Astroviruses:Astroviruses: AA ConcernConcern forfor thethe MeatMeat Industry?Industry? StaceyStacey SchultzSchultz--CherryCherry AssociateAssociate MemberMember Department of Infectious Diseases St. Jude Children’s Research Hospital Memphis, TN MajorMajor identifiedidentified foodbornefoodborne pathogens,pathogens, UnitedUnited StatesStates –– circacirca 20022002 • Bacterial: • Bacterial, continued: ¾ Bacillus cereus ¾ Vibrio vulnificus* ¾ Brucella ¾ Vibrio, other* ¾ Campylobacter* ¾ Yersinia ¾ Clostridium botulinum enterocolitica* ¾ Clostridium perfringens ¾ E. coli O157:H7* • Parasitic: ¾ E. coli, non-O157 STEC* ¾ Cryptosporidium* ¾ E. coli, other ¾ Cyclospora* diarrheagenic* ¾ Giardia* ¾ Listeria monocytogenes* ¾ Toxoplasma* ¾ Salmonella Typhi ¾ Trichinella Prions* ¾ Salmonella non-typhoidal ¾ Shigella • Viral: ¾ Staphylococcus ¾ Norwalk-like viruses* ¾ Streptococcus ¾ Rotavirus* ¾ Vibrio cholerae, ¾ Astrovirus*? toxigenic* ¾ Hepatitis A * Recognized as foodborne in last 30 years (Zoonotic reservoir) TheThe NewNew FoodborneFoodborne ZoonosesZoonoses ¾ The infected food animal looks healthy ¾ Sustained or repeated infections in animals ¾ Contaminated food looks normal ¾ Pathogen survives standard processing and preparation ¾ Missed by current inspection strategies ¾ Spreads silently around the globe ¾ Requires new control strategies

¾ More to be discovered – avian influenza? Hepatitis E virus? FoodborneFoodborne VirusesViruses

• CDC estimates 67% of all FB illness due to viruses.

• Growing awareness

• Improved Diagnostics

• Enteric viruses – transmission? Where? How? FoodborneFoodborne VirusesViruses Names 1.Stomach flu NOTNOT influenzainfluenza virus!virus! 2.Viral gastroenteritis 3.Acute gastroenteritis 4.Non-bacterial gastro 5.Food poisoning 6.Calicivirus FACTOIDS 1. Highly contagious (physical contact, sharing, kissing) 2. Age related disease 3. Seasonal occurrence – cool weather 4. Incredible stability 5. Symptoms within 48 hpi Persist 1 to 10 days FoodborneFoodborne VirusesViruses SymptomsSymptoms 1. Diarrhea 2. Cramps 3. Dehydration - severe 4. Nausea, vomiting 5. Low grade fever (specific to viral) 6. Muscle spasms VehiclesVehicles DiagnosisDiagnosis 1. Shellfish 1. Difficult – food? 2. Fresh salads 2. Exclusion 3. No-peel fruits 3. Differentiate 4. Pastry, icing 4. Clinical signs 5. Sewage/water contamination 5. EM, ELISA, PCR 6. Seroconversion FoodborneFoodborne VirusesViruses

WhatWhat isis aa virus?virus?

VirusesViruses andand HumanHuman HealthHealth

“Here’s a fun fact” 1898 – Filterable agent smaller than bacteria Fax machine invented Daquiri invented Pepsi invented WhatWhat ISIS aa virus?virus? A virus is a small, infectious, obligate intracellular parasite, capable of replicating itself in a host cell. Size Respiratory tract – Most common rhinovirus, influenza, adenovirus, MMR, VSV

Urogenital tract HIV, hepatitis B, herpes, papilloma

Eyes herpes simplex virus

Skin hepatitis B and C, togavirus, flavivirus,

Gastrointestinal tract

FOODBORNE ILLNESS ViralViral EnteritisEnteritis Reoviridae (Rotavirus) Picornaviridae (Hep A, entero) HumansHumans Noroviridae (Norwalk) Astroviridae

Caliciviridae Parvoviridae MammalsMammals Flaviviridae Coronaviridae Hepatitis E

Astroviridae Reoviridae BirdsBirds Coronaviridae Astroviridae ImportantImportant DifferencesDifferences FromFrom FoodborneFoodborne BacterialBacterial Infections:Infections:

1.1. MinimalMinimal numbersnumbers requiredrequired 2.2. LotsLots shedshed inin pooppoop 3.3. WonWon’’tt ““growgrow”” inin foodfood 4.4. EnvironmentallyEnvironmentally stablestable 5.5. CanCan bebe zoonoticzoonotic

FoodborneFoodborne VirusesViruses AstrovirusAstrovirus

•• PositivePositive sense,sense, nonnon--enveloped,enveloped, RNARNA virusvirus ~6.8~6.8 -- 7.57.5 Kb.Kb.

•• KnownKnown toto causecause entericenteric diseasedisease inin mammalsmammals andand birds.birds. – Second leading cause of childhood diarrhea – Fatal duckling hepatitis – Widespread in poultry – Oysters and shellfish TAstV-2

TAstV-1 OtherOther Astroviruses:Astroviruses: •• DeerDeer ANV •• MinkMink •• ChickensChickens • Canine OAstV • Canine •• GuineaGuinea FowlFowl PAstV NEW!!NEW!!

FAstV •• BatsBats

HAstV-6 HAstV-2 •• CheetahsCheetahs

HAstV-7 HAstV-5 •• WeirdWeird humanshumans HAstV-3 HAstV-8 HAstV-1 HAstV-4 100 changes AstrovirusAstrovirus FactoidsFactoids 1975, worldwide distribution • Primarily affects children, elderly, and immunocompromised people. • Transmission - fecal/oral, fomites, contaminated food and water. VERY STABLE • Numerous serotypes in humans and cows – other animals? • Infection – species specific? Disease?

Ú Immunity? Probably serum neutralizing antibodies (IgG) in humans. Innate in turkeys? PATHOGENESIS?PATHOGENESIS? PoultPoult EnteritisEnteritis MortalityMortality SyndromeSyndrome (PEMS)(PEMS) // GrowthGrowth DepressionDepression // MortalityMortality // DiarrheaDiarrhea // LymphoidLymphoid AtrophyAtrophy // ImmunosuppressionImmunosuppression CommercialCommercial FlockFlock -- 66 weeksweeks ofof AgeAge

TAstVTAstV--22 PathogenesisPathogenesis

Control 400 120 TurkeyTurkeyAstro PoultsPoults 350 – ControlMortality – 0-50% 100 300 Astro 80 250 – Growth depression 200 60 150

40 % Diarrhea

Body Weight (g) Weight Body 100 20 50 0 0 Day 0 Day 3 Day 5 Day 12 Days Post-Infection TAstVTAstV--22 PathogenesisPathogenesis

SystemicSystemic Tissue RT-PCR Isolation InIn --situsitu Intestine √√ √√ √√ Feces √√ √√ Blood √√ √√ Spleen √√ √√ Thymus √√ √√ Bursa √√ √√ Kidney √√ √√ Skeletal Muscle √√ √√ TAstVTAstV--22 PathogenesisPathogenesis TurkeyTurkey PoultsPoults – Extensive diarrhea – No inflammatory response – Mild to no lesions

Control Infected HowHow doesdoes astrovirusastrovirus causecause diarrhea?diarrhea? TypesTypes ofof InfectiousInfectious DiarrheaDiarrhea

•• InvasiveInvasive DiarrheaDiarrhea –– InvasionInvasion ofof MucusMucus MembraneMembrane

•• SecretorySecretory DiarrheaDiarrhea www.med.sc.edu:85/ fox/shig-dk.jpg –– UnregulatedUnregulated IonIon oror NutrientNutrient FluxFlux –– DisruptionDisruption ofof IntestinalIntestinal BarrierBarrier

www.designthatmatters.org Intestinal Epithelial Cell Barrier Function CacoCaco--22 CellsCells asas anan InIn VitroVitro ModelModel SystemSystem •• HumanHuman intestinalintestinal carcinomacarcinoma cellcell lineline •• ExhibitExhibit characteristicscharacteristics ofof enterocytesenterocytes inin vitrovitro •• SpontaneouslySpontaneously differentiatedifferentiate •• SupportSupport HAstVHAstV replication!replication!

Li, et al. (2004) Am J Physiol Gastrointest Liver Physiol 287: G726-G733. IntestinalIntestinal BarrierBarrier PermeabilityPermeability

Fluorescent Flux Assay Transepithelial Electrical Resistance (TER)

TER = Apical permeability

Basal HAstVHAstV--11 IncreasesIncreases BarrierBarrier PermeabilityPermeability

160 175 140 Mock 150 * 120 HAstV-1 125 * 100 80100 6075 * % Time Zero 4050 Mock

% No Cell Control 2025 HAstV-1 * * 0 0 04 8121620243248 4 8 12 24 36 48 Hours Post Infection CharacteristicsCharacteristics ofof AstrovirusAstrovirus--IncreasedIncreased BarrierBarrier Permeability:Permeability:

1.1. ApicalApical

2.2. TimeTime andand concentrationconcentration--dependentdependent

3.3. SubtypeSubtype--independentindependent

4.4. NeutralizedNeutralized

RequireRequire viralviral replication?replication? ProducedProduced aa ViralViral--LikeLike ParticleParticle PurifiedPurified CapsidCapsid IncreasesIncreases BarrierBarrier PermeabilityPermeability

120

100

80

60 Mock 40 PBS HAstV-1 20 % Time Zero Capsid 0 0 4 8 12 16 20 24 28 Hours Post Infection

InIn Vivo?Vivo? AstrovirusAstrovirus CapsidCapsid isis aa NovelNovel ViralViral EnterotoxinEnterotoxin

100 TAstV-2

80

60 15 μg capsid

40 % Diarrhea 20 7.5 μg capsid PBS 0 12 24 36 48 60 Hours Post Infection SignificantSignificant Findings:Findings:

1.1. AnimalAnimal model(s)model(s)

2.2. NovelNovel enterotoxin?enterotoxin?

3.3. ReplicationReplication isis speciesspecies--specific;specific; DiarrheaDiarrhea isis not!not! ModelModel ForFor AstrovirusAstrovirus--InducedInduced PermeabilityPermeability

Key Claudin Occludin ZO-1 Actin HAstV-1 Viral Enterotoxin Viral Enterotoxin

– –

New concept to consider New concept to consider AreAre AstrovirusAstrovirus AA ProblemProblem inin Meat?Meat? BAD NEWS

GOOD1. Widespread NEWS in poultry and shellfish – cows? 1. Infectious dose in meat? 2. Infectious virus found in meat 2. Shouldn’t survive cooking 3. Environmentally stable

4. Become “activated” in the gut

5. Low Infectious Dose FoodborneFoodborne VirusesViruses -- PreventionPrevention At Source 1. Responsible sewage disposal 2. Control shellfish harvesting 3. Depuration for 48 hr

At Processing Level Food Service/Individual-level 1.Water chlorination 1. TRAINING!!! 2. Pasteurization 2. Proper hand-washing (55C for 30 min)? 3. Thorough cooking 3. Freeze-drying • Meat: 70C for 30 min 4. Heat • Shellfish: 100C for 30 min 5. Radiation?? (infectivity may remain) DesperatelyDesperately NeededNeeded……

1. DIAGNOSTIC TESTS • Humans • Water • Food

2. INACTIVATION • Disinfectants • Ways to inactivate

3. YOUR THOUGHTS??