation and
subsequent RNA purification.
Practical assays to detect intact norovirus or hepatitis A virus are not available,
primarily because there are no cell culture methods for the propagation of norovirus
or most strains of hepatitis A virus. Virus detection requires that the RNA be
extracted and tested by RT-PCR. To release viral RNA from within the viral capsid,
capsids must be digested, usually by the addition of phenol and guanidinium isothiocyanate, in compounds like TRIzol
® or TRI Reagent
® , along with chloroform. The
RNA may then be concentrated by binding it to a variety of commercially available
silica or glass fiber matrix columns or filters, such as NucliSens (bioM´ erieux,
Durham, NC), RNeasy Mini Kits or QIAamp Viral RNA Mini Kits (Qiagen,
Valencia, CA), and Roche High Pure Viral Nucleic Acid Kit (Roche Diagnostics,
Indianapolis, IN). Viruses are also concentrated by immunomagnetic beads (L´ opezSabater et al. 1997) and poly dT magnetic beads (Kingsley and Richards 2001;
Kingsley et al. 2002), porcine gastric mucin conjugated to magnetic beads (Tian
et al. 2008), or histo-blood group antigens conjugated to magnetic beads (Harrington
et al. 2004).
G.P. Richards et al.
General Principles of Virus Extraction
Oysters, clams, mussels and cockles are filter-feeding bivalve molluscs which
can concentrate viruses to high levels within their tissues. The first step in virus
extraction is the collection and transport of samples from the environment, food
distribution center, or from the consumer. Since enteric viruses are incapable
of growth within shellfish, no increases in numbers will occur during transport;
however, samples should be refrigerated or frozen to prevent product degradation.
Viruses may be extracted from whole shellfish or from digestive tissues dissected
from the shellfish. The latter has the benefit of reducing the volume of sample.
Tissues may be diluted in a high salt and high pH buffer before homogenization
for 2–3 min in a blender. The homogenized sample is centrifuged and the viruscontaining supernatant is retained, while the pelleted tissues are discarded. Viruses
may be precipitated using polyethylene glycol followed by centrifugation. Additional chemical treatments may be applied to remove potential assay inhibitors
from the shellfish extracts, like the addition of cetyltrimethyl-ammonium bromide
(CTAB), which can be used to reduce polysaccharides from the sample, and Freon
TF (DuPont, Wilmington, DE), Vertrel XF (DuPont), or chloroform-butanol to
remove lipids, as previously described (Richards et al. in press). Protein may also be
removed using Pro-Cipitate (Biotech Support Group LLC, North Brunswick, NJ).
At this point in the purification, intact viruses may be present. Intact viruses may
also be separated from the mix by immunomagnetic capture of whole viruses on
antibody-conjugated beads. A simpler method involves homogenization of shellfish
digestive tissues followed by enzyme (protease K) digestion, centrifugation and
subsequent RNA purification.
Practical assays to detect intact norovirus or hepatitis A virus are not available,
primarily because there are no cell culture methods for the propagation of norovirus
or most strains of hepatitis A virus. Virus detection requires that the RNA be
extracted and tested by RT-PCR. To release viral RNA from within the viral capsid,
capsids must be digested, usually by the addition of phenol and guanidinium isothiocyanate, in compounds like TRIzol
® or TRI Reagent
® , along with chloroform. The
RNA may then be concentrated by binding it to a variety of commercially available
silica or glass fiber matrix columns or filters, such as NucliSens (bioM´ erieux,
Durham, NC), RNeasy Mini Kits or QIAamp Viral RNA Mini Kits (Qiagen,
Valencia, CA), and Roche High Pure Viral Nucleic Acid Kit (Roche Diagnostics,
Indianapolis, IN). Viruses are also concentrated by immunomagnetic beads (L´ opezSabater et al. 1997) and poly dT magnetic beads (Kingsley and Richards 2001;
Kingsley et al. 2002), porcine gastric mucin conjugated to magnetic beads (Tian
et al. 2008), or histo-blood group antigens conjugated to magnetic beads (Harrington
et al. 2004).