Mechanisms of Interspecies Transmission of Avian Influenza

Viruses at the Human-Animal Interface: Examination of Animal Factors

David E. Swayne*Exotic and Emerging Avian Viral Diseases

Research Unit Southeast Poultry Research Laboratory

USDA/Agricultural Research ServiceAthens, Georgia

*Detailed as OFFLU Scientific OfficerWorld Organisation for Animal Health (OIE)

Paris, France

28 HPAI Epizootics1. 1959-Scotland, H5N1 2. 1961-S. Africa, H5N33. 1963-England, H7N34. 1966-Canada, H5N95.1975-Australia, H7N76. 1979 –Germany, H7N77. 1979-England, H7N7*8. 1983-84 - USA, H5N29. 1983-Ireland, H5N810. 1985-Australia, H7N711. 1991-England, H5N112. 1992-Australia, H7N313. 1994-Australia, H7N3*§14. 1994-95-Mexico, H5N2§15. 1995 & 2004 –

Pakistan, H7N3

16. 1997-Australia, H7N417. 1997-Italy, H5N2**§18. 1996-2010 – Eurasia & Africa, H5N1*19. 1999-2000 - Italy, H7N1*20. 2002 - Chile, H7N3*21. 2003 – Netherlands, H7N7*22. 2004 – USA, H5N2*23. 2004 – Canada, H7N324. 2004, 2006 – S. Africa, H5N2§25. 2005 – N. Korea, H7N7*26. 2007 – Canada, H7N327. 2008 – England, H7N728. 2009 – Spain, H7N7*LPAIV HPAIV**Largest epizootic in 50 yrs

§Vaccine used in the control strategy

Portals of Entry (Routes of Exposure)• Experimental evidence for AIV infections in various animal models:

• Intranasal, intratracheal & bioaerosol (P-149 [131]; O-813 [p.12]) – various birds, mice, ferrets, pigs, guinea pigs• Oral or intragastric – chicken, ducks, gulls, ferrets, pigs, cats• Ocular exposure – mice• Parenteral (e.g. intravenous injection) – chickens, ducks and turkeys

Human-Animal Interface

Human-Animal InterfacePotential Modes of Transmission:

• Inhalation (airborne virus): • Contaminated dust from rearing or slaughter• Fine water droplets generated during

household/LPM slaughter process

• Contact with oral/nasal mucus membrane or conjunctiva:

• Hand-transplantation of virus from contaminated surface, or dust/feathers to eye and mucus membranes of nose

• Swimming/bathing in contaminated household pond (Vong et al. JID 199:1744-52, 2009)

• Consumption of raw or undercooked infected birds or their products?

• Natural cases in carnivorous mammals, raptors and scavenging birds – includes consuming infected wild birds

Transmission

“…What is most important in influenza transmission is not just the feasible, but the

practical…” Robert Couch

Consumption of Infected Birds/Products

• Natural cases: tigers, domestic cats, dogs, mustelidae, humans (one case: VN dk blood)

• Experimental cases: cats and red foxes (Reperant et al. EID 14:1835-41, 2008; Rimmelzwaan et al. AJP 168:176-83, 2006)

(Lipatov et al., PLOS Pathogens 7:e1000102, 2008)

Feeding pigs WS/05 infected chicken meat (100g, 1010 EID50) No lesions• No weight loss• No changes in food consumption • No behavioralabnormalities• No virus rectal swabs or GI tissues

• Transmission by consumption of infected meat• Respiratory infection, no evidence of digestive infection

System IN IG Meat Meat-IGTonsil + - + -Respiratory

Nasal Turb +++ - +++ ++Lung +++ - + -

DigestiveSI - - + +LI + - +++ ++

Liver + - +++ +++Systemic

Spleen + - + +Brain ++ - + +

Olf bulb + - - -

5 DPI – virus titer- = no detection+ = 1.5-2.5++ = >2.5-3.5+++ = >3.5

(Lipatov et al, JID:717-25, 2009)

•Meat consumption:• MDk/Vietnam/05 – mild respiratory infection• WS/Mongolia/05 – infection & mild respiratory disease: 2 dpe – virus in tonsil and nasal turbinates, 5 dpe – virus negative• VN/1203/04 – Lethal systemic disease

Ferret Model: Results

Can cooking kill avian influenza viruses in chicken meat?

• Cooking 70 C will kill HPAI virus in less than 5 sec

Time (s)

-40 -20 0 20 40 60

Tem

pera

ture

(oC

)

20

30

40

50

60

70

80

Viru

s T

iter

(Log

10E

ID5

0/gr

am)

1

2

3

4

5

6

7

8

Temperature CurveKorea/03 - ThighKorea/03 - BreastPA/83 - ThighPA/83 - BreastVirus Isolation Limits

Swayne, Int. J. Food Microbiol, 2006

Chicken Eggs and HPAIV• HPAIV and chicken eggs

– High incidence of HPAI virus in eggs laid by infected hens before death – Moderate concentration of HPAI virus in such eggs– Standard pasteurization temperatures and times kill HPAI virus in

liquid and dried egg products– Cooking kills HPAI virus

Swayne & Beck, Av. Pathol. 33(5):512-518, 2004

• Conclusions:• Most poultry products are consumed cooked or pasteurized• H5N1 HPAI has not been a Food Safety issue for humans

BID50, SPF WL chickens, A/Whooper Swan/Mongolia/244/2005 (H5N1) HPAIV

1. Intranasal - 103.3 and 102.8 EID50

2. Crop Gavage of liquid - 106.2 and 106.3 EID50

3. Crop Gavage of Meat – > 108 and 107.1

4. Oral Feeding of Meat – > 106.0, 107

5. Oral Water - approx. 106.7

Conclusion: Upper respiratory exposure requires lower dose of virus than oral or digestive tract exposure in chickens

Individual verses group housed

Portals of Entry: Exposure Dose

Kwon and Swayne, Avian Diseases, in press

Day 0 Day 3 Day 6 Day 14

Challenge106 EID50

HarvestTissues

Observe Daily

Lung, Nose,Eye

Morbidity/Mortality

Viral Titers (EID50/mL)

Corneal Scarification

(Belser et al., J. Virology 83:7075-84, 2009)

• H7N7 and H7N3 HPAIV replicated to significant titer in mouse eye , but some H5N1 HPAIV, also replicated in the eye, but usually to lower titers

• Pronounced morbidity and mortality w/NL/219 (H7N7), HK/483 (H5N1), & Thai/16 (H5N1)

• Growth in human respiratory and ocular epithelium (Belser et al., O-882 [p.66])

Mechanism: Inhalation of contaminated airborne particles

Preliminary Study: Air Sampling at Necropsy

1. IN inoculated 4 wk-old SPF Chickens with WS/Mongolia/05 HPAIV

2. Three birds that died (2DPI) necropsied 3. Air sampled – negative air ionizing sampler

a. 60 cm from birds b. 10 minc. 104.1 EID50 of virus

Emphasizies need for respiratory protection for necrospy of H5N1 HPAIV suspect birds

LPM: inhalation or contact exposure

Exposure to Wet Markets in Developing World

Experimental Approach:

1. Simulated home slaughter (exception anesthetized the birds before slaughter process began)

2. 10 asymptomatic H5N1 HPAI virus infected chickens in 1 hr (inoculated 24hrs previously)

3. Measurement of virus in the air (negative air ionizing sampler)

4. Determine particle size and number (none-infected)

5. Transmission to caged chickens

LPM: airborne virus study

Table

Isolators

2

3

4

1. Processing site2. Sample point – 300 cm3. Sample point – 150 cm4. Sample point – 75 cm

1

8.3 air changes/hr (340 m3/hr)

Home Slaughter Simulation: Airborne Virus Generation

Home Slaughter Simulation: Airborne Virus Generation

LPM Slaughter Simulation: Airborne Virus Generation

Particle size

(microns)2.5 ft

(log10) 5 ft (log10)10ft

(log10)>25 3.5 3.8

10-24.9 4.6 4.95-9.9 5.4 5.71-4.9 6.6 6.7

0.5-0.9 6.7 6.70.3-.049 7.5 6

Droplet Distribution – non-infected layers

LPM Slaughter Simulation: Airborne Virus Generation

75 cm 300 cm

WL Adult Layers, 106 IN Mongolia/05, Slaughtered 1 DPI (preclinical)

Sampler (Ft)

Virus (log10) Dead Birds

2.5 2.1 5/5 (4.4)5 1.4 5/5 (3.6)

10 1.1 5/5 (5)

LPM Slaughter Simulation: Airborne Virus Generation

Conclusions: • Home slaughter of chickens generates aerosols & large droplets• Home slaughter of infected chickens generates airborne virus• Can transmit H5N1 HPAIV to chickens & ferrets when exposed in same air space as slaughter of infected asymptomatic chickens• Takes lower dose to infect than via IN inoculation (IN-CID50 = 103 EID50)

• Ferrets exposed at 150 cm for 1 hr to slaughter of VN/1203/04 infected chickens; 3 of 4 ferrets became infected & died

75 cm150 cm300 cm

Intervention Strategies

Determine what interventions will reduce H5N1 HPAIV in air samples and reduce transmission of the virus to ferrets in relation to the location where slaughter of asymptomatic H5N1 HPAIV-infected chickens is performed:

•Standard home slaughter process versus the kill step in a plastic bag (Andrew Clark and Mohammad Hassan)

• Non-vaccinated versus vaccinated chickens

Study Design

• White leghorn chickens, 24 hr post-H5N1 HPAIV inoculation intranasally (106 EID50, A/VN/1203/2004): do home slaughter •1 hr, 5 chicken study per treatment group• Measure virus in air – high volume air sampler

•Repeat and expose 3 ferrets in same airspace

Study Design

• 5 steps in halal slaughter process:• Kill (anesthetized)• Hard-scald• Defeathering• Evisceration• Clean-up

Study Design

• Kill step:• Tie feet• Place in bag• Incise carotid and jugular• Retract head, place in can & allow muscle contractions• Contains blood & feathers

Table

HEPA Isolators with infected chickens

2

3

4

1. Processing site2. Scald site3. Kill can 4. Air sampler/particle

sizer/ferret cage (<75 cm)5. 8.3 air changes/hr (340 m3/hr)

1

BSL-3Ag Certified Room

Results

Air Samples During Slaughter

* Number positive/total tested (virus titer, EID50/ml)

Group Method Kill* Scald* Defea.* Evisc* Clean-up*

Non-vax Standard 5/5 (2.2) 5/5 (1.9) 5/5 (1.8) 4/5 (1.4) 4/5 (1.5)

Bag 2/5 (1.3) 2/5 (1.4) 2/5 (0.97) 0/5 1/5 (0.97)

Vax Standard 0/5 0/5 0/5 0/5 0/5

  Bag 0/5 0/5 0/5 0/5 0/5

• Non-vax cks: average oropharyngeal titer 105.25, 5.75 EID50/ml, cloacal titer 104.0, 4.1 EID50/ml at 24 hrs post-challenge

• Vax cks: ave. oropharyngeal titer 103.15 EID50/ml, cloacal titer <100.97 EID50/ml at 24 hrs post-challenge

Results

Ferret Exposure During Slaughter

* Number infected/total exposed

GroupProcess Method

Ferret Infected*

Non-vax chickens Standard 3/3

Bag 1/3

Vax ck Standard 0/3

  Bag 0/3

LPM Simulation: Mitigation Strategies

• Egyptian Islamic Council approval

• Miniaturize air sampler

LPM Simulation: Mitigation Strategies

• Future: Repeat using a 5 gallon plastic bucket and kitchen pan/lid (sustainable and consistent)

• Developing a 12 panel cartoon for CDC/WHO to communicate the new process

• Examining the LPM processes that produce airborne virus and develop mitigation steps

• Transmission occurs through various routes of exposure, mechanisms of infection and vehicles

• In mouse model, conjunctival infection has been demonstrated, especially feasible with H7 AIV

• In the pig model, consumption of WS/05 H5N1 infected meat caused respiratory infection via tonsil and pharynx exposure

• In ferret model, consumption of two H5N1 HPAIV caused non-fatal respiratory infection while one H5N1 HPAIV caused lethal infection with initiation of infection in both respiratory and digestive tracts

• When processing H5N1 HPAI infected chickens, home slaughter process produces aerosols & large droplets which contain virus

Conclusions

Conclusions

• Using Bag technique for the kill step on asymptomatic H5N1 HPAIV-infected chickens, there was a reduction in number of virus+ air samples, reduced quantity of virus in air samples and reduced transmission of HPAIV to ferrets• Using H5N1 HPAIV-infected but vaccinated chickens, prevented virus+ air samples and prevented transmission of H5N1 HPAIV to ferrets• Simple intervention strategies may prevent transmission of HPAIV from birds to humans

Contributions

USDA/SEPRL:Joan Beck

Kira Moresco

James Doster

Justin Brown

Grady Witcher

Y.B. Kwon

A. Lipatov

AHRI (Egypt):

Mohammed Hassan

USDA/APHIS:Andrew Clark

CDC:Terry Tumpey

FUNDING:

DHS

CDC

USDA/ARS