15 Detection of Enteric Viruses in Shellfish
179
Limitations of Shellfish Extraction
The extraction of viruses from shellfish is typically a slow process which is labor
intensive and costly. The procedure produces some hazardous chemical waste,
and the virus yield and reproducibility are not always certain. The procedures are
prone to contamination and can produce seasonal and shellfish species variability,
depending on the composition of the shellfish. For instance, high glycogen and
lipid levels in shellfish just before spawning may interfere with virus extraction
or lead to the transfer of inhibitory substances in the final extract. Extracts often
contain RT-PCR inhibitors which must be monitored. Extraction controls consist
of a positive process control, such as added Mengovirus to monitor virus recovery
efficiency during extraction. A negative process control of known non-contaminated
shellfish should also be extracted to monitor for cross-contamination.
Virus Extraction Methods for Shellfish
One method for virus detection from shellfish is the United States Department of
Agriculture (USDA) method developed by Kingsley and Richards (2001), which
has been validated for use in Canada and can be used for regulatory purposes.
Another procedure under evaluation for use in the European Union is known as
the TAG4 method (Anon 2010; Lees and CEN-WG6-TAG4 2010). Other methods
are also available. The USDA method, known as the GPTT method uses a glycine
buffer elution of virus from shellfish tissues, PEG precipitation of virus, TRI-reagent
extraction of viral RNA, and poly dT magnetic bead capture of viral RNA. This
method was used to identify both hepatitis A virus and norovirus in clams imported
from China, which were implicated in a restaurant-associated outbreak of illness in
New York state (Kingsley et al. 2002). It was also used in identifying the source
of contaminated shellfish traced to an outbreak of norovirus illness in Canada.
The method was validated for use in Canada after a multi-laboratory evaluation
sponsored by Health Canada. Labs participating in the validation included Health
Canada, the Canadian Food Inspection Agency, Agriculture and Agri-Food Canada,
British Columbia Centre for Disease Control, Centre qu´ eb´ ecois d’inspection des
aliments et de sant´ e animale, University of California at Davis, U.S. Food and Drug
Administration, and the Alaska Environmental Health Laboratory. This method was
published in Health Canada’s Compendium of Analytical Methods (Trottier et al.
2010). Regulatory actions can be taken based on this method in the presence of other
supporting information, such as epidemiological evidence. Noteworthy steps in this
extraction procedure are pictured in Fig. 15.1. A more comprehensive overview of
this procedure is shown in the diagram in Fig. 15.2.
In 2004, the European Committee on Standardization (CEN) established a
Technical Advisory Group for Viruses (TAG4). Its purpose was to develop and
publish standard virus extraction and assay procedures for food surfaces, soft fruit
and salad vegetables, bottled water, and bivalve molluscan shellfish for the European
179
Limitations of Shellfish Extraction
The extraction of viruses from shellfish is typically a slow process which is labor
intensive and costly. The procedure produces some hazardous chemical waste,
and the virus yield and reproducibility are not always certain. The procedures are
prone to contamination and can produce seasonal and shellfish species variability,
depending on the composition of the shellfish. For instance, high glycogen and
lipid levels in shellfish just before spawning may interfere with virus extraction
or lead to the transfer of inhibitory substances in the final extract. Extracts often
contain RT-PCR inhibitors which must be monitored. Extraction controls consist
of a positive process control, such as added Mengovirus to monitor virus recovery
efficiency during extraction. A negative process control of known non-contaminated
shellfish should also be extracted to monitor for cross-contamination.
Virus Extraction Methods for Shellfish
One method for virus detection from shellfish is the United States Department of
Agriculture (USDA) method developed by Kingsley and Richards (2001), which
has been validated for use in Canada and can be used for regulatory purposes.
Another procedure under evaluation for use in the European Union is known as
the TAG4 method (Anon 2010; Lees and CEN-WG6-TAG4 2010). Other methods
are also available. The USDA method, known as the GPTT method uses a glycine
buffer elution of virus from shellfish tissues, PEG precipitation of virus, TRI-reagent
extraction of viral RNA, and poly dT magnetic bead capture of viral RNA. This
method was used to identify both hepatitis A virus and norovirus in clams imported
from China, which were implicated in a restaurant-associated outbreak of illness in
New York state (Kingsley et al. 2002). It was also used in identifying the source
of contaminated shellfish traced to an outbreak of norovirus illness in Canada.
The method was validated for use in Canada after a multi-laboratory evaluation
sponsored by Health Canada. Labs participating in the validation included Health
Canada, the Canadian Food Inspection Agency, Agriculture and Agri-Food Canada,
British Columbia Centre for Disease Control, Centre qu´ eb´ ecois d’inspection des
aliments et de sant´ e animale, University of California at Davis, U.S. Food and Drug
Administration, and the Alaska Environmental Health Laboratory. This method was
published in Health Canada’s Compendium of Analytical Methods (Trottier et al.
2010). Regulatory actions can be taken based on this method in the presence of other
supporting information, such as epidemiological evidence. Noteworthy steps in this
extraction procedure are pictured in Fig. 15.1. A more comprehensive overview of
this procedure is shown in the diagram in Fig. 15.2.
In 2004, the European Committee on Standardization (CEN) established a
Technical Advisory Group for Viruses (TAG4). Its purpose was to develop and
publish standard virus extraction and assay procedures for food surfaces, soft fruit
and salad vegetables, bottled water, and bivalve molluscan shellfish for the European