2 6NOVO

Molecular Epidemiologyof Noroviruses: 

Molecular Epidemiology of Noroviruses Steve Monroe, Ph.D. Division of Viral and Rickettsial Diseases Infectious Diseases 1

Slide2: 

Steve Monroe, NCID Williams Lake, NM 2

Role of Viruses in Sporadic Foodborne Illness: 

Role of Viruses in Sporadic Foodborne Illness Source: Mead, et al Emerging Infectious Diseases 5:607-625:1999 3

Sporadic Norovirus Gastroenteritis:Emergency Department Patients (1999-2001): 

Sporadic Norovirus Gastroenteritis: Emergency Department Patients (1999-2001) 3 FoodNet sites (CT, NY, OR) 364 subjects enrolled 152 subjects with stool sample tested for all pathogens Norovirus most common pathogen detected 20% positive by RT-PCR Additional 12% positive when serologic diagnosis is included 1

Foodborne Outbreak Surveillance: 

Foodborne Outbreak Surveillance Source: Foodborne Outbreak Reporting System http://www.cdc.gov/foodborneoutbreaks/ 1

Enhanced Foodborne Outbreak Surveillance:CA, MD, TN; 2001-2002: 

Enhanced Foodborne Outbreak Surveillance: CA, MD, TN; 2001-2002 Active Outbreak Detection Aggressive sample collection (In-home kits) Comprehensive pathogen testing N=27 1 1

Viruses:The “Other” Enteric Pathogens: 

Viruses: The “Other” Enteric Pathogens No replication outside of human host No animal reservoir Must detect directly in clinical or env samples Detection = adulteration Vigorous replication inside of human host Low infectious dose High yield (excrete 1010 infectious doses) Small on the inside, hard on the outside Difficult to disinfect 1

Viruses of Gastroenteritis: 

Viruses of Gastroenteritis Rotavirus Groups A, B, and C Adenovirus Primarily Group F (types 40 & 41) Astrovirus Calicivirus Norovirus (NoV) aka: Norwalk-like viruses (NLV), small round structured viruses (SRSV) Sapovirus (SaV) aka: Sapporo-like viruses (SLV), "classic human caliciviruses" Poorly Characterized Viruses Coronavirus, Picobirnavirus 1

Taxonomy Update: Family Caliciviridae: 

Taxonomy Update: Family Caliciviridae Genus Norovirus (NoV) Type species Norwalk virus Aliases: “Norwalk-like Virus” (NLV), Small Round Structured Viruses (SRSV) Genus Sapovirus (SaV) Type species Sapporo virus Aliases: “Sapporo-like Virus” (SLV), “Classic Human Caliciviruses” Genus Vesivirus Type species: feline calicivirus Genus Lagovirus Source: ICTV, Seventh Report; Minutes of XII ICV 1

Enteric Viruses in Finnish ChildrenPlacebo Group (N=788): 

Enteric Viruses in Finnish Children Placebo Group (N=788) Source: Pang, et al, Pediatr Infect Dis J, 18:420-426, 1999 1

Norovirus Diagnostic Gap:Why is such a common illness so rarely diagnosed?: 

Norovirus Diagnostic Gap: Why is such a common illness so rarely diagnosed? Sporadic cases aren’t reportable Outbreaks rarely investigated Samples aren’t collected Routine testing isn’t available (no clinical or commercial labs) No cell culture system or small animal model 1

Norovirus Detection by RT-PCR: 

Norovirus Detection by RT-PCR 5' 3' Hel Pro Pol ORF 1 ORF 2 ORF 3 1 5 5358 5374 6950 6950 7588 7654 1789 530 212 Capsid A B C Source: GenBank M87661 123 bp 213 bp 322 bp CDC Primers Others 1

Why Sequencing?: 

Why Sequencing? Confirmation of standard RT-PCR results Limitations to RT-PCR Does not identify genogroup (Region B) or cluster information Clarification of the epidemiology of transmission in outbreaks 1

A General Caution!: 

A General Caution! Molecular sub-typing is a bit like the parable of the blind men and the elephant – you can get an entirely different picture of what you’re dealing with depending on which part of the beast you’re examining! 1

Different PCR Targets, Different Answers!: 

Different PCR Targets, Different Answers! GI-1-NORWALK GI-2-SOUTHAMPTON 629 653 615 GI-4-NEWORLEANS 487 772 611 GI-5-APALACHICOLABAY GI-6-HESSE 579 661 GI-3-DESERTSHIELD 488 560 665 736 464 684 GIII-1-JENA GII-5-WHITERIVER 535 562 607 604 643 GII-2-MELKSHAM 536 682 565 GII-1-HAWAII 577 596 564 642 762 710 663 649 GII-J-TOLEDO GII-4-BRISTOL 737 759 542 692 677 708 GII-3-TORONTO 506 654 707 549 712 741 567 634 668 GII-8-IDAHOFALLS 740 683 734 GII-7_GWYNEDD GII-6-FLORIDA 715 723 GIV-1-FORTLAUDERDALE 765 5.0 GI-1-NORWALK GIII-1-JENA GII-2-MELKSHAM GII-5-HILLINGDON GII-N-ERFURT 642 762 GII-1-HAWAII 710 GII-1B-WORTLEY 663 604 643 697 607 649 GII-J-TOLEDO GII-4-BRISTOL 542 737 708 692 677 759 GII-3-TORONTO GII-8-AMSTERDAM GII-7-LEEDS 712 741 506 549 567 654 707 GII-6-SEACROFT 683 734 740 634 668 GIV-1-ALPHATRON 5.0 GII/16 GII/10 GII/1 GII/13 GII/4 GII/7 GII/6 GI/6 Region B Region C 1

29 Clusters of Noroviruses(Analysis of Capsid Proteins): 

Clusters differ by ≥ 20% amino acid pairwise distance Genogroups differ by 44-55% amino acid pairwise distance 29 Clusters of Noroviruses (Analysis of Capsid Proteins) 1

Characteristics of NoV Epidemiology: 

Characteristics of NoV Epidemiology Transmitted through multiple routes Foodborne, Waterborne, Person-to-Person, Mixed Outbreaks difficult to control due to: Very common Very low infectious dose Viral shedding can occur for a prolonged period of time and in the absence of clinical illness Environmental persistence Resistance to common disinfectants Substantial strain diversity 1

Recent Examples of Norovirusesin Foods: 

Recent Examples of Noroviruses in Foods On the "Farm" Oysters contaminated with NoV (FL, LA & CA) Raspberries contaminated with NoV (Canada, Europe) At your Table Deli meats at university cafeteria (TX) Pastries in an Army Mess Hall (TX) Potato salad at catered luncheon (AK) "Banquet-in-a-Box" sent to automobile dealers Frosting on cakes at grocery store (GA) Catered luncheon (Netherlands) Wedding cakes (MA) 1

Molecular Epidemiology of Foodborne Norovirus Outbreaks (1): 

Molecular Epidemiology of Foodborne Norovirus Outbreaks (1) University Dining Hall (TX, March 1998) Acute AGE associated with lunch or dinner from dining hall deli-Bar 23 students hospitalized 9 / 18 stools positive for NoV Foodhandler's child symptomatic, RT-PCR positive Ham sample from deli bar RT-PCR positive, sequence matched that from patient stools Source: Daniels, et al. JID, 181:1467-1470, 2000 Schwab, et al. App. & Envir. Microbiol. 66(1):213-218, 2000 1

Slide20: 

Let Them Eat Cake ! 1

Molecular Epidemiology of Foodborne Norovirus Outbreaks (5): 

Molecular Epidemiology of Foodborne Norovirus Outbreaks (5) Grocery Store Cakes (GA, Feb 2000) AGE associated with eating specialty cakes prepared in a grocery store bakery 153 of 195 attendees from 38 events ill Decorated cakes implicated vehicles (OR=22.2) One foodworker admitted to being symptomatic 15 of 15 stool samples positive for NoV by RT-PCR Sequences of 3 PCR products identical Food samples negative by RT-PCR 1

Molecular Epidemiology of Foodborne Norovirus Outbreaks (6): 

Molecular Epidemiology of Foodborne Norovirus Outbreaks (6) Catered Luncheon - Netherlands, 2001 250 / 800 sick Illness associated with eating lunch rolls (OR=1.7) Baker vomited in sink before hand-slicing rolls 24 of 27 stool samples positive by RT-PCR Identical sequence in baker, family and cases 1

Cases of AGE by 6-hourly Time Period, the Netherlands, 2001: 

n=231 Median incubation = 33 h Cases of AGE by 6-hourly Time Period, the Netherlands, 2001 1

Molecular Epidemiology of Foodborne Norovirus Outbreaks (7): 

Molecular Epidemiology of Foodborne Norovirus Outbreaks (7) Wedding Cakes (MA, Apr 2002) AGE associated with eating wedding cakes prepared by a single bakery 332 of 850 attendees from 12 events ill (Projected total ~2,700 cases from 46 events) Wedding cakes implicated vehicles (OR=4.5) Two foodworkers admitted to being symptomatic Sequences of 4 PCR products identical 1

Multi-State Outbreak Potential: 

Multi-State Outbreak Potential NoV is so common that we can not identify multi-state outbreaks without molecular epidemiology Point Source Individuals infected with NoV in one location and then travel to another area (e.g. conference or cruise ship) Wide-spread common exposure Multiple individuals infected with NoV over large geographic regions (e.g. contaminated produce or oysters) 1

Cruise Ship Outbreaks: 

Cruise Ship Outbreaks Multiple points of exposure and transmission methods Brought on by passengers and crew Contaminated food or water Multiple co-circulating strains within the population (“mixing bowl”) 1

AGE in Passengers and Crew during one 5-day cruise on ship A: 

AGE in Passengers and Crew during one 5-day cruise on ship A Cruise Pax Crew GI/7 GII/4 GII/4 GII/4 GII/4 GII/4 1

Cruise Ship A: 

Cruise Ship A Five-day Caribbean cruise Reached outbreak levels: 5.8% pax and 7.8% crew Overall symptom profile: 83% vomiting 76% diarrhea 7% fever Multiple strains detected On subsequent 4-day cruise: 0.8% pax and 3.7% crew Identical GII/4 sequence from previous cruise 1

Slide29: 

Cruise 1 Cruise 2 CLEANING Cruise 3 GII/4 GII/4 GII/3 GII/4 GII/4 1

Slide30: 

Cruise Ship B Seven-day Caribbean cruises First cruise: 4% pax Subsequent cruise: 8% pax and 2.3% crew One strain detected in pax and crew One case from the first cruise was the suspected index case in a nursing home (126 residents and 49 staff ill) Ship was docked for one week for extensive cleaning On third cruise, three strains of NoV detected Including the GII/4 sequence from the previous two cruises 1

Common Source Outbreak: 

Common Source Outbreak Several sushi chefs visited a hotel in Nevada for food preparation lessons in March 2004 During this time, an acute gastroenteritis outbreak was occurring among hotel patrons Three chefs became ill during their visit Upon their return to Hawaii, a waitress and patrons of the restaurant became ill after eating the sushi prepared by one of the chefs who visited the Nevada hotel 1

Common Source Outbreak: 

Common Source Outbreak Stool was collected from the ill restaurant patrons in Hawaii, sushi chef, and Nevada hotel patrons Identical nucleotide sequence in the sushi chef, Hawaiian restaurant patrons, and several persons in the Nevada hotel outbreak 1

Imported Raspberries: 

Imported Raspberries Canada, 1997* Several clusters of disease in Quebec City in July-August 1997 GI illness linked to consumption of raspberry mousse with raspberry sauce (2 cohorts) More than 200 people ill after eating a raspberry dessert Common ingredient: raspberries imported from Bosnia *Gaulin et al, Can J Public Health 1999 Jan-Feb; 90(1):37-40 1

Imported Raspberries: 

Imported Raspberries Canada, 1997 Identical NoV strain sequences found in stools of cases and raspberry sauce Similar outbreak traced to frozen raspberries in Finland (1998) and France (2005) 1

Slide35: 

Source: http://www.eurosurveillance.org/ew/2005/050428.asp 1

Linking Outbreaks: 

Linking Outbreaks Norovirus outbreaks that are clustered in temporally and geographically 1

Slide37: 

GA KY Feb. 17-19 Feb. 9 Feb. 5-26 Cluster of GII/4 NoV in a 2-week period, Feb. 2004 34-bed nursing facility 88% of residents ill Duration of OB: 21 days 61% attack rate Duration of OB: ~15 days 210 reported ill 6 hospitalizations 1

Slide38: 

Cluster of GII/4 NoV in a 3-week period, Jan. 2005 17.8% pax 6.2% crew Jan. 3-15 Jan. 15-29 Jan. 7-22 Jan. 17-28 Caribbean Itinerary Caribbean Itinerary S. America Itinerary 5.7% pax 1.2% crew 40% attack rate 4.6% pax 0.7% crew 96% diarrhea 46% vomiting 1

Interpretation: 

Interpretation Questionable genuine clusters No later outbreaks were found where that particular norovirus strain was implicated Difficult to interpret with limited information, but does offer tantalizing information about the potential for linking outbreaks to a common source 1

Strain Emergence: 

Strain Emergence In late 2002, a sharp increase in the number of outbreaks was observed on both land and on cruise ships One strain found to predominate during this season Provisionally named the “Farmington Hills” strain 1

Slide41: 

Spread of the Farmington Hill Strain: April 2002 - February 2004 Cruise Ships 1

Slide42: 

Farmington Hills strain by setting, 2002-2004 (N=25 outbreaks) 1

Symptom Frequency: 

Symptom Frequency Compared frequency of diarrhea and vomiting of OBs due to the Farmington Hills strain (n=28) to outbreaks due to other norovirus strains (n=89) Individuals in the 28 outbreaks caused by the Farmington Hills strain were slightly more likely to be ill with diarrhea (87% vs. 80%, p=.03) but no difference in frequency of vomiting (73% vs. 76%, ns) 1

Limitations: 

Limitations Only analyzing a small portion of the NoV genome Region B: difficult to differentiate strains Region D: only a portion of the capsid gene Predominant emerging strains limits the ability to link outbreaks Difficulties in obtaining sequencing information for foods and water 1

Conclusions: 

Conclusions Norovirus is the leading cause of sporadic cases and outbreaks of AGE in adults Transmitted by multiple routes Multiple strains co-circulate; genetic characterization of strains is essential for distinguishing and linking cases NoV is so common that we can not easily identify multi-state outbreaks without molecular epidemiology Future goals: Rapid Dx assays (e.g. real-time RT-PCR; increased surveillance; increased strain characterization 1

Acknowledgements: 

Acknowledgements Viral Gastroenteritis Lab Susan Adams Suzanne Beard Leslie Hadley Amanda Newton Angie Trujillo Gang Wei Du-Ping Zheng Viral Gastroenteritis Epi Joe Bresee Lenee Browne Marc-Alain Widdowson Many, Many State & Local Lab & Epi 1