Avian Influenza David J' Henzler, D'V'M', Ph'D' Public Health Veterinarian West Virginia Dept of Hea

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Avian InfluenzaDavid J. Henzler, D.V.M.,
Ph.D.Public Health VeterinarianWest Virginia
Dept of Health and Human ResourcesBureau of
Public Health

• Presented at West Virginia University,
  Department of Community Medicine, Public Health
  Grand Rounds
• October 11, 2006

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Outline

• Interaction of influenza viruses among different
  hosts
• Become familiar with the clinical morbidity and
  mortality of LPAI and HPAI viruses
• Learn survival times of AIV in the environment
• Understand global progression of the Eurasian
  HPAI
• Recognize the ecology of influenza viruses
• Whats in the future ???

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Acknowledgements

• Dr. Loretta Haddy, WVDHHR, Director Division of
  Surveillance and Disease Control
• Dennis Senne, USDA, NVSL, Ames, Iowa
• Dr. Tony Joannis, National Veterinary Laboratory,
  Jos, Nigeria

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The Influenza Virus

• Orthomyxovirus
• Small-medium size, enveloped
• RNA
• Negative sense
• Single stranded
• 8 segments, codes 10 proteins
• Major surface proteins
• Hemagglutinin (H1-H16)
• Neuraminidase (N1-N9)
• 144 potential combinations
• Virulence varies (LPAI, HPAI)

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Type A Influenza Surface Antigens
Hemagglutinin (H) Subtypes
1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16



Human
Equine



Swine
Avian
Neuraminidase (N) Subtypes
1 2 3 4 5 6 7 8 9
Human

Equine

Swine
Avian
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H3
Ecology of Influenza
Genetic Reservoirs
H3, H7
H1, H2, H3
Intermixing
H5N1
Commercial, LBMs Others
H10
H1-12 H14-15
H1-2, 4-7, H9-13, 15-16
Other Aquatic Birds?
H1, H3, H4, H7, H13
H1, H3
Modified from D. Swayne
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AI Viruses Change Frequently

• Antigenic Drift mutations in H gene cause minor
  antigenic changes to H protein
• Continuous process
• Limits protective immunity, virulence shift
• Antigenic Shift Replacement of H or N
• Emergence of novel (pandemic) strains
• Genetic reassortment (human and animal viruses)
• Recombination
• Insertion of large chunk of extraneous RNA near
  the HA cleavage site virulence shift

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Asian H5N1 Bird FluWhere Did it Come From?

• 1996 HP H5N1 isolated from geese in Guangdong
  Province, China

• 1997 - Hong Kong
• 18 human cases, 6 deaths
• 1.5 M birds depopulated

D. Swayne
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Cumulative Number of Confirmed Human Cases of
Avian Influenza A/(H5N1) Reported to WHO 3
October 2006
Total number of cases includes number of
deaths.WHO reports only laboratory-confirmed
cases.
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Clinicopathologic Forms of AI
Highly Pathogenic (HPAI)
Low Pathogenicity (LPAI)
Localized infection no, mild disease (any
subtype)

• Acute, systemic
• disease
• High mortality
• H5 H7 subtypes

H5 and H7 subtypes are notifiable
International reporting
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AI Factors Influencing Pathogenicity

• Only H5 or H7 subtypes (most are LPAI)
• Polygenic
• HA plays dominant role
• Multiple basic amino acids at
• HA cleavage site
• Glycosylation sites near HA cleavage site
• Recombination - insertion of extraneous RNA near
  HA cleavage site

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Pathogenesis of AI
Replication at point of entry
LP strains
HP strains
HA cleaved
HA cleaved
by furin, PC6 (ubiquitous)
by trypsin (localized)
Viremia
Respiratory / Intestinal
replication
Systemic infection
Multiple dibasic
Few dibasic
amino acids
amino acids
(B-X-X-R/)
(B-X-B-R/)
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HPAI outbreaks in poultry since 1959

• 25 outbreaks, 11 (H5), 14 (H7)

A/chicken/Scotland/59 (H5N1) A/turkey/England/63
(H7N3) A/turkey/Ontario/7732/66
(H5N9) A/chicken/Victoria/76 (H7N7) A/chicken/Germ
any/79 (H7N7) A/turkey/England/199/79
(H7N7) A/chicken/Pennsylvania/1370/83
(H5N2) A/turkey/Ireland/1378/83
(H5N8) A/chicken/Victoria/85 (H7N7) A/turkey/Engla
nd/50-92/91 (H5N1) A/chicken/Victoria/1/92
(H7N3) A/chicken/Queensland/667-6/94 (H7N3)
A/chicken/Mexico/8623-607/94 (H5N2) A/chicken/Paki
stan/447/94 (H7N3) A/chicken/NSW/97
(H7N4) A/chicken/Hong Kong/97 (H5N1) A/chicken/Ita
ly/330/97 (H5N2) A/turkey/Italy/99
(H7N1) A/chicken/Chile/2002 (H7N3) A/chicken/Nethe
rlands/2003 (H7N7) A/chicken/SE Asia/2003
(H5N1) A/chicken/USA-Texas/2004
(H5N2) A/chicken/Canada-BC/2004
(H7N3) A/Ostrich/South Africa/2004 (H5N2)
Largest outbreak in last 50 years
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Serological Epidemiology (serum)

• 34 of 47 flocks had serum evaluated by AGID
• 24 flocks had antibodies present in gt 50 of
  birds (71) - 10 flocks had antibodies present
  in lt 50 of birds (29)
• Multiple serum antibody measurements were taken
  in 17 flocks of which 11 flocks (64.7) had
  decreasing serum antibody
• 5 (45.5) of these 11 flocks had rapid and steep
  decreasing antibody over four to five months

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Serological Epidemiology (egg yolk)

• Egg samples were collected from nine flocks -
  three of theses flocks had multiple egg
  collections which allowed for some assessment of
  antibody response as monitored by AGID on egg
  yolks
• In a period of six weeks measurable antibody
  decreased rapidly from 97, 93 and 76
  respectively to 10, 20 and 12
• In two of three flocks no detectable antibody was
  present from a sample of 30 eggs each at 11 weeks
  from initial detection of yolk antibody

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Serological Epidemiology(serum and egg yolk)

• This H7N2 (MPAI) virus did not result in full
  conversion of birds when sampled for antibody-
  many of which experienced declining antibodies on
  a flock basis
• These data also suggest eggs can be used as a
  monitor for a flocks AI status but for a limited
  period relatively early in infection
• Eggs may not have detectable antibodies in the
  first weeks following infection with (MPAI)

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Wild-Caught and Experimental Mouse Studies

• 141 wild House mice, Mus musculus, trapped from
  eighteen poultry houses in the early stages of AI
  infection and were not involved as biological
  vectors of the H7N2 (MPAI) virus
• Experimental studies with two strains of H7N2
  (MPAI) isolates in influenza susceptible mice did
  replicate in the respiratory tract and produce
  infrequent mild lesions evident on histopathology
• H5N1(HPAI) isolated from poultry and humans in
  Hong Kong did replicate to high titers in
  influenza resistant mice resulting in clinical
  signs, and severe lesions in the respiratory
  tract and death

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Traditional Depopulation Methods Spread the
Virus

• In the process of traditional depopulation
  methods much organic debris including feathers,
  dust, and other organic material became airborne
• New farms located within 1 to 1 ¼ miles were
  diagnosed with AI within a few days of
  depopulating infected flocks with (MPAI)
• The pattern of spread became predictable and six
  premises were thought to be infected with these
  methods prior to trial field experiments of
  allowing birds to remain in production

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Depopulation Methods
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Surveillance and Control Measures Applied to
Flocks Remaining in Production

• Quarantine, strict biosecurity, and controlled
  marketing of products
• Nest run eggs were held in farm coolers during
  early stages of infection and then sent to
  further processing (breakers) via dedicated
  trucks on dedicated days and a closed loop of egg
  handling and transport materials
• Surveillance mortality cultures, serum and egg
  yolk antibody, environmental culture and sentinel
  hen serology

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Extent of OutbreakPennsylvania 2001-02 Broilers

• Nine broiler flocks affected (172,518 birds)
• Influenza isolated from seven flocks (142,816
  birds)
• Includes two broiler breeders (15,939 birds)
• Index case (12/4/01), others through 1/3/02
• Spread over two county area containing 3.5
  million poultry
• Five of seven cases within one-mile area, one
  case 17 miles away and separated by two mountain
  ranges

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Hemorrhagic trachea with caseous plugs
Hemorrhagic wet edematous lungs
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FLOCK A
Fig. 1.  Daily mortalities () of hens and
roosters in broiler breeder flock A before
(12/01/01), during and after the clinical phase
of avian influenza H7N2 infection. 
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FLOCKS A B
Fig. 3. Daily egg production () in two broiler
breeder flocks A and B before (12/01/01), during
and after the clinical phase of avian influenza
H7N2 infection.
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PA Overall Surveillance
1985 85,000 tests 2004 211,000
tests 2005 240,000 tests (estimate) AI
Monitored Flock Program 390 flocks Tested
Flocks Several hundred Meat Bird Surveillance
20 flock at slaughter Layers 12 to 30
eggs/flock/month Diagnostic Cases Dealer and
truck or crate wash facilities
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Molecular Diagnostics

• Real Time RT-PCR (RRT-PCR)
• Smart Cycler System Cepheid, Sunnyvale, CA
• Results in 2.5 hr
• Matrix assay detects all subtypes
• Can differentiate H5 and H7 subtypes

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Cases of non- H5 or H7 AI

• 2004 19 cases (2 commercial layer complexes
  (H2N2)
• Frequently in muscovey ducks
• Duck flock with H4,N4,N2 antibodies (raccoon
  captured on premise also with H4N2 antibodies)
• 2005 21 cases (H4N2 from environmental sample
  on dealer premise) H6N8 guineas and H11N9
  environments in Northeast LBM (11/15/2005)-
  depopulation of 535 birds

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21 Base Insertion in BC LPAI

• February 2004 in two-aged broiler breeder farm in
  Frazer Valley, BC a 21 base AA insertion sequence
  at hemagglutinin cleavage site over 3-wk period
  HPAI
• Resulting 17 million poultry depopulated
• Open air tissue grinding/composting after
  depopulation procedures to another operation 12
  days post

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Frazer Valley HPAI cont.

• Phylogenetic epidemiology of index case LPAI
  (H7N3) to A/TU/Ontario/2000 (H7N3)
• Lack of comprehensive AIV monitoring program in
  Canada may have resulted in failure to detect
  circulating virus
• Bowes, VA, Ricthie SJ, Byrne S et al, Avian Dis.
  48928-934, 2004

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Risk Factors Spread of AI H7N2 in 2002 VA
Outbreak

• Rendered Poultry Carcasses (Corrected OR of 7.3)
• Birds over 1019 wks (OR 4.9)
• Non-family caretakers (OR 2.1)
• Observation of wildlife (OR 1.9)
• McQuiston JH, Garber LP, Porter-Spalding, BA et
  al, JAVMA, Vol 226, No. 5, March 1, 2005

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Stability of Hong Kong H5N1 Virus
in Feces at Environmental Temperatures
5
Wet Feces
(4 C)
4
3
Virus Titer (log 10)
2
Wet Feces
Wet Feces
(35 C)
(25 C)
1
Dry Feces
(25 C)
0
0
1
2
3
4
5
8
17
21
28
40
Days at Condition
Source Shortridge et al. 1998
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HPAI H5 Experimentally Recovered from Chicken Meat

• 2 LPAI and 2 HPAI viruses intranasally inoculated
  in 3-4 wk chickens
• HPAI (H5N1) Korea 2003 and (H5N2) PA 1983 virus
  detected in blood, bone marrow, breast and thigh
  meat
• Korea virus infected breast meat caused resulted
  in AI infection and death
• David E. Swayne and Joan R. Beck, Avian Dis.
  4981-85, 2005

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Highway or Flyway? Wild Bird Species Infected
with H5N1
Bar headed goose
Whooper swan
Greater black-headed gull
Grey heron
Brown-headed gull
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Danube River/ Black Sea
?
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Index Case Nigeria, Africa February 8, 2006
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Poultry worker should put on face mask also motor
bike should be located further from .. THE
BUILDING (Biosecurity Measure)
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Keep sick birds away from feed mill (Biosecurity
may track virus on bottom of shoes and feed bags
to healthy poultry)
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Chickens in Kaduna State
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Hemorrhage in intestines (can also be END,
Coccidiosis, other)
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Chickens in Kaduna State
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Coloration and swelling
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Blue/purple coloration of leg shanks. Also swollen
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Ovarian follicles may be hemorrhagic
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Ostriches also infected
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Human Influenza Pandemics
Antigenic Shift
1918 H1N1
1957 H2N2
1968 H3N2

• 1918-19 Spanish Flu (H1N1)
• - 20-50 million deaths worldwide - 500,000 U.S.
  deaths
• 1957-58 Asian Flu (H2N2)
• - 2-4 million deaths worldwide
• - 70,000 U.S. deaths
  
• 1968-69 Hong Kong Flu (H3N2) - 1 million deaths
  worldwide -34,000 U.S. deaths

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Closing Thoughts

• International perspective visible (WHO)
• Poultry/birds are primarily infected (humans are
  sentinels)
• Extremely limited human-to-human transmission
• Antiviral therapy
• LHD planning
• Development of human vaccines

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David J. Henzler, D.V.M., Ph.D. State Public
Health Veterinarian Bureau for Public
Health Division of Surveillance Disease
Control Infectious Disease Epidemiology
Program 350 Capitol St., Room 125 Charleston, WV
25301 Phone [phone] E-mail
davidhenzler_at_wvdhhr.org
Snake River Hells Canyon, Idaho