Antiviral resistance of influenza viruses in Singapore

3rd Meeting of National Influenza Centres

in the Western Pacific and South-East Asia Regions

May 5-8, 2009

Background

We started HA and NA fragment sequencing from Sept 2007 to fulfill the objectives of influenza surveillance

At the mean time, we looked at particular mutations related to virulence and antiviral resistance in order to

Collect information to assist in making recommendations for antiviral usage

Provide scientific evidence for pandemic plan

Methods

Conventional Sequencing (HA and M) Pyrosequencing (HA and M, April 2009)

Markers of admantanes resistance

PositionWild type

Mutant

26 L F27 V A30 A V or T31 S N34 G E

* Alone or combined (e.g V27A and S31N);

* Confers cross-resistance to amantadine and rimantadine

Adamantanes (amantadine and rimantadine)Block the ion channel formed by M2 protein, stop infection by preventing release of viral RNA

Results of S31N in 2008

Subtype No. testedNo. of Mutant

% of Mutant

H1N1 6 1 17%

H3N2 32 32 100%

Markers of neuraminidase inhibitors NA 274 (actually position 275 in N1)

His (H) is susceptible Tyr (Y) is resistant Actually at position 255 of H5N1 genotype Z NA with 20 AA

deletion

NA 294 (actually position 295 in N1) Asn (N) is susceptible Ser (S) is partially resistant Actually at position 275 of H5N1 genotype Z NA with 20 AA

deletion

Others including: N2 E119V and R292K B: D198E or N, R152K, R371K and I222T ……..

Results of H275Y in H1N1

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total

No. tested 1 4 2 2 9 4 7 7 11 7 15 19 88

No. with H275Y 0 0 1 0 4 1 7 4 4 5 14 19 59

% with H275Y 0% 0% 50% 0% 44% 25% 100% 57% 36% 71% 93% 100% 67%

2008

2009

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total

No. tested 3 7 3 13

No. with H275Y 3 7 3 13

% with H275Y 100% 100% 100% 100%

NAI results from WHO CC

Sender Sample ID

Spec. Date

Date Received

Type Sub type

Designation Zanamivir IC50 (nM)

Oseltamivir IC50 (nM) Result

K046D/N525 18/06/2008 8/08/2008 A H1 A/SINGAPORE/227/2008 1.73 2.17 Sensitive

K157F/N475 21/05/2008 8/08/2008 A H1 A/SINGAPORE/226/2008 5.56 4.43 Sensitive

K236G/N465 8/05/2008 8/08/2008 A H1 A/SINGAPORE/225/2008 2.98 1869.00

Highly reduced sensitivity to oseltamivir

K799Z/N446 8/05/2008 8/08/2008 A H1 A/SINGAPORE/223/2008 1.20 0.89 Sensitive

K9181/N460 8/05/2008 8/08/2008 A H1 A/SINGAPORE/224/2008 4.51 1660.00

Highly reduced sensitivity to oseltamivir

P362E/N526 18/06/2008 8/08/2008 A H1 A/SINGAPORE/228/2008 2.87 1599.00

Highly reduced sensitivity to oseltamivir

Results from WHO CC Melbourne

Future plan

Pyrosequencing (collaboration with Singapore Polytechnic)

M segment HA segment

Neuraminidase inhibition assay (NAI assay)

Oseltmivir was in place already Will request pure zanamivir from GSK (MTA) Workshop in Melbourne

Acknowledgements

Influenza surveillance team, NPHL, Singapore Virology Laboratory, Singapore General Hospital Ministry of Health, Singapore Melbourne WHO CCs Singapore Polytechnic