1. Testing for viruses – the way forward with PCR
James Lowther

2. Viruses in shellfish; the problems
Viruses (esp. norovirus and hepatitis A virus) are recognised as the principle cause of illness following bivalve shellfish consumption worldwide
No known recent outbreaks of HAV following consumption of British shellfish
Several outbreaks or incidents of norovirus food poisoning related to shellfish consumption reported annually
Occasional large outbreaks may cause significant economic impact on producers
No viral standards for shellfish in UK or EU legislation; requirement for robust fit-for purpose tests AND more data on background and outbreak events

3. Detection of norovirus in shellfish
Straub et al. reported culture of human norovirus from faeces in 2007; other labs unable to reproduce results
No realistic short- to mid-term prospect of culture methods for shellfish
Detection of virus particles using microscopy or virus coat proteins using immunology (e.g. ELISA) currently lacks sensitivity required for low virus levels in shellfish – no reports of successful application to “real” food samples
Identification of virus in shellfish currently only possible through “molecular methods” - specific detection of viral nucleic acid (PCR)

4. Real-time PCR (TaqMan)
In last 5 years most labs worldwide have replaced “conventional” PCR-based methods for norovirus detection with real-time PCR (TaqMan) -based methods
TaqMan technology enables quantitative analysis of data
Simultaneous amplification and detection of virus sequence allows more rapid sample throughput
In-built probe confirmation eliminates necessity for costly sequencing-based confirmation of positive PCR results

5. Principles of TaqMan
Fluorescence released by TaqMan probe is proportional to the amount of PCR product amplified
Fluorescence data is collected at every cycle by the real-time PCR machine
The threshold cycle (Ct value), at which the fluorescence rises above a specified threshold level is identified for each reaction by the detection software
The greater the initial amount of target DNA, the sooner exponential amplification is detected, and the lower the Ct value will be

6. Principles of TaqMan
Most concentrated
Least concentrated

7. Principles of TaqMan

8. Principles of TaqMan

9. Variety of virus detection methods
23 international labs involved in 2006 ring trial organised by Cefas as European Community reference lab (detection of norovirus in contaminated oysters)
Virus extraction; 13 methods – most targeting digestive gland
Viral RNA extraction – 29 methods
RT-PCR - one and two-step, conventional single round, nested and semi-nested and real-time RT-PCR formats used
Primers/probes – at least 13 different sets
Development of standardised methodology desirable

10. European efforts towards development of a standard method for detection of viruses in food: CEN/TC275/WG6/TAG4
CEN is ISO equivalent body
CEN/TC 275– Food analysis – Horizontal methods
WG 6 – Microbial contamination
TAG 4 – Detection of viruses in food
Working group comprised of European food and water virology experts, circa 30 participants from 12 countries

11. CEN standard method
TAG4 will circulate draft standard to parent bodies within next 6 months
Hope to receive funding for full characterisation/validation of method soon
Standard stipulates:-
Treatment of 2g digestive glands with proteinase K solution followed by extraction of viral RNA with silica (recommends use of Nuclisens magnetic silica technology)
Use of “one-step” TaqMan RT-PCR (recommends Invitrogen kit)
Stringent criteria for primer selection (recommends particular sets)
Use of particular classes of controls; positive and negative process controls, positive and negative RT-PCR controls etc.
Method for quantification and expression of test results
Many labs both within and outside EU (Australia, NZ, Peru) adopting or preparing to adopt CEN procedure

12. Prevalence
Results of Cefas studies using TaqMan detection have detected norovirus RNA in 45-68% of samples
Similar results in equivalent studies in e.g. France, Ireland, Japan
Legal standards based on absence of detectable norovirus RNA is most simple approach but
likely to be problematic (cf. Singapore, HK import requirements)
Major industry impact
*may* overestimate risk (further data required)
Quantitative element of testing very important
Necessity for proper systematic information on background levels and levels in outbreaks for accurate assessment of risk

13. Prevalence
Unit of measurement stipulated in CEN standard will be detectable virus genome copies/g digestive gland (cf. Ct values, PCR units)
Limit of detection method dependent but ~15 copies/g
Since adoption of approach (Mar 08) levels detected at Cefas range from LOD to 1500 copies/g
Based on previous data levels up to 3000 copies/g may be found routinely during winter
Levels >5,000 copies/g are more unusual
Note: these are preliminary estimates only – may change as we generate more data

14. Seasonality
All studies at Cefas demonstrate distinct seasonality both in terms of prevalence and levels

15. Seasonality

16. Seasonality Possible causes of seasonality
Increased levels of norovirus in community during winter; “winter-vomiting disease”
Increased persistence of virus particles at lower temperatures and lower solar irradiation levels
Decreased clearance of virus by shellfish due to lower metabolism during winter
Observed seasonality consistent with predominance of shellfish-related outbreaks in winter period
Caution!! Shellfish sampled in summer can return very high results e.g. ~10,000 copies/g from oysters harvested end of June

17. Correlation with illness
Data on virus levels in shellfish causing illness incomplete
Shellfish from relevant batch available/collected for testing infrequently following illness incidents/outbreaks
Where “from the restaurant” shellfish have been available, sample sometimes norovirus positive at reasonably high levels, sometimes positive at low levels or negative
Cause of illness (toxin, virus, other pathogen) not always established
Epidemiological link with shellfish consumption not always confirmed
Requirement for more systemic epidemiological data

18. Correlation with illness
“Dubious” post-harvest practises can increase risk of illness (not in Scotland!!)
Outbreak Malta, Jan 2007:-
50+ people ill with gastroenteritis (21 faecal samples norovirus positive) following consumption of oysters (grown in France) at 2 hotels, 1 private home
Cefas supplied with 2 samples, 1 from hotel, 1 from importer
Norovirus sequence match between oyster and faecal samples
Shellfish probably stored in contaminated water by the importer prior to sale

19. Correlation with illness
Shellfish contaminated with low levels of norovirus CAN pose risk of infection
Outbreak Cardiff, Dec 2004:-
6 people ill with gastroenteritis following consumption of oysters (grown in Netherlands) at restaurant
Oysters from restaurant tested, positive for norovirus at low levels ~LOD
Sequence matches for both GI and GII norovirus (unusual strains) found between oysters and faecal sample
Strong evidence for oysters as route of infection

20. Correlation with illness
Cefas study with industry collaborators to systematically correlate reports of illness with norovirus levels in oysters
Shellfish supplied to collaborating restaurants during winter tested for norovirus at Cefas
Customer reports of illness following oyster consumption screened for similarity of symptoms, time of onset to norovirus
Using restaurant and supplier traceability, illness reports linked to particular batches of oysters
Correlation with test results analysed

21. Correlation with illness
Majority of samples (59%) and batches (74%) tested positive for norovirus
Levels generally low, one batch provided exceptionally high results for norovirus GII
Majority of batches (77%) NOT linked to illness reports
No illness reports for batches where norovirus not detected

22. Correlation with illness
“Attack rates” calculated for each batch associated with illness
One batch (high norovirus results) linked to illness in 70+ people (21 incidents) – calculated attack rate ~4% (likely underestimate)
Other incidents reported to local health authorities – several norovirus positive results for faecal samples
Maximum attack rate for other batches ~1%, maximum number of linked incidents 4 (12 people)

23. Correlation with illness
Positive correlation between attack rate and detected norovirus levels

24. Correlation with illness
Correlation less clear cut at lower norovirus levels

25. Correlation with illness
Conclusions of industry collaboration
No detection of norovirus RNA = low health risk
Detection of “low” levels = may be some risk, but unlikely to cause large outbreaks?
Detection of “high” levels = risk likely to be severe
Detection of “medium” levels = ???
For more effective risk management more data is required

26. Why not more illness reported??
Infectious dose of norovirus often quoted as <10 virus particles
Most samples of shellfish tested contain >15 copies/g, often many times more
Why are illness reports relatively scarce??
Discrepancy between norovirus RNA copies and viable virus particles?
Strain-related effects?, e.g. GI norovirus as common as GII in SF, GII causes more than 90% of outbreaks
Host-related effects. Blood group confers sensitivity to certain strains only?
Under-reporting? – e.g. HPA IID study suggests 1:1500

27. Future work Improvements/refinements to methodology
Validation of standard method
Build up data on background levels through surveillance
Continue to monitor harvesting areas in Scotland at 1-3 monthly intervals as part of FSAS-funded sanitary surveys
Upcoming UK-wide long-term surveillance of 50 sites funded by FSA
Continue to generate data on health risks of norovirus levels
Data from sporadic illness outbreaks etc.
Further industry collaborations??
Volunteer studies……

28. Acknowledgements The CEN/TC275/WG6/TAG4
The Cefas molecular virology team
Industry collaborators