Influenza from Genome to Vaccine. Influenza from Genome to Vaccine. Danny Thomas Lecture Series Director s Rounds. Influenza from genome to vaccine

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1 St. Jude Director s Rounds: St. Jude Director s Rounds: Robert G. Webster, PhD Clayton Naeve,, PhD Jie Zheng,, PhD Richard Webby, PhD Introduced by William E. Evans, PharmD Director St. Jude Children's Research Hospital Danny Thomas Lecture Series Director s Rounds genome to vaccine The virus hunters. Ca 1968 The CPU hunter. Ca 1984 EVOLUTION 1

2 The virus hunters. Ca 1968 St. Jude Director s Rounds: Robert G. Webster, PhD Infectious Diseases St. Jude Children's Research Hospital Evolution of Influenza A Viruses Point mutations Reassortment Insertions and deletions Recombination Influenza A Virus Host Range H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 2

WHO Collaborating Centers for Reference & Research on Influenza 4 Human/1 Animal

Lower Animals: WHO FAO OIE Annual Epidemics Vaccines H1N1, H3N2, B Pandemics 2 to 3

Geneva Rome Paris The Ecology of Influenza Viruses That there are a limited number of

Eurasian and American lineages Laos China Spread of H5N1 Influenza in Asia 2004-2006

Reemergence and South Korea spread in Asia 2005 May H5N1 Vietnam kills Barheaded

3 WHO Collaborating Centers for Reference & Research on Influenza 4 Human/1 Animal Memphis Atlanta London Melbourne Tokyo Global: Humans: Influenza Global Organizations Lower Animals: WHO FAO OIE Annual Epidemics Vaccines H1N1, H3N2, B Pandemics 2 to 3 per century Origins and Spread in Wild Birds Disease in domestic poultry, pigs, horses Geneva Rome Paris The Ecology of Influenza Viruses That there are a limited number of host specific lineages of influenza viruses There is geographical separation into Eurasian and American lineages Laos China Spread of H5N1 Influenza in Asia H5N1 in geese in Guangdong Japan of 18 humans die of H5N1 2002/3 Reemergence and South Korea spread in Asia 2005 May H5N1 Vietnam kills Barheaded Cambodia geese in Qinghai China Current status: Human Cases: 224 Indonesia Human Deaths: 127 Thailand Spread of H5N1 3

How Did the H5N1 Virus Spread Westwards So Quickly Migratory

700 wild bird outbreaks 4 commercial bird outbreaks Africa Introduction

The Trojan Horses How Pathogenic Is This Virus?

in 1-2days High risk of death in humans Diarrhea Respiratory symptoms

paralysis Does H5N1/04 Replicate and Transmit in Pigs?

Vietnam/1203/04 lll 0 Ck/Vietnam/C-58/04 lll 0 DK/TH/D4AT/04

4 How Did the H5N1 Virus Spread Westwards So Quickly Migratory birds/globalized poultry industry Cold winter, Baltic Sea froze Europe 700 wild bird outbreaks 4 commercial bird outbreaks Africa Introduction to Nigeria Not in wild birds Will it burn out? The Trojan Horses How Pathogenic Is This Virus? A/Vietnam/1203/04 (H5N1) Kills chickens in less than one day Kills ducks in 1-2days High risk of death in humans Diarrhea Respiratory symptoms High Risk of death in ferrets Respiratory symptoms Diarrhea Hind Leg paralysis Does H5N1/04 Replicate and Transmit in Pigs? Expanding Host Range for Influenza Infected Contact H5N1 in Thailand Vietnam/1203/04 lll 0 Ck/Vietnam/C-58/04 lll 0 DK/TH/D4AT/04 GS/TH/G7CS/04 lll lll 0 0 Experimental transmission in domestic cats Choi et al 2005 Kuiken et al, Science 2004 Will H5N1 Acquire Transmissibility? HAPPY NEW YEAR OF THE DOG 4

St. Jude Children s Research Hospital Acknowledgement Support: AI95357, s NIAID, ALSAC St.

Hulse, Katharine Sturm-Ramirez, Aleksandr Lipatov and The Influenza Support Staff Hong

Dr. Amin Soebandrio Vietnamese Ministry of Agriculture and Rural Health Development Dr.

Chantanee Buranathai Kasetsart University, Kamphaengsaen Campus Dr. Thaweesak Songserm St.

Jude Children s Research Hospital The first large-scale bird flu genomics effort -

Robert Webster s avian influenza virus collection Hartwell Center

5 St. Jude Children s Research Hospital Acknowledgement Support: AI95357, s NIAID, ALSAC St. Jude Children s Research Hospital Richard Webby, Elena Govorkova, Erich Hoffmann, Diane Hulse, Katharine Sturm-Ramirez, Aleksandr Lipatov and The Influenza Support Staff Hong Kong University Drs. Yi Guan, Malik Peiris, Leo Poon, K.Y. Yuen, Honglin Chen Influenza Research Group Indonesian Ministry of Research & Technology Dr. Amin Soebandrio Vietnamese Ministry of Agriculture and Rural Health Development Dr. TD Nguyen Thailand Bureau of Disease Control and Veterinary Services Dr. Chantanee Buranathai Kasetsart University, Kamphaengsaen Campus Dr. Thaweesak Songserm St. Jude Director s Rounds: Clayton W. Naeve,, PhD Hartwell Center St. Jude Children s Research Hospital The first large-scale bird flu genomics effort - launched 11/1/2005 Dr. Robert Webster s avian influenza virus collection Hartwell Center biotechnology/bioinformatics expertise/resources Computational Horsepower Clayton W. Naeve, Ph.D. Director s Rounds June 2, 2006 SJ Influenza Virus Repository 76,000 cpu-hours on 420-cpu linux cluster Sampled viruses viruses (all (all known known serotypes) Synthesized segment-specific primers primers Sequenced 124,000 templates Produced 58,550,436 bp bpraw data data Assembled 2,823 2,823 complete genes genes (4,719,005 bp bpfinished sequence) Assembled complete genomes. NCBI 5

6 PB2 ROOT AJ410498 32 7 PB2 AY651729 AY585516 31 5 PB2 313PB2 M21851 020PB2 067PB2 111PB2 077PB2 213PB2 240PB2 23 7 PB2 257PB2 061PB2 019PB2 241PB2 092PB2 253PB2 181PB2 178PB2 191PB2 17 7 PB2 A

162PB2 128PB2 244PB2 255PB2 164PB2 271PB2 103PB2 23 3 PB2 056PB2 A Y633315 052PB2 053PB2 215PB2 055PB2 06 2 PB2 198PB2 102PB2 125PB2 199PB2 364PB2 15 0 PB2 153PB2 154PB2 001PB2 119PB2 36 3 PB2 09 9

225PB2 22 3 PB2 182PB2 172PB2 014PB2 25 1 PB2 173PB2 013PB2 22 0 PB2 231PB2 232PB2 166PB2 163PB2 203PB2 254PB2 155PB2 117PB2 042PB2 044PB2 362PB2 10 0 PB2 23 0 PB2 165PB2 366PB2 082PB2 124PB2 029PB2

024PB2 030PB2 022PB2 021PB2 025PB2 026PB2 031PB2 027PB2 373PB2 M73514 006PB2 080PB2 160PB2 13 4 PB2 10 7 PB2 106PB2 249PB2 009PB2 01 0 PB2 112PB2 008PB2 089PB2 039PB2 114PB2 219PB2 086PB2 185PB2

6 6 PB2 ROOT AJ PB2 AY AY PB2 313PB2 M PB2 067PB2 111PB2 077PB2 213PB2 240PB PB2 257PB2 061PB2 019PB2 241PB2 092PB2 253PB2 181PB2 178PB2 191PB PB2 A Y A Y AY AF AF PB PB2 065PB2 064PB2 069PB2 239PB PB PB PB2 143PB2 260PB2 110PB2 180PB2 081PB2 079PB2 236PB2 250PB2 221PB2 222PB2 174PB2 259PB2 162PB2 128PB2 244PB2 255PB2 164PB2 271PB2 103PB PB2 056PB2 A Y PB2 053PB2 215PB2 055PB PB2 198PB2 102PB2 125PB2 199PB2 364PB PB2 153PB2 154PB2 001PB2 119PB PB PB2 365PB2 229PB2 023PB2 054PB PB2 AF AF AF AF PB2 357PB2 015PB2 105PB2 016PB2 141PB2 167PB2 168PB2 176PB PB2 108PB2 270PB2 209PB2 207PB2 AY PB2 226PB2 225PB PB2 182PB2 172PB2 014PB PB2 173PB2 013PB PB2 231PB2 232PB2 166PB2 163PB2 203PB2 254PB2 155PB2 117PB2 042PB2 044PB2 362PB PB PB2 165PB2 366PB2 082PB2 124PB2 029PB2 151PB2 041PB PB2 242PB2 076PB2 126PB2 127PB2 214PB2 256PB2 004PB2 188PB2 096PB2 146PB2 147PB2 149PB2 206PB2 140PB2 132PB2 130PB2 057PB2 058PB2 059PB2 135PB2 060PB2 121PB2 123PB2 152PB2 183PB2 024PB2 030PB2 022PB2 021PB2 025PB2 026PB2 031PB2 027PB2 373PB2 M PB2 080PB2 160PB PB PB2 106PB2 249PB2 009PB PB2 112PB2 008PB2 089PB2 039PB2 114PB2 219PB2 086PB2 185PB2 248PB PB2 216PB2 217PB2 091PB2 245PB2 218PB2 005PB2 072PB2 116PB PB2 043PB2 003PB2 161PB PB PB PB2 047PB2 048PB2 159PB2 157PB2 158PB2 156PB PB2 228PB PB PB PB2 148PB2 344PB2 097PB2 040PB2 145PB2 187PB2 144PB2 122PB PB2 120PB2 131PB2 118PB2 349PB2 011PB2 083PB2 202PB2 AF AF AF AF A J AB A B AJ AJ AJ AJ AJ AJ A J AF PB2 273PB2 301PB2 360PB2 M PB2 AJ PB2 AB AJ AJ AJ AY AY AF PB2 305PB PB2 M73516 M PB2 113PB2 007PB2 095PB2 169PB2 205PB2 361PB2 345PB PB2 314PB2 269PB2 323PB2 324PB2 322PB2 316PB2 317PB2 302PB2 318PB2 304PB2 306PB2 295PB PB PB2 M PB2 291PB2 AF AF PB2 279PB2 281PB2 AY AY AY AF AY AY A Y A Y A Y AY AY AY A Y AY AY AY AY DQ DQ DQ DQ AY AY AY AB A Y AB AB AY AB AB AY AY AY A Y AY AY AY AY AY AY AY AY AY AY AY AY A Y A Y AY AY AY AY AY A Y AY A Y AY AY AY AY AY AY AY AY AY PB PB PB2 288PB2 A Y AY PB PB PB2 AY AY AY A Y AY AY AY AF AY PB2 311PB PB2 297PB2 300PB PB2 298PB2 308PB2 303PB2 310PB2 312PB2 341PB2 321PB2 329PB2 331PB2 M PB2 PB1 RO OT 042PB PB1 118PB1 AY PB1 317PB1 315PB PB1 323PB1 324PB1 312PB1 313PB PB1 327PB PB PB1 072PB1 116PB PB1 151PB1 150PB1 194PB1 195PB1 363PB1 AF AF AF PB PB1 020PB1 111PB1 094PB1 AY AY A Y AY PB1 254PB1 260PB1 075PB1 213PB1 064PB1 179PB1 014PB1 065PB PB PB PB PB1 AF AF PB1 017PB1 067PB1 180PB1 182PB1 071PB1 253PB1 018PB1 016PB1 019PB PB1 143PB1 079PB1 358PB1 357PB1 240PB PB1 224PB1 223PB1 081PB1 172PB1 060PB1 164PB1 103PB1 002PB1 165PB PB PB1 003PB1 202PB PB1 166PB1 AY PB1 259PB1 238PB1 239PB1 252PB1 015PB1 141PB PB PB1 237PB PB PB PB1 176PB1 140PB1 177PB1 251PB1 191PB1 135PB1 174PB1 173PB1 160PB1 123PB1 257PB1 110PB1 270PB1 126PB1 206PB1 249PB1 114PB1 219PB1 086PB1 185PB1 009PB1 008PB1 010PB1 038PB1 091PB1 089PB1 216PB PB1 234PB1 218PB1 039PB1 169PB1 264PB PB1 113PB1 205PB1 248PB1 011PB1 262PB1 085PB PB1 366PB1 132PB PB1 250PB1 221PB1 222PB PB1 233PB1 364PB1 047PB1 048PB PB PB1 051PB PB PB PB PB PB1 057PB1 058PB1 059PB1 220PB1 247PB1 271PB1 207PB1 005PB PB1 100PB1 244PB1 242PB PB1 255PB PB1 012PB PB PB1 167PB1 168PB1 171PB PB PB1 230PB1 076PB1 127PB1 256PB1 214PB1 128PB1 365PB1 036PB1 131PB1 044PB1 045PB1 229PB1 041PB1 121PB1 023PB1 153PB1 373PB1 M PB1 043PB1 184PB1 198PB PB PB1 148PB1 145PB1 119PB1 183PB PB PB1 125PB1 021PB PB1 022PB1 024PB1 025PB1 027PB PB1 006PB1 112PB1 130PB1 029PB1 122PB1 120PB PB1 099PB1 037PB1 199PB1 004PB1 192PB1 344PB1 M PB1 144PB1 080PB1 157PB PB PB PB1 155PB1 097PB1 096PB1 146PB1 147PB1 149PB1 188PB1 M PB1 331PB PB1 341PB1 346PB1 AJ AJ AJ AJ AJ AJ AF AB AB AJ AF A J AJ AF AF AY AY AY AY AY AY AY AY AY AY AY AY A Y A Y AY AF AF AF AF AY A Y AY AY AY AY AY AY AY AY AY AY DQ DQ DQ DQ DQ DQ AY A Y AY AY AB A B AB AB AB AY AB AY AY AY AY AY A Y AY AY AY PB PB1 AY AY PB1 282PB1 288PB1 AF AF PB1 AF PB1 290PB1 AF AF AF AF AF AJ AY A Y AY AY AY AF PB1 286PB1 293PB1 292PB1 AY PB1 278PB1 279PB1 AF PB1 AJ AY AY AY PB1 359PB PB1 335PB PB1 296PB1 302PB1 311PB PB1 300PB1 301PB1 295PB1 348PB1 333PB1 AY AY AY PB1 297PB1 306PB1 305PB1 309PB1 304PB1 303PB1 308PB1 307PB PB1 007PB1 095PB1 261PB PB1 320PB1 319PB PB1 PA PA ROOT AF AF AF A F PA 291PA AF AF AF AF AF AF AF AF PA 33 5P A AJ AJ AJ AJ A J AJ A J AF A F A J AF AJ A B A B AF AF AF AF AF AF AF AF AF PA 299PA AY AY AY AF AY AY AY A Y A Y AY P A 33 3P A AY AF AF AY PA 292PA 289PA 279PA 27 8P A AF AF A Y A Y A Y AY AF AF A F AF AY AY AY AY AY A Y P A 27 4P A AJ AY A Y AY A B AB A B AB AB AY AY AB AY AF PA 340PA 36 0P A 226PA 227PA AY AF P A A F AF AF AY A Y AY A Y AY A Y A Y AY AY A Y AY AY AY A Y AY A Y AY A Y A Y AY A Y AY A Y AY A Y A Y AY A Y A Y A Y A Y A F AY A Y AY PA AF A F PA 28 2P A 288PA 09 4P A A Y PA 306PA 32 6P A 329PA 32 8P A 330PA 331PA 32 5P A 31 6P A 315PA 269PA 323PA 324PA 318PA 31 7P A 314PA 313PA 31 2P A 30 1P A 302PA 31 1P A 30 9P A 26 6P A 29 6P A 30 3P A 295PA 310PA 30 8P A 305PA AF AF PA AF AF AF PA 16 0P A M PA 106PA 057PA 058PA 059PA 179PA 06 2P A 01 4P A 189PA 11 3P A 263PA 264PA 205PA 00 7P A 26 1P A 09 5 PA AF AF AF PA 27 0P A 167PA 00 4P A 053PA 05 6P A 215PA 052PA 23 4P A 25 4P A 09 0P A 22 4P A 22 5P A 064PA 143PA 10 5P A A Y AY A Y PA 180PA 01 5P A 141PA 23 6P A 17 5P A 176PA 257PA 11 4P A 219PA 171PA 112PA 091PA 218PA 08 9P A 216PA 24 5P A 00 8 PA 03 9P A 16 8P A 249PA 08 6P A 185PA 00 9 PA 010PA 217PA 060PA 207PA 20 6 PA 247PA 251PA 061PA 108PA 24 4 PA 243PA 242PA 161PA 16 2P A 23 3P A 10 0P A 12 8P A 231PA 232PA 230PA 15 3P A 122PA M PA 187PA 363PA 367PA M PA 05 5P A 19 4P A 19 5P A 19 6P A 145PA 148PA 002PA 01 1P A 262PA 00 6P A 003PA 093PA AY PA A F P A 08 3P A 06 5P A 18 1P A 18 2P A 252PA 178PA 271PA 16 5P A 25 5P A 214PA A Y PA 076PA 12 6P A 256PA 054PA 20 3P A 202PA 012PA 22 1P A 250PA 22 2P A 132PA 13 1P A 190PA 043PA 18 8P A 066PA M PA 04 5P A 023PA 04 2P A 22 9P A 02 9P A 078PA 228PA 36 4P A 192PA AJ PA 072PA 116PA 082PA 348PA 30 0P A 307PA 321PA 319PA 320PA 345PA 34 6 PA 332PA M X P A M P A 260PA 239PA 240PA 087PA 08 8P A 18 6P A 019PA 111PA 092PA 241PA 079PA 02 0P A 016PA 23 8P A 23 7P A 209PA 01 3 PA 17 3P A 00 5P A 366PA 144PA 22 0P A 13 5P A 19 1P A 017PA 01 8P A 17 4P A 166PA 150PA 154PA 15 2P A 044PA 344PA 119PA 183PA 151PA 096PA 146PA 147PA 149PA 041PA 12 1P A 097PA 201PA 184PA 155PA 157PA 158PA 156PA 08 0P A 10 2P A 120PA 04 7P A 050PA 048PA 049PA 365PA 021PA 02 2 PA 02 5 PA 02 4P A 36 2P A 051PA 099PA 349PA AY AY HA RO OT D90305 AY AY A Y AY AY AY AB AB AY AY AF AF AF AF AY AY AF AF AF A F AY A Y AY AF AF HA 293HA AY A Y HA DQ A Y AF AF A F A Y AF AF AF AY AY AY AY AY AY AY AY AY AY AY AF AF AF A F A Y A Y AY AF AF HA AY AF AF AF AY A Y HA A Y AF A Y A Y A Y HA 226HA 328HA AY AY AY HA AY HA 114HA 217HA 216HA 218HA 089H A 220HA AY HA AY HA AY H A 214H A AY H A 363H A 188HA 187HA D HA 256HA D HA 260HA 083HA 257H A 024HA 027HA 021HA 022HA 025HA 026HA 031HA 032HA 033HA 034HA 037HA 042HA 045HA 044HA 364HA 047HA 048HA 049HA 050HA 051HA AY AY AY AF AF AY HA 052HA 215HA AY H A AY H A 054H A 057HA 058HA 059HA 106HA 107HA AY HA 023H A 046HA 029H A AJ AJ AJ AJ AJ AJ AJ AJ AF A F A F AF A F AF AJ AJ AY AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ D HA AJ H A AJ H A AY HA 060HA AY HA AF AY AJ AJ AJ AJ AJ AJ AJ A F AF AF AF AF AF AF AF HA A Y AY AY HA AJ AY AY A Y D A F HA 105HA 111HA 362H A L HA 102HA D10477 AF HA 119H A 096HA 097HA AY H A 110HA 108H A AF AF L H A AF HA A F A F A F A F L11141 L HA L HA 009HA 112HA L HA L11139 L11140 A Y L1136 L HA L HA AF HA 011HA 002HA 270HA 005HA L HA A Y HA A Y HA A Y AY HA AY HA 019H A 020H A L HA 004HA L11138 L HA J04325 M H A AY AY AY AY AY AY HA 186HA U67783 AF HA AF AF U69277 A F HA M18450 M H A AF AY AY AY AY AB AY AY AB AB AB AB AY AY AY AY AY AY AY AY AY AY AY AY AJ AY AY AY AY AY AY AY AY AY AY AY AY DQ AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY A Y DQ AY AY AY AY AY AY AY AY A Y A Y AY AY AY AY AY AY A Y AY AY AY AY A Y A Y A Y AF A F AF AF AY AY AY AY AY A Y A Y AF AY A Y A Y AY A Y A Y A Y A Y A Y AY AY AF AF AF AF A F A F A F AF AF AF AF AF A F A F AF AF AY AY DQ AY AY AF AF AF AY AF AJ AF AF AY AY AF S68489 AF HA 302H A AY X07826 X07869 U D90306 AY DQ HA 081HA 253HA 244HA 080HA 250HA AY A Y A Y HA AY D90308 M H A 095HA 007HA 094HA M AY AY M HA M A F AF AF D90302 M HA 316HA M HA 287HA 180HA 178HA 179HA 160HA M HA 174HA M25288 AY HA 167HA 182HA 175HA 173HA 162HA M H A 156HA 157HA 158HA 151HA 154HA 153HA 155HA M HA 159HA 183H A 148HA 150HA 146HA 147HA 149HA AY AY HA 297HA AY H A V01087 A Y H A 278H A AJ AJ HA AJ H A A Y HA 276HA A Y AY A Y HA 143HA AY A Y HA AY H A AY HA 132HA 128H A 120HA 068HA 126HA 041HA 121HA 043HA 123HA 201HA 122HA 116HA 144HA 330HA M HA 092HA 241HA 079HA 240HA 239HA 237HA 236HA 231HA 232HA 078HA 228HA 229HA 268HA L HA 324HA 269HA 323HA L43917 AY AY AY AY AY AY AY A F A Y AF AF AF AF HA AY AY AY AY AY AY AY HA 202HA 207HA 088H A M HA 194HA 196HA 195HA M17735 M17736 AF AF20231 AF AF AF AF AF AF AF AF AF AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ H A AJ AJ AJ AJ AJ AJ A Y A Y AJ AY AY AY AY AJ AJ AY AY AF AF A F A F A F AF A F AF U20465 U20469 AF AF AF L43914 L43913 U20459 L43915 AF AF AF DQ H A U20461 U20462 M24457 M24458 Z12617 L HA NP ROOT 342NP 272NP A Y M NP 028NP A Y AY NP 3 20N P 3 19N P AY AB AJ A Y AF AF AY NP 29 0NP AY AY AY AF A Y NP M N P 2 96N P 317N P M NP 304N P 308 NP AY A B A Y A J A Y NP 274 NP AJ AY AY AY N P A Y AY AB AB AB AB AB AY A Y A Y A Y AY AY AJ AY DQ AY AY AY AY AY AY A Y AY A Y AY A Y AY AY AY AY A Y A Y A Y A Y A Y A Y AY AY AY AY AY A Y A Y A Y AJ A B A J AJ AJ AJ AJ AF AJ A J AF AF A F A F AF AF AF AF AF A F A F A F AF AY NP 287 NP A F A F NP 281N P AJ AJ NP 28 4NP AY AJ AJ NP AY NP DQ N P 293 NP AF NP A Y AY AY AY AY A Y AY A F A F AY A Y AY A Y AY A F AY A Y A Y A Y A Y AY AY AY A F A Y A Y A F A F A Y A Y A Y AY A Y A F A F A F AF AY A Y A Y AF AF NP 294 NP AF N P M NP 265 NP M NP M M N P M NP Z N P 34 0NP 3 60N P 335NP 33 3NP 303 NP AF NP 295NP Z M N P M M M M30767 M M30763 M M NP 345 NP AJ NP 300NP 30 9NP AF AF NP M M63780 M M21937 M M M M M A Y A Y A F A F A F AF A F A F NP 143 NP 014N P 23 7NP 01 6NP 079NP 238NP 241N P AY N P 181 NP 252NP 180 NP 17 9NP AY NP 069NP 105N P 240NP 17 3NP 209N P AY N P 0 90N P 223 NP 224NP 226 NP 10 6NP 10 7NP 13 4NP 015 NP 358 NP 35 7NP A F A F A F AF AF NP 1 72N P 04 7NP 048 NP 049N P 050N P 051 NP 25 4NP 22 7NP 260NP 11 1NP 09 2NP 23 9NP 182NP 07 7NP A Y A Y A Y AY N P 087NP 088NP 186N P 14 1NP 11 0NP 019 NP 067NP 236N P 25 7NP 160 NP M N P 004N P 0 44N P 16 6NP 104N P 255 NP 1 00N P 162N P 362 NP A F N P 1 32N P 108 NP 165 NP 20 6NP 207N P 364N P 145 NP 19 4NP 19 5NP 1 96N P 003 NP 057 NP 058N P 059N P 0 13N P 1 64N P 22 0NP AY N P 2 47N P 2 02N P 0 54N P 26 2NP 011N P 1 67N P 1 68N P 2 44N P 2 42N P 2 31N P 23 2NP 040 NP 15 5NP 1 83N P 11 9NP 102 NP 04 2NP 03 2NP 03 3NP 0 35N P 188N P 00 2NP 0 43N P 201N P 230NP 149 NP 096NP 14 6NP 147NP 363 NP 365 NP 12 3NP 023N P 1 59N P 0 52N P 0 53N P 056N P 215N P AY AY NP 09 7NP 02 1NP 022 NP 024 NP 025 NP 027 NP 030 NP 031 NP 15 0NP 343NP 070 NP 144 NP M N P 0 82N P 153 NP 154 NP 151N P 199NP 029 NP 075 NP 213N P 01 8NP 08 1NP 211NP 017 NP AY A Y NP 178N P 253N P 0 62N P 251N P 06 5NP 20 3NP 005 NP 191NP 175N P 176 NP 2 71N P 13 5NP 0 12N P 36 6NP 373NP M NP 04 1NP 14 8NP 152NP 099 NP M A Y NP 221 NP 25 0NP 131 NP 127 NP 12 8NP 076N P 12 6NP 2 14N P A Y NP 229NP 156 NP 157N P 158N P 072NP 116NP D M NP 122N P 120 NP 066N P 074NP 19 2NP 22 8NP 07 8NP M N P M N P 11 4NP 21 9NP 03 9NP 248 NP 249N P 00 9NP 01 0NP 086N P 185N P 112 NP 03 8NP 171N P 216N P 089 NP 21 7NP 218 NP 006 NP 344 NP M M M NP 26 9NP 32 4NP 316NP 32 3NP 31 5NP M NP 314 NP 312 NP 31 3NP 326N P 327NP 328 NP 329N P M N P M30753 A Y AY M M M M NP M NP 11 3NP 26 3NP 084N P 095 NP 2 61N P 205 NP 169 NP 264 NP 00 7NP NP 0.05 NA ROOT 342 NA 268 NA 3 12N A L N A 068 NA A Y L N A 282 NA A Y A Y A Y A Y A Y NA L NA 010 NA 038N A L NA 199N A 041N A 121N A 06 5NA A Y L AY A Y N A 143 NA A Y NA 1 80N A 17 9NA A Y NA 1 54NA 0 42NA AY NA L L A J AY L L06573 L N A 141N A 06 0NA AY NA 2 60N A 064N A 08 3NA 25 7NA 089N A 216N A 091N A 25 5NA 00 4NA 12 8NA 1 24NA 1 03N A 0 23N A 192N A 08 2NA 318N A M AY N A 259 NA AY NA 19 0NA 15 9NA 051 NA 18 8NA 145 NA AY A Y A Y NA A Y A J A J A Y A F A Y A F A Y AY AY AY AY AY AF A Y A Y A Y A Y AY AY A Y A Y A Y A Y A Y AY A Y A Y A Y A F A F A Y A Y A Y A Y A Y A Y A F A F A Y A F A F AY A F A F N A 340N A A Y A Y NA A J K A Y AJ AJ NA AY AJ N A AJ NA A Y NA A Y AF NA A F NA A Y NA 090 NA 111 NA AY NA 213N A 23 6NA AY NA 076N A 127N A 126N A 256N A A Y AF A F NA A Y N A 10 0NA 0 46N A 10 2NA AY AY N A 183N A 0 66N A 1 44N A 3 43N A AF NA 09 7NA 32 8NA AY N A A Y N A 249N A 093 NA 2 18N A A Y A Y A Y A Y A Y A Y A Y AY A Y AY AY AY N A A Y NA A Y A Y A Y A Y A Y A Y NA 018N A AY AY N A AY A Y NA A Y NA A Y A Y NA 057NA 058NA 059NA 054NA 005NA 086N A 185N A 203N A 202N A AY A Y NA 194NA 195NA A Y NA A Y A Y NA AY N A 22 9NA 150 NA A Y A Y N A 2 77N A A Y AY A Y A Y AY A Y A Y A Y AY N A AY NA 327NA A Y NA A Y A Y AY AY A Y A Y A F NA 301N A 295 NA A F A F N A AY NA A F A F AF A F A Y A Y A Y A Y A Y A Y A Y A Y A F A F A F A Y AY A Y A Y A Y A Y A Y A Y A Y AY AF N A 293N A D Q NA 28 9NA A F AF NA 290 NA A F A F AJ AF A F A F A F A B AB A F AF AF A F A F AF AY AY AY AY AY AY AY AY A F NA DQ A F AF AY AF A Y A Y A Y A Y AY A Y N A 247N A 3 73NA 007 NA 095 NA 20 5NA 05 2NA 05 3NA 05 5NA 05 6NA 21 5NA 072 NA 116 NA 028N A 025N A 026N A 021N A 024N A 027N A 155N A 184N A 157N A 158N A 156N A 1 23NA 3 63N A 0 40N A 16 2NA 366N A 358N A 35 7NA A Y A Y A Y A Y A Y A Y A Y A Y A Y A Y A Y A Y NA 25 0NA A Y N A 107N A 134N A 22 0NA A Y AY A Y A Y A Y A Y A Y M M N A 044N A 045N A 273 NA 274 NA 080NA 002 NA 003NA 161NA M N A 242NA 09 4NA 254 NA 016 NA AY AY N A 08 1NA 142N A 269NA 324NA 323NA 296N A 36 0NA 16 0NA 07 0NA 149NA 096N A AY NA 147NA 271 NA AY NA 163 NA 182 NA AY AY AY AY N A AY NA AY N A AY AY N A 175N A 176N A 164N A 2 31N A 2 32N A A Y NA 1 53N A 122 NA 1 66N A 36 4NA 0 47N A 0 48N A 0 49N A 0 50N A A Y NA 152NA 316N A AY N A A Y A Y NA 168 NA 114 NA 219 NA 264 NA 169 NA A Y A Y A Y N A 278N A 281N A 2 80N A 2 86NA 2 87NA A Y A Y N A 300N A 2 99N A 31 0NA 079 NA 087N A 241N A 186NA 239N A 240N A 092NA 237N A 006 NA 011 NA 262 NA 078 NA 228 NA 233NA 207NA 234 NA 2 06N A M A Y A Y A J AY AY NA 0.2 M NS M RO OT 342M AF AF A F AF AF AF AF AF AF AF AJ AY AY A Y AJ A F M 084M 007M 095M 094M 113M 263M 169M 205M 361M M63539 M M63538 AY AY AY A Y A Y AF AY M AY M AY49713 A Y30967 AY A Y M AY AY AY M 092M AY M AY AF AF AF AF AF AF AF AF AF AF AF AF AF AF AF AF M 253M 181M AF M AY M AF AF M AY AY M 077M 079M 143M 182M 019M 252M 062M AY M 087M 088M 186M 241M AY A Y30969 AY AY M 142M AY M AY AY AY M AY M A Y M AF AF AF M 111M AY AY A Y AY AY M 069M 237M AY AY AY AY63313 AY A Y M 206M 190M 257M 164M AY M 171M 201M 103M 271M 154M 188M 365M 120M 119M 183M AF M 16M 072M 078M 228M 229M 097M 118M 096M 146M 147M 149M 150M 373M M M 023M 029M A F M 180M 233M 100M 166M 189M 230M 255M 074M 093M 247M A Y63237 A Y63325 A Y63381 AY M 018M AY A Y63269 AY M 020M 081M 250M 251M AY M 054M 132M 131M 220M AY M 135M 160M 168M 165M 203M 159M 176M 175M 140M 191M 057M 058M 059M 047M 048M 049M 050M 051M 364M 032M 033M 034M 042M 052M 053M 055M 056M 106M 107M 215M AY AY AY AY AY M M M 037M AF AF AY AY AY A Y AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY AY A Y AY AY A Y AF AF AF AF AY AY AY A Y AY AY AF M AF AY AY AY AY AY AY AY AY AY M 221M 22M AY AY M AF AF A F AF AF AF A F AF M 227M 090M 060M 067M 172M 177M AF M 259M 083M 128M 242M 202M 231M 232M 234M 091M M M 022M 024M 026M 027M 030M 031M 025M 244M 076M 126M 127M 214M 256M AY M 082M 122M 102M 046M 117M AF M 013M 045M 04M 184M 362M 163M 187M 363M 003M 085M 161M 152M M A F AF M AY AY AY AY AY AY M 043M 153M 148M 115M 194M 195M 197M AF M 15M 001M 06M 144M 070M 366M 124M 041M 121M 068M 080M 099M 192M 156M 158M 157M 162M 319M 320M 321M M38299 L3797 M L37796 L37795 M23917 M55474 M55475 Z M 325M M M 303M 305M 297M 298M Z M 345M AF AF AF AF AY AY AY AY AY AY AF M 275M A F AY AY AY AY AY AY AY AY AY AY AY AF AF AF AF M 291M AF AF AF AF A F AF AY M AF AF AF AF AY M AY AF AF M AF AF AF A J AB AB AF AF AJ AJ AF AF A F AF AY AY AJ AF AJ AJ AJ A J AJ AY M 328M 30M 329M AJ U M 296M AF AF AF M 300M 307M 306M 309M 301M 308M U49119 AF25048 AF AJ M 274M A Y AY AF AY M AJ A J AJ M 339M AJ M AJ AY M M 266M M M 314M 269M 323M 324M 316M 327M 313M 312M 315M 304M AF M L M 011M 006M 008M 010M 249M 039M 086M 089M 245M 216M 218M 009M 112M 038M 114M 219M 248M 217M 310M AY AY AY A Y AY A Y AF AY AY AY AY AY A Y DQ DQ DQ AY AY AY AY AY AY AY AY AY AY AY A Y AY AY AM AY AY AY AY AY AY AY AY AY AY DQ AY AB16865 A B A Y AY AB AB AB AY AY A Y AY AY DQ DQ A Y AY DQ DQ DQ DQ DQ DQ DQ DQ DQ DQ DQ DQ DQ DQ DQ AY AY AY AY AY A Y AY AY AY AY AY AY AY AY A Y AY AY AY AY AY AY AY AY AY AY AY AY AY AB AY AY AY AY AY AY AF AY AF AF AY AY AF AF AF AF AF A F AY AY AF AF AF AY AY AY05951 AY AY AY AY A F AF AF AF AF AF AF AF AF A F AF AF AY A F AY A F AF AY AF AF AY AY AY AY AY AY AF A Y AY A Y AF14306 AF M 278M AF AF A Y M 333M 360M Z26860 M M A F M 294M U49117 AJ AY AY AY A Y AY AY AY A Y AY AY AY AY AF AF20378 AF AF AY AY NS ROOT 342NS AY AY AY AY AY AY A Y A Y AY AY A Y AY A Y AY AY AY AY AY A Y A Y A Y A Y AY AY A Y AF AF AY AY AY A Y A Y A F AF AF AY AY AY U NS 140N S 175NS AY NS 111 NS 081N S AY NS 250NS 25 1NS 163N S A Y AY AY A Y A Y A Y A Y AY AY A Y A Y AY NS AY AY AY AY U85387 U85388 AF AY AY AY A Y U AY AY N S AY AY AY A Y A Y AY AY AY A Y A Y AY AY AY AY AY AY AY U85389 U85390 A Y A Y AF AY AY AY AY AY A Y U A F A F AF AF AF AF AF AF AF AY AY NS AY NS 239N S AY NS 067NS AF AF AF A F AF A F AF AF A F AY AY AY AY AY AY AY AY AY AY AY NS 075N S 213N S 1 77NS 1 41NS 242N S A Y NS AY AY N S A Y NS AY NS 088N S 176NS AY NS 162N S 19 0NS U N S 202NS 110NS 1 48NS 243N S 363NS U NS 131NS 132 NS M NS M N S 1 23NS 201NS M NS 102NS 125NS 115N S 144NS 153N S 068N S 146NS 147NS 066NS 070NS 07 8NS 22 8NS 0 82NS J N S 154N S 198NS U N S U NS X66524 X U85378 A Y M55468 AY A Y A Y A Y A Y A Y AY AY A F AY AY AY AY A Y AY AY A F AF AF AF U N S 35 9NS 340NS 33 9NS A F AF NS 33 0NS U49494 U49495 A F AF NS 303NS 301NS 297NS 072NS 116NS M25371 M NS M55467 AF M NS 094NS 169NS 264NS 263NS 113NS 20 5NS 26 1NS 09 5NS 007NS M80956 M M U U96743 M U U NS L L L37800 V01101 AF AJ AB AB AF AJ AJ AJ A J AJ A Y AF AF AF A F A F AJ A J AF AF AF A F NS 292NS 291N S 290N S 360NS Z26864 Z U N S M AF NS AJ NS A J NS 302NS 267 NS 265 NS 332NS AY AF A F A F AF A F AJ AY AY A F AY A Y A Y AY NS 223N S AF A J AF AF A Y A Y AF AF NS AY AY A Y A Y AY AY AY AJ N S 286N S 287N S 273NS 274NS AY AY A Y NS 279NS A Y AF NS 283NS A F NS U N S AJ NS 305NS AJ NS 311N S 310NS AF NS 298NS 345NS A F NS AF AF AF AF AF A F A F N S A Y NS AY NS 253NS 092N S 077NS 259NS A Y A Y A Y AY A Y AY NS AY N S AY NS 237NS AF NS AY A Y U A F NS 224NS 225NS 254NS 182N S 181NS AF NS A F N S A Y A Y A Y NS A F NS 065N S A Y A Y NS 108NS 207NS 209N S 083NS AY NS AY NS A Y NS 191NS U NS 058NS 059NS A F NS 221N S 222N S A Y A Y NS 107NS AY A Y NS 240NS 135N S 17 3NS 174NS 003NS 161NS 247NS 365N S 076NS 127N S 214 NS A Y N S 256NS 167N S 168N S 231NS 232NS 004NS 128NS 160NS M N S 166NS 235NS A F M NS 244NS 364NS 00 1NS 012N S 203N S U NS 053NS 055NS 056NS 215NS AY AY NS M M N S 119NS 047NS 048NS 049N S 050NS 051NS 19 4NS 19 5NS 19 6NS 187N S 34 3NS 080NS 1 56NS 1 57NS 1 58NS 041NS 121NS 097N S U NS 087NS 241NS 044NS U NS 093NS 171N S U NS 010N S 249NS 1 12NS 008N S 218N S 039 NS 091NS 217NS 114NS 219NS M NS 216NS 245NS 248NS 011NS 2 62NS M N S 00 6NS 149NS 271NS U NS 042NS 124NS 362N S 002 NS 120N S M NS 150NS U N S 023 NS 1 59NS 028NS 027NS 021NS 024NS 025NS 030NS 155NS 031NS M NS 192NS 188N S 099NS 032NS 033NS 034NS 152NS 029N S U N S 043NS 268NS 313N S 314N S 312N S 269N S 322N S 323N S 324N S 315 NS 316NS 318NS 321NS 319N S 320NS M N S 346NS 348NS L NS 32 8NS 33 1NS 327NS 325NS NS plays an important role in virus biology. Identified 8 new clades in PB1, PB2, PA, and NP genes. Identified virus families that share specific combinations of genes. NS plays an important role in virus biology. Identified 8 new clades in PB1, PB2, PA, and NP genes. Identified virus families that share specific combinations of genes. HA All Avian ML tree 692 sequences 57.7% conserved H9 H8 H12 H6 H1 H2 H5 H11 H16 H4 H3 H10 H15 H7 ML tree clade/serotype amino acid signature proteotype H13 p9.1 p9.2 p8.1 p12.1 p6.1 p6.2 p6.3 p6.4 p6.5 p6.6 p1.1 p2.1 p5.1 p5.2 p5.3 p5.4 p11.1 p16.1 p13.1 p4.1 p3.1 p10.1 p15.1 p7.1 p7.2 p7.3 HA M HA M A B PB1 PB2 PA HA NP NA M NS Sorted by NS proteotype NSp1.1 NSp1.4 NSp1.5 C Sorted by HA genotype (left) and proteotype (right) Proteotyping identified genes that co-reassort (i.e. travel together). Proteotyping identified 50+ protein-protein pairs. Proteotyping identified virus families that share specific combinations of genes. Proteotyping identified proteins exhibiting compensatory mutations. Proteotyping identified genes that co-reassort (i.e. travel together). Proteotyping identified 50+ protein-protein pairs. Proteotyping identified virus families that share specific combinations of genes. Proteotyping identified proteins exhibiting compensatory mutations. >002NS MDSNTVSSFQVDCFLWHVRKRFADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGKQIVERILE EESDEALKMTIASVPASRYLTDMTLEEMSRDWFMLMPKQKVAGSLCIRMDQAIMDKNIILKANFSVIFDR LETLILLRAFTEEGAIVGEISPLPSLPGHTDEDVKNAIGVLIGGLEWNDNTVRVSETLQRFAWRSSNEDG RPPLPPKQKRKMARTIESEV >002NS MDSNTVSSFQVDCFLWHVRKRFADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGKQIVERILE EESDEALKMTIASVPASRYLTDMTLEEMSRDWFMLMPKQKVAGSLCIRMDQAIMDKNIILKANFSVIFDR LETLILLRAFTEEGAIVGEISPLPSLPGHTDEDVKNAIGVLIGGLEWNDNTVRVSETLQRFAWRSSNEDG RPPLPPKQKRKMARTIESEV High level of variability relative to other core proteins. Many examples of specific proteotype pairs (non-random reassortment and/or compensatory mutations) among core proteins. Surprising conservation of core gene combinations revealed by specific proteotype pairs. Normal function: abrogate the host cell antiviral response (IFN, NF-κB, PKR). NS1 contains a PDZ ligand Distribution of PL in 1,196 NS1 proteins Avian NS1 (ESEV) Human NS1 (RSKV) A. I II III IV B. M r V V+aNS1 M r V V+hNS1-30kDa Binding of His-tagged ans1 and hns1 to PDZ domains

A completely novel mechanism by which avian influenza interacts with cells. ans1 in combination with HA, and perhaps other genes, contributes to high virulence of H5N1 strains.

mutagenesis Identification of cell NS1 binding partners using proteomics methods.

, Large Scale Sequence Analysis of Avian Influenza Isolates Science 311:1576-1580, 2006 Hartwell Center for Bioinformatics & Biotechnology John Obenauer, Ph.D. Jackie Denson Perdeep Mehta, Ph.D. Xiaoping Su, Ph.

Webster, Ph.D., Infectious Diseases Virology Division Erich Hoffman, Ph.D. Scott Krauss Jie Zheng, Ph.D., Structural Biology Ziwei Zhang Funded by ALSAC, CCSG, US Public Health Service, and the Hartwell Foundation St.

7 A completely novel mechanism by which avian influenza interacts with cells. ans1 in combination with HA, and perhaps other genes, contributes to high virulence of H5N1 strains. Bioinformatics analysis Comparative genomics of avian and human influenza Co-evolution analyses SNP analyses Role of PDZ proteins in influenza infection Characterization of PDZ ligand by NS1 mutagenesis Identification of cell NS1 binding partners using proteomics methods. Effect of avian vs human NS proteins on cell pathways (alone and in combination with other viral proteins) Obenauer et al., Large Scale Sequence Analysis of Avian Influenza Isolates Science 311: , 2006 Hartwell Center for Bioinformatics & Biotechnology John Obenauer, Ph.D. Jackie Denson Perdeep Mehta, Ph.D. Xiaoping Su, Ph.D. Suraj Mukatira, Ph.D. David Finkelstein, Ph.D. Tony Xu, Ph.D. Jinhua Wang, Ph.D. Jing Ma, Ph.D. Yiping Fan, Ph.D. Karen Rakestraw Jianling Armstrong Stephanie Tate Caroline Aldridge Momodou Sanyang Scott Olsen Patrick Rodriquez Bob Cassell Robert G. Webster, Ph.D., Infectious Diseases Virology Division Erich Hoffman, Ph.D. Scott Krauss Jie Zheng, Ph.D., Structural Biology Ziwei Zhang Funded by ALSAC, CCSG, US Public Health Service, and the Hartwell Foundation St. Jude Director s Rounds: Jie Zheng,, PhD Structural Biology St. Jude Children s Research Hospital PDZ domains Chemical Shift Perturbation PDZ domains are one of the most commonly found protein-protein interaction domains in organisms from bacteria to humans. Typically, PDZ domains play important roles assembling signal pathway components into large supramolecular complexes. The PDZ domain is a small protein interaction module that specifically recognizes the carboxyl terminus of proteins. In our laboratory, we have studied the PDZ domain of Dishevelled, the first PDZ domain of PSD95 and the seventh PDZ domain of GRIP1. H N N H + H 15 N 15 N H 7

8 PSD95 PDZ1 / GSESEV NMR studies showed that the C-terminal peptides of NS1 proteins interact with PDZ domains at the conversional peptide binding site with different binding affinities. Future studies Functional studies - In humans, there are about 800 PDZ domains within about 400 proteins. Using computational, protein array, NMR and other biophysical approaches, we want to identify the true binding partner(s) of NS1 proteins. St. Jude Director s Rounds: Diagnostic tool - Combining mutagenesis with our structural biology studies, we would like to design super PDZ domains to recognize specific NS1 proteins with high binding affinity. Richard Webby, PhD Virology/Infectious Diseases St. Jude Children s Research Hospital H5N1 pathogenicity Viral virulence Host immunity Host genetics 8

Inactivated flu vaccines Problems with H5N1 1)

Pathogenicity Seed strains to H5N1 A polygenic

9 Inactivated flu vaccines Problems with H5N1 1) kills embryonated eggs Formulation Formulation Problems with H5N1 1) kills embryonated eggs 2) highly pathogenic to humans Formulation H5N1 Pathogenicity Seed strains to H5N1 A polygenic trait PB2 PB1 PA HA NP NA HA PQRERRRKKRGLF M NS 9

GMP Processing Rooms at St Jude C Summary Can respond rapidly to influenza threats with reverse genetics

Need to determine best use of vaccines and antivirals Influenza is not going away HA Acknowledgments End

Safety Biostatistics ARC Pathology Legal Counsel Technology Licensing Richard Webster, PhD Clayton

print and download content for personal educational use only.

10 GMP Processing Rooms at St Jude C Summary Can respond rapidly to influenza threats with reverse genetics Requires -containment facilities -access to validated cell bank -access to virus -production facilities Need to determine best use of vaccines and antivirals Influenza is not going away HA Acknowledgments End Infectious Diseases GMP folks (St Jude, LLC, and St Jude/LLC) Hartwell Center Environmental Health and Safety Biostatistics ARC Pathology Legal Counsel Technology Licensing Richard Webster, PhD Clayton Naeve,, PhD Jie Zheng,, PhD Richard Webby, PhD More medical education materials are available at: You may print and download content for personal educational use only. All material is copyrighted by the author of the content or St. Jude Children s Research Hospital. See legal terms and conditions at 10

Influenza: Ecology and Continuing Evolution

Influenza: Ecology and Continuing Evolution Robert G. Webster, PhD Division of Virology Department of Infectious Diseases St. Jude Children s s Research Hospital Influenza Virus Negative sense RNA virus