1. Norovirus Testing: What Happens at “The Lab”
Dr. Amy Woron
Molecular Biologist
TN Dept. of Health
Laboratory Services

2. Norovirus GI and GII Assay
Differentiates between GI and GII
Can have results in as little as 5 hours
2009 data below
TN Grand Region
County
GI pos
GII pos
GI and GII pos
East
Hamilton 5 9
Henderson 2
Knox 1
Middle
Davidson
Robertson
Sumner
Williamson 3
Wilson
West
Gibson
Out of State
N/A GI
GII

3. CaliciNet Maybe. There are multiple patients all with norovirus GII.
Are they all related?
Maybe.

4. National Norovirus Outbreak Network
CaliciNet USA
Best tool is the epi investigation …..
CaliciNet can help
National Norovirus Outbreak Network

5. Current Pitfalls in Norovirus Surveillance
No national laboratory surveillance system
Tracking multi-state (international) outbreaks with a potential common source is a challenge
Only a fraction of the states report to the CDC
No standard protocol among laboratories for sequence analysis and genotyping 5

6. Objectives of CaliciNet
Improve surveillance of norovirus (sapovirus, other GI viruses)
Allow for real-time data exchange at the state and national level
Rapid genotyping and compare norovirus sequences
Link geographically different clusters of illness to a common (food) source
Facilitate the identification of new emerging norovirus strains

7. States Participating in CaliciNetWA WA ME MT MT ND ND ME ME OR OR MN MN ID VT NH SD SD WI WI NY NY MA WY WY MI MI RI CT IA IA PA PA NE NE NJ NV NV OH OH MD MD DE UT UT IN IN CO CO CA CA IL IL WV WV VA VA DC KS KS MO MO KY KY NC NC TN AZ AZ OK OK AR AR SC SC NM NM MS MS AL AL HI GA GA TX TX LA LA AK AK FL FL
States with CaliciNet certified participants March 2010 (n = 16) 7

8. Confirmed and Suspected Etiology of 1270 Foodborne Outbreaks Reported to CDC, 2006
Other/Multiple 2%
Unknown
29%
Norovirus
40%
Chemical 5%
Parasites 1%
Bacteria
23%
CDC 2009 MMWR

9. From Stool Cup to Sequence
Norovirus testing:
From Stool Cup to Sequence
Stool specimen
RNA Extraction
RT-PCR testing
Real-time RT-PCR
Conventional RT-PCR
positive
Compare norovirus sequence with database
Cycle sequencing 9

10. Norovirus regions targeted by detection and typing RT-PCR assays
Hel
Pro
Pol
VP1
VP2
7654 1 5' 3'
ORF 3
ORF 1
ORF 2
RT* C D
85/98 bp
330/344 bp
177/253 bp
* RT = TaqMan realtime RT-PCR
Norovirus detection
Norovirus typing

11. Required fields for CN upload
Epi
Lab
Page 8; CN Manual v.3

13. Multiple Alignment

14. Dendrogram

15. Nucleotide Polymorphisms

16. GI Reference Strains

17. GII Reference Strains

18. Similarity to PulseNet
BioNumerics
Encourage state level participation
Increase real-time information flow
Ability to view/compare outbreaks at the national level
Another tool in the epidemiology tool belt

19. CaliciNet Summary
A novel national surveillance system for linking norovirus outbreak clusters potentially to a common source
Network for standardized norovirus genotyping and classification
Potential to identify new emerging norovirus strains 19

20. Leslie Barclay lbarclay@cdc.gov (CaliciNet scripts)
CDC CaliciNet Team Contact information:
Leslie Barclay (CaliciNet scripts)
Nicole Gregoricus (Laboratory protocols)
Jan Vinjé (Supervisor CaliciNet) 20 20

21. Future Study Human Astrovirus testing … Human Sapovirus testing …
Food testing for norovirus …
Slides with the CDC CaliciNet logo courtesy of Dr. Jan Vinjé, CaliciNet Team Leader, CDC

22. Questions?