1. Update of Antiviral Resistance in Seasonal and Pandemic Influenza Viruses 3 rd NIC Meeting, Beijing, China August 18-20 th 2009 Aeron Hurt WHO Collaborating Centre for Reference and Research on Influenza, Melbourne

2. Overview Neuraminidase Inhibitors Neuraminidase Inhibitors –Emergence of oseltamivir resistance in A(H1N1) in 2008 –Resistance monitoring of pandemic H1N1 2009 viruses Adamantanes Adamantanes –Current levels of resistance in seasonal and pandemic viruses Summary Summary

3. Overview Neuraminidase Inhibitors Neuraminidase Inhibitors –Emergence of oseltamivir resistance in A(H1N1) in 2008 –Resistance monitoring of pandemic H1N1 2009 viruses Adamantanes Adamantanes –Current levels of resistance in seasonal and pandemic viruses Summary Summary

4. Influenza antiviral drugs Neuraminidase (NA) inhibitors Zanamivir and Oseltamivir (Relenza™ and Tamiflu ™) Used since 1999 Large volumes stockpiled for pandemic use Effective for influenza A and B Prior to 2007, resistance was uncommon Inhibit neuraminidase

5. Emergence of oseltamivir resistance European 2007/2008 influenza season European 2007/2008 influenza season –Oseltamivir resistant isolates first detected in France, UK and Norway in late 2007 H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance

6. Emergence of oseltamivir resistance European 2007/2008 influenza season European 2007/2008 influenza season –Oseltamivir resistant isolates first detected in France, UK and Norway in late 2007 H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance Change in IC 50 Oseltamivir  1500-fold Zanamivir No change What impact does the H274Y have on resistance?

7. Emergence of oseltamivir resistance European 2007/2008 influenza season European 2007/2008 influenza season –Oseltamivir resistant isolates first detected in France, UK and Norway in late 2007 H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance –From untreated patients - low oseltamivir usage in Europe Source : IMS Rx data Germany: 49 % France: 38 % Greece: 1 % Finland: 3 % Belgium: 7 % Austria: 2 % Other countries with negligible use

8. Emergence of oseltamivir resistance European 2007/2008 influenza season European 2007/2008 influenza season –Oseltamivir resistant isolates first detected in France, UK and Norway in late 2007 H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance –From untreated patients - low oseltamivir usage in Europe –Subsequent testing revealed spread of mutant throughout Europe

9. Emergence of oseltamivir resistance Meijer et. al., EID, 15 (4), 2009

10. Emergence of oseltamivir resistance European 2007/2008 influenza season European 2007/2008 influenza season –Oseltamivir resistant isolates first detected in France, UK and Norway in late 2007 H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance –From untreated patients - low oseltamivir usage in Europe –Subsequent testing revealed spread of mutant throughout Europe –Variable resistance seen in different countries

11. Emergence of oseltamivir resistance 47 % 1 % Compared with < 1% resistance in previous seasons across all countries!! 67 % Meijer et. al., EID, 15 (4), 2009

12. Emergence of oseltamivir resistance European 2007/2008 influenza season European 2007/2008 influenza season –Oseltamivir resistant isolates first detected in France, UK and Norway in late 2007 H274Y mutation in NA responsible for the resistanceH274Y mutation in NA responsible for the resistance –From untreated patients - low oseltamivir usage in Europe –Subsequent testing revealed spread of mutant throughout Europe –Variable resistance seen in different countries –“Fit” oseltamivir resistant virus circulating not driven by drug usage USA 2007/2008 influenza season USA 2007/2008 influenza season –Oseltamivir resistant viruses being detected

13. 25% 16% ?

14. 25% 16% WHO CC Melbourne received H1 viruses from these countries during 2008 season

15. % Resistance in A(H1N1) viruses in 2008 0%(n=5) 6%(n=34) 30%(n=10) 32%(n=22) 44%(n=34) 100%(n=26) 86%(n=111) 100%(n=7) 91%(n=11) 75%(n=4)

16. Emergence of H274Y mutant viruses in different countries. Australia Macau Malaysia New Zealand New Caledonia Philippines Singapore South Africa Taiwan Thailand 2007 2008 Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Oseltamivir resistant H274Y virus Oseltamivir sensitive virus

17. Emergence of H274Y mutant viruses in different countries. Australia Macau Malaysia New Zealand New Caledonia Philippines Singapore South Africa Taiwan Thailand 2007 2008 Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Predominantly H274Y mutants

18. Meijer et. al., EID, 15 (4), 2009 20 24 28 32 36 40 44 48 52 90100 = prevalence of oseltamivir- resistant A(H1N1) H274Y mutants in Oceania, South East Asia and South Africa Speed of H274Y global spread From 0 to 100% in one year!

19. ..and now in 2009… Australia 97% (n=33) Thailand 100% (n=18) Singapore 100% (n=17) Philippines 100% (n=9) Fiji 100% (n=7) New Zealand 100% (n=6) Tahiti 100% (n=1) Virtually 100% of seasonal A(H1N1) viruses are oseltamivir resistant Virtually 100% of seasonal A(H1N1) viruses are oseltamivir resistant

20. Regional publication – WHO CC and NIC collaboration It was great opportunity for an NIC / CC collaboration and co-publication – 27 authors!!!

21. M N P N Q K…………………N A P N F H Y E 1 2 3 4 5 6 ………………………270 271 272 273 274 275 276 277 N1 amino acid sequence H = Oseltamivir Sensitive Y = Oseltamivir Resistant Question: If this is residue 275 why do people refer to the mutation as H274Y? Answer: Because they are referring to the residue based on N2 numbering H274Y or H275Y ?

22. M N P N Q K…………………N A P N F H Y E 1 2 3 4 5 6 ………………………270 271 272 273 274 275 276 277 N1 amino acid sequence M N P N Q K………………….... G S A Q H V E 1 2 3 4 5 6 ………………………….. 270 271 272 273 274 275 276 N2 amino acid sequence Equivalent Histidine residue H274Y or H275Y ?

23. Traditionally N1 residues have been numbered based on the equivalent residue in the N2 neuraminidase Traditionally N1 residues have been numbered based on the equivalent residue in the N2 neuraminidase However, either H274Y or H275Y are acceptable for reporting, publication, etc However, either H274Y or H275Y are acceptable for reporting, publication, etc Important that: Important that: –State which numbering system you are using eg H274Y (based on N2 numbering)eg H274Y (based on N2 numbering) –Ensure that you are looking at the correct residue!!! H274Y or H275Y ?

24. Overview Neuraminidase Inhibitors Neuraminidase Inhibitors –Emergence of oseltamivir resistance in A(H1N1) in 2008 –Resistance monitoring of pandemic H1N1 2009 viruses Adamantanes Adamantanes –Current levels of resistance in seasonal and pandemic viruses Summary Summary

25. As of 31 July, 162,000 cases confirmedAs of 31 July, 162,000 cases confirmed Unprecedented volumes of oseltamivir (and zanamivir) used to treat infected patients and prophylax contactsUnprecedented volumes of oseltamivir (and zanamivir) used to treat infected patients and prophylax contacts Only eight reported cases of oseltamivir resistanceOnly eight reported cases of oseltamivir resistance Denmark, 4 x Japan, Canada, Hong Kong, Singapore Denmark, 4 x Japan, Canada, Hong Kong, Singapore All resistant viruses contained the H274Y mutationAll resistant viruses contained the H274Y mutation Pandemic H1N1 2009

26. Only eight reported cases of oseltamivir resistanceOnly eight reported cases of oseltamivir resistance Denmark, 4 x Japan, Canada, Singapore, Hong Kong Denmark, 4 x Japan, Canada, Singapore, Hong Kong Patients under oseltamivir prophylaxis No evidence of transmission of resistant virus to contacts Patient did not receive oseltamivir Of more concern if resistant viruses are occurring in the absence of drug selective pressure Oseltamivir resistant cases Already infected? Suboptimal dose? Patient received full oseltamivir dose

27. Current resistance testing at WHO CC, Melbourne Clinical specimens (that have not been cultured) Cultured isolates NA enzyme inhibition assay (Fluorescence- based) Pyrosequencing or conventional sequencing

28. NA enzyme inhibition assay Neuraminidase enzyme inhibition assay (fluoresence- based) testing of cultured isolates Neuraminidase enzyme inhibition assay (fluoresence- based) testing of cultured isolates AprilMayJuneJulyAugTOTAL New Zealand40221027 Australia01569270111 Philippines010001 Singapore0470011 Fiji003003 TOTAL420101280153 None of the isolates tested have demonstrated resistance to either oseltamivir or zanamivir

29. Genetic analysis Conventional sequencing Conventional sequencing Pyrosequencing Pyrosequencing –Mutation detection H274YH274Y –Rapid: following PCR, can analyse 96 samples in less than one hour –Dr Yi-Mo Deng, WHO CC, Melb None of the clinical specimens tested have contained the H274Y mutation

30. Monitoring for H274Y mutation Advantages Advantages –Most likely resistance mutation to arise in N1 under oseltamivir pressure –Quick, most labs have PCR / sequencing expertise Disadvantages Disadvantages –Other oseltamivir or zanamivir mutations which can confer resistance may be missed Just because it has a H @ 274, doesn’t mean that it is sensitive to oseltamivir!Just because it has a H @ 274, doesn’t mean that it is sensitive to oseltamivir! –New strain unsure of which other resistance mutations may occur (eg N294S in H5N1)

31. Overview Neuraminidase Inhibitors Neuraminidase Inhibitors –Emergence of oseltamivir resistance in A(H1N1) in 2008 –Resistance monitoring of pandemic H1N1 2009 viruses Adamantanes Adamantanes –Current levels of resistance in seasonal and pandemic viruses Summary Summary

32. Influenza antiviral drugs Adamantanes Inhibit M2 channel protein Amantadine and rimantadine (Symmetrel ™ and Flumadine ™) Used since 1967

33. Influenza antiviral drugs Adamantanes Inhibit M2 channel protein Amantadine and rimantadine (Symmetrel ™ and Flumadine ™) Used since 1967 Effective only for influenza A Rapidly select for resistance

34. Adamantane resistance A(H3N2) Overall – Australasia and South East Asia Overall – Australasia and South East Asia

35. Adamantane resistance A(H3N2) A(H1N1) seasonal A(H1N1) pandemic 2009 Overall – Australasia and South East Asia Overall – Australasia and South East Asia

36. Overview Neuraminidase Inhibitors Neuraminidase Inhibitors –Emergence of oseltamivir resistance in A(H1N1) in 2008 –Resistance monitoring of pandemic H1N1 2009 viruses Adamantanes Adamantanes –Current levels of resistance in seasonal and pandemic viruses Summary Summary

37. Summary Virtually 100% of current seasonal A(H1N1) viruses are oseltamivir resistant Virtually 100% of current seasonal A(H1N1) viruses are oseltamivir resistant However the number of seasonal A(H1N1) viruses circulating has decreased significantly since the emergence of pandemic A(H1N1) However the number of seasonal A(H1N1) viruses circulating has decreased significantly since the emergence of pandemic A(H1N1) –Will the seasonal A(H1N1) continue to circulate?

38. Summary Virtually 100% of current seasonal A(H1N1) viruses are oseltamivir resistant Virtually 100% of current seasonal A(H1N1) viruses are oseltamivir resistant However the number of seasonal A(H1N1) viruses circulating has decreased significantly since the emergence of pandemic A(H1N1) However the number of seasonal A(H1N1) viruses circulating has decreased significantly since the emergence of pandemic A(H1N1) –Will the seasonal A(H1N1) continue to circulate? 20 July27 July3 August10 August A(H1N1) pandemic120747535 A(H3N2)7930 A(H1N1)1020 Influenza B0000 Influenza specimens analysed from Victoria, Australia, by VIDRL Data kindly provided by Dr Chris Birch, VIDRL, Melbourne

39. Summary Virtually 100% of current seasonal A(H1N1) viruses are oseltamivir resistant Virtually 100% of current seasonal A(H1N1) viruses are oseltamivir resistant However the number of seasonal A(H1N1) viruses circulating has decreased significantly since the emergence of pandemic A(H1N1) However the number of seasonal A(H1N1) viruses circulating has decreased significantly since the emergence of pandemic A(H1N1) –Will the seasonal A(H1N1) continue to circulate? Large volumes of NA inhibitors have been used to treat pandemic A(H1N1) infections Large volumes of NA inhibitors have been used to treat pandemic A(H1N1) infections –Encouragingly only a few cases of resistance have been detected with no evidence of further transmission Pandemic A(H1N1) and seasonal A(H3N2) are all resistant to adamantanes Pandemic A(H1N1) and seasonal A(H3N2) are all resistant to adamantanes Continued monitoring of pandemic A(H1N1) viruses is essential, particularly in patients under treatment and their contacts Continued monitoring of pandemic A(H1N1) viruses is essential, particularly in patients under treatment and their contacts

40. WPRO / SEARO Antiviral Workshop ‘Hands-on’ laboratory based workshop ‘Hands-on’ laboratory based workshop Melbourne WHO CC, November 9-13 th 2009 Melbourne WHO CC, November 9-13 th 2009 Phenotypic assays Phenotypic assays –Fluorescence-based –Chemiluminescence-based Genetic assays Genetic assays –Real-time PCR Information to get testing established Information to get testing established –SOPs, Sourcing antivirals, control viruses

41. Submission of influenza isolates and specimens Thank you to all WHO National Influenza Centres and other submitting laboratories for the provision of influenza isolates and epidemiological data WHO CC Influenza, Melb staff Jo Ernest – NA inhibition assays Yi-Mo Deng – Pyrosequencing (rapid confirmation of mutations) Pina Iannello and Naomi Komadina – Conventional sequencing Robert Shaw and Helen Sjogren – Culture of viruses The WHO Collaborating Centre for Reference and Research on Influenza, Melbourne is supported by the Australian Government Department of Health and Ageing Acknowledgements