FUTURE ROAD MAP IN DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA

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FUTURE ROAD MAP IN DIAGNOSIS AND CONTROL OF AVIAN
INFLUENZA

• H K PRADHAN
• NATIONAL CONSLTANT (AI)
• WHO INDIA COUNTRY OFFICE,
• NEW DELHI
• 
  10-7-09

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Ecology of Influenza Viruses and Interspecies
Transmission, (Peiris JSM et al,2007)
Human H1N1, H3N2, H1N2,H2N2
Pig H1N1, H3N2, H1N2,H3N1
Terrestrial Poultry (Chicken, Turkey, Quail) HA-
4,5,6,7,9,10 NA-1,2,4,7 Eg, H9N2,H5N2,H7N3,H5N1
Equine H3N8,H7N7
Waterfowl and Shore Birds H1-16, N1-9
Sea Mammals (Seals, Whales) H7N7,H4N5,H4N6, H3N3
,H3N2,H13N9
Canine H3N8
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HISTORICAL BACKGROUND

• 1918-1919-Spanish flu (H1N1)-30-50 million people
  died
• 1957-Asian flu (H2N2)-Over 5 million people died
• 1968-Hongkong flu (H3N2)-over 1 million people
  died
• 1977-Russian flu (H1N1)
• 1997- Hong kong (H5N1) direct transmission
• Present outbreaks with H5N1-2003-continuing in 65
  countries (China-epicenter)

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IMPORTANCE OF H5N1

• Rapidly mutates (genetic drift and shift)
• Can acquire genes from viruses infecting
• other animals ( 1997 virus-half H5N1 half
  H9N2)
• Emergence of novel subtype with human
• genes for efficient transmission( man-man)
• Human, pig, quail act as mixing vessel
• Highly pathogenic to man (61) and poultry (100)
• Affects human health and food security
• Fear of next pandemic

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HA clades of HPAI H5N1 in the World
2.2
2.1 2.2 2.3 2.4 2.5
2.32.4 2.5
2.2
?
2.2
2.2
2.2
1 2.3
1 2.3
2.1
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Cumulative Number of Confirmed Human Cases of
Avian Influenza A/(H5N1) Reported to WHO
(1.07.09)
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AI DIAGNOSIS

• REAGENTS
• Reference antigen and antiserum for HA
• sub-typing (H1 to H16)
• Reference antigen and antiserum for NA
• sub-typing (N1 to N9)
• New Castle disease antigen and antiserum
• Receptor destroying enzyme (RDE)

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AI DIAGNOSIS (contd..)

• Primers for RT-PCR, cloning and
  sequencing
• WHO primers of HA gene 219 bp
• OIE (Lee) primers of HA gene 545 bp
• Primers for NA sub-typing
• Primers for Real time RT-PCR-Taqman assay
• Primers for Sybr green assay for
• determination of pathogenicity (HA
  gene)

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TESTING METHODS

• Testing for antibody AGID, HI, SNT in cells /
  embryos, indirect ELISA, cELISA
• Testing for virus/viral antigen
• Isolation of virus in chicken embryos and MDCK
  cells
• Immuno-fluorescence (direct, indirect)
• HA and HI tests (with subtype specific serum)
• AGID test
• RT-PCR with WHO and OIE (Lee) primers-HA gene
• Multiplex RT-PCR-both for HA NA
• Real time RT-PCR (Taqman and Sybr Green assay)
• NA sub-typing
• Sequencing
• Western blot
• Pathogenicity test- IVPI test, polybasic amino
  acid at cleavage site of HA gene, Sybr Green
  assay (Tm value)

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TaqMan assay for H5 sub-typing of Avian Influenza
Virus
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HA1-HA2 Junction
Tm 77.5
Nucleoprotein
Tm 82.4
Multiplex Sybr Green Real-Time Assay for Avian
Influenza virus Typing and H5 subtyping
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Dissociation Curve
H5_AIV_OP1_pJunction_RT_SYBR Green, 01-04-2006,
15Hr 23Min.mxp
TM 77.5
No Template
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SITUATION IN INDIA

• Since 2003- H9N2 has been detected
• First outbreak of H5N1-18th Feb.,2006
• States affected- Maharastra, Gujurat and Madhya
  Pradesh
• Introduced through Migratory birds-From China to
  Europe and then to India
• Heavy mortality in chickens
• Culling of 1.36 million birds, destruction of
• 1.475 million eggs, 8600 MT feed
• DISEASE CONTROLLED

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SITUATION IN INDIA contd..

• Second outbreak-Manipur-25th July 2007
• No of birds culled 3,31,606. controlled
• Third outbreak-West Bengal 15th Jan 2008
• No of birds culled-40.03 lakhs
• No of eggs destroyed-14 lakhs
• Controlled but resurfaced in 1st week March,
  Declared free on 31st October 2008.
• Fourth outbreak-Tripura-8th April 2008
• Virus isolated from chicken, duck and geese
• Introduction to WB and Tripura from Bangladesh
  which experienced 286 outbreaks(25-4-08),
• to Manipur from china/Russia/Mangolia
• Assam-27th Nov2008,WB-15th Dec.2008, Sikkim-19th
  Jan 2009

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SITUATION IN INDIA contd...

• West Bengal Scenario
• Gap between onset declaration-about 10days
• Incomplete culling lack of awareness
• Delay in deciding quantum of compensation
  payment
• Incomplete or no sanitization
• Restocking before 3 months
• Recirculation of virus among chicken-duck-water
  fowl (frequent mixing)
• Contamination of water bodies
• High density poultry(382)duck(184) per sq km
• Topography similar to Vietnam,Thailand

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HPAI(H5N1) outbreaks in India
summary (Feb 2006 June2009)

• Year No outbreak susceptible dead
  destroyed
• --------------------------------------------------
  ---------------
• 7 - -
  1367419
• 1 144 133
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• 42 8570688 127911
  4325832
• 28 672615 3562
  669073
• --------------------------------------------------
  ---------------
• Total 78 92,43,447 1,31,606
  63,62,335
• States affected Maharastra, Gujurat, MP,
  Manipur,
• WB, Tripura, Assam,
  Sikkim

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Important features of Indian virus

• H5N1 virus- Clade 2.2 (MMB)
• Navapur viruses (7972,7966,7964) reassortants HA
  from clade 2.2 and NA from clade 1(VTM)
• HA protein- polybasic amino acids-341 RRRKKR 346
• NA protein-119 glutamic acid, 274 histidine,
• 292 argenine, 294 asparagine sensitive to
• oseltamivir (Tami flu)
• NS1 protein -92 glutamic acid- highly virulent to
  mammals
• PB2 protein-627 lysine adaptation to mammals
• 2nd chicken virus with lysine next to
  Nigerian virus
• West Bengal and Bangladesh viruses genetically
  similar
• but not similar to Maharastra and Manipur
  viruses
• CPE in MDCK,CEF and Vero cells
• IVPI index-2.9/3.0 (100 mortality within 48 Hrs)
• Manipur virus-RRRRKR at HA cleavage site

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Analysis of HA gene

• Leu 8, Asn 170 and Asn 289 are present in all the
  Indian isolates - related to low pathogenicity of
  the H5N1 isolates in mammalian models (Lipatov
  et. al., 2007).
• Gln at position 238 and Gly at position 240,
  S148, W165, H195, N198, E202 and L206 - known to
  be associated with binding to ?(2,3) linked
  sialic acid receptors.

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HA gene
Ck/HK/220/97
99
HK/97/98
HK/156/97
Environment/HK/437-10/99
Gs/HK/ww26/00
Gs/Cambodia/28/04
THA/1(KAN-1)/04
Ck/Laos/7192/04
79
VTM
THA/16/04
Ck/MYS/5858/04
99
Hatay/04
90
VNM/1194/04
Ck/VNM/27/03
Dk/GY/497/06
FJ
Dk/HN/856/06
100
ZJ/16/06
Dk/FJ/1734/05
100
IDN/5/05
IDN
Ck/Wajo/BBVM/05
95
Ck/Yogjakarta/BBVet-IX/04
Ck/Wonosobo/BPPV4/03
swan/Slovenia/760/06
Dk/Egypt/2253-3/06
96
domestic Gs/Iraq/812/06
Whooping swan/Mongolia/244/05
Ck/Sudan/1784-10/06
Ck/Nigeria/641/06
Ty/Suzdalka/12/05
Ck/Crimea/08/05
Gf/ST/1341/06
Ck/Manipur/India/59001/07
Ck/Manipur/India/59007/06
great crested grebe/Denmark/7498/06
MDk/JX/2136/05
BHGs/QH/65/05
QH
Ck/India/15053/06
100
Ck/India/7964/06
Ck/India/8767/06
Ck/India/13746/06
Ck/Adygea/203/06
cygnus cygnus/Iran/754/06
swan/Italy/179/06
Ck/Afghanistan/1207/06
Ck/India/7979/06
Ck/India/7972/06
Ck/India/8362/06
Ck/India/13766/06
Ck/India/13777/06
Ck/India/8361/06
Ck/India/18760/06
Ck/India/9257/06
Ck/India/9255/06
Ck/India/8824/06
Ck/India/15339/06
0.05
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Analysis of NA gene

• Highest percent identity matrix to isolates from
  India (99.8), Thailand (98.5), Malaysia
  (98.0), Qinghai (97.7), Nigeria (97.5)
• 20-amino acid deletion in the stalk region
  (positions 49 to 68)
• Histidine residue at position 274 and Asparagine
  at 294 which are linked to susceptibility to
  Oseltamivir (Tami flu) are present.

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NA gene
100
Gs/HK/ww26/00
Environment/HK/437-10/99
THA/1(KAN-1)/04
Ck/MYS/5858/04
99
Gs/Cambodia/28/04
Ck/Laos/7192/04
VTM
THA/16/04
Ck/VNM/27/03
Dk/VNM/40/04
99
VNM/1194/04
Ck/India/7964/06
Hatay/04
100
Ck/India/7966/06
Ck/India/7972/06
FJ
Dk/GY/497/06
Dk/Laos/3295/06
100
100
ZJ/16/06
Dk/HN/856/06
82
Dk/FJ/1734/05
IDN
Ck/Yogjakarta/BBVet-IX/04
Ck/Wajo/BBVM/05
97
IDN/5/05
Qa/ST/911/05
BHGs/QH/65/05
BHGs/QH/59/05
Ck/Manipur/India/59007/07
87
Ck/Manipur/India/59001/07
great crested grebe/Denmark/7498/06
QH
Ck/Sudan/1784-10/06
Ck/Nigeria/641/06
Ck/Crimea/08/05
Whooping swan/Mongolia/244/05
swan/Slovenia/760/06
Dk/Egypt/2253-3/06
Gf/ST/1341/06
Ty/Suzdalka/12/05
Ck/Afghanistan/1207/06
cygnus cygnus/Iran/754/06
Ck/India/8292/06
Ck/India/8824/06
Ck/India/9254/06
Ck/India/8829/06

Ck/India/9253/06
Ck/India/13766/06
Ck/India/8361/06
Ck/India/8362/06
Ck/India/7979/06
Ck/India/9257/06
Ck/India/13767/06
Ck/India/18760/06
Ck/India/9386/06
Ck/India/15053/06
Ck/India/13777/06
Ck/India/9255/06
Ck/India/8299/06
Ck/India/13746/06
Ck/India/15339/06
0.05
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Analysis of NS gene

• There is a deletion of 5 amino acid residues
  between 80-84 positions in the NS1 protein.
• Presence of amino acid Alanine at position 144
  indicates virulence to chicken.

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NS gene
Ck/HK/220/97
100
HK/97/98
HK/156/97
99
Ck/India/8292/06
VTM
Hatay/04
Ck/MYS/5858/04
THA/1(KAN-1)/04
86
Ck/VNM/27/03
Dk/VNM/40/04
MDk/JX/2136/05
IDN
Ck/Yogjakarta/BBVet-IX/04
Ck/Wonosobo/BPPV4/03
70
IDN/5/05
Ck/Wajo/BBVM/05
Dk/FJ/1734/05
FJ
DkGY497 06
100
ZJ/16/06
Dk/HN/856/06
Ck/Sudan/1784-7/06
Ck/Nigeria/641/06
92
Swan/Slovenia/760/06
Dk/Egypt/2253-3/06
Gf/ST/1341/06
Ty/Suzdalka/12/05
Qa/ST/911/05
BHGs/QH/65/05
Ck/Crimea/08/05
Ck/India/18760/06
92
Ck/Afghanistan/1207/06
Ck/India/8829/06
QH
Ck/India/8824/06
Ck/India/8829/06
cygnus cygnus/Iran/754/06
Ck/India/15339/06
Cygnus olor/Italy/742/06
Ck/India/9253/06
Ck/India/7972/06
Ck/India/7966/06
Ck/India/8362/06
Ck/India/8361/06
Ck/India/9386/06
Ck/India/13767/06
Ck/India/9257/06
Ck/India/15053/06
Ck/India/13746/06
Ck/India/13777/06
Ck/India/9255/06
Ck/India/13766/06
Ck/India/7979/06
0.05
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Analysis of PB2 gene

• Indian isolates sequenced at PB2 gene have Lysine
  at position 627 of PB2 protein, indicating that
  these viruses might be able to cross the host
  barrier and infect mammals
• This finding was confirmed by the ability of the
  Indian viruses to infect various mammalian cell
  lines viz. MDCK, Vero and CEF cells.

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PB2 gene
HK/220/97
100
HK/156/97
HK/97/98
Ck/Yogjakarta/BBVet-IX/04
IDN
100
Qa/Yogjakarta/BBVet-IX/04
CkWajo/BBVM/05
IDN/5/05
ZJ/16/06
FJ
Dk/HN/856/06
Dk/FJ/1734/05
99
Dk/GY/497/06
100
Ck/HN/999/05
THA/1(KAN-1)/04
Ck/MYS/5858/04
Ck/THA/9.1/04
VTM
Qa/THA/NakhonPathom/QA-161/05
THA/16/04
Ck/VNM/C57/04
90
VNM/CL115/05
Dk/VNM/40/04
VNM/1194/04
Qa/ST/911/05
QH
greatcrestedgrebe/Denmark/7498/06
100
Ty/Suzdalka/12/05
BHGs/QH65/05
96
Gf/ST/1341/06
Ck/Egypt/3/06
Swan/Slovenia/760/06
Ck/Sudan/1784-10/06
Ck/Nigeria/641/06
Ck/Crimea/08/05
grebe/Tyva/Tyv06-1/06
Ck/Afghanistan/1207/06
cygnus cygnus/Iran/754/06
Ck/India/15053/06
Ck/India/18760/06
Ck/India/7972/06
Ck/India/9256/06
Ck/India/8824/06
Ck/India/13840/06
0.02
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CONTROL STRATEGIES

• Stamping out within 3 km (could be
  extended-depending on situation)
• Surveillance zone-3 to 10 km
• Safe disposal of dead/killed birds, eggs, feed
• Proper sanitization
• Strict bio-security including ban on transport
• Vaccination with effective monitoring-DIVA
• Combination of stamping out vaccination
• Countries controlled AI-Rep Korea,India(2006
  2007),Mayanmar
• Countries using vaccine- Southern Russia
  (backyard), France,Holland(Backyard,sanctuary),Chi
  na,Vietnam,Thailand,Mexico(H5N2),,Cote d Ivoire,
  Indonesia, Hongkong, Italy, Sudan, Nigeria, Egypt

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TYPES OF VACCINES

• Inactivated Homologous vaccine (field strain)
• Inactivated heterologous vaccine (same HA,
  different NA-DIVA), H5N2, H5N8, H5N9
• Recombinant live viral vector vaccines - Fowl
  pox, Adeno (HA gene insert).
• Vaccines developed by reverse genetic
  (inactivated).
• Recombinant H5 HA antigen using prokaryotes or
  baculovirus expression system.
• DNA vaccine (H5 HA).
• Peptide vaccine.

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ADVANTAGES OF VACCINATION

• Protect against clinical sign and death.
• Greatly reduces shedding of virus. If good
  vaccine no virus shedding.
• Prevents contact transmission.
• Protects against challenges with high dose of
  field virus.
• Protects against changing virus .
• Increases resistance to AI infection.
• Reduces AIV replication.
• Decreases the risk of human infection.
• No virus in meat of vaccinated birds even
• after challenge.
• Massive vaccination can eradicate HPAI in short
  time in high density commercial poultry.
• Cost effective

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DISADVANTAGES OF VACCINATION

• Fear for mutation due to continuous circulation
  of virus or immunological pressure.
• Emergence of vaccine resistant strain (China,
  Mexico, Iwami et al.2009)
• Silent spreading of virus if shedding continues.
• Difficult to differentiate between vaccinated and
  infected birds (serological) now possible.

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ISSUES NEED TO BE RESOLVED BEFORE VACCINATION

• Choice of vaccine-Homologous
• Heterologous
• Antigenic similarity between vaccine and field
  virus
• Standardization of manufacturing practices.
• Proper storage, distribution administration.
• Exit strategy to prevent permanent use.
• Selective or mass vaccination.
• For broilers or for layers including breeders.
• Dosage-gt3 weeks 0.5ml,lt3 weeks 0.25ml

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WHAT INDIA SHOULD DO

• AI surveillance to continue (poultry and ducks)
• Sampling should be adequate and proper.
• Choice of quick reliable diagnostic methods
• If small areas are affected-culling is best.
• If widespread outbreaks-vaccination to create
  immune zone.
• Some birds be left unvaccinated to act as
  sentinels.
• Detect circulation of wild type virus .
• Confirmation of AI infection in immune zone
  warrants culling
• Post vaccination monitoring to ensure efficacy
• Creation of immune zone bordering Bangladesh.
• WHO and OIE also recommend vaccination

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Vaccination Strategies

• Routine vaccination in endemic areas.
• Emergency vaccination in the face of epidemic to
  create immune zone around infected area( ring
  vaccination).
• Preventive vaccination when high risk virus
  incursion is identified.
• Targeted vaccination of defined categories
  of birds on the basis of risk analysis

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CHOICE OF VACCINE

• Inactivated heterologous vaccines
• H5N2, H5N8, H5N9
• Inactivated homologous vaccine (H5N1) developed
  in India.( antigenic ally good, WHO)
• Recombinant fowl pox vaccine
• (from H5N8, H5N1).
• Recombinant adeno virus vaccine.

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• Thank You