Update of Antiviral Resistance
in Seasonal and Pandemic
Influenza Viruses
3rd NIC Meeting, Beijing, China
August 18-20th 2009
Aeron Hurt
WHO Collaborating Centre for Reference and Research on Influenza,
Melbourne
Overview

Neuraminidase Inhibitors
– Emergence of oseltamivir resistance in A(H1N1) in
2008
– Resistance monitoring of pandemic H1N1 2009
viruses

Adamantanes
– Current levels of resistance in seasonal and
pandemic viruses

Summary
Overview

Neuraminidase Inhibitors
– Emergence of oseltamivir resistance in A(H1N1) in
2008
– Resistance monitoring of pandemic H1N1 2009
viruses

Adamantanes
– Current levels of resistance in seasonal and
pandemic viruses

Summary
Influenza antiviral drugs
Neuraminidase (NA) inhibitors
Inhibit neuraminidase
Used since 1999
Zanamivir and Oseltamivir
(Relenza™ and Tamiflu ™)
Effective for influenza A and B
Large volumes stockpiled for pandemic use
Prior to 2007, resistance was uncommon
Emergence of oseltamivir resistance

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 resistance
Emergence of oseltamivir resistance

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 resistance
What impact does the H274Y have on resistance?
Change in IC50
Oseltamivir
 1500-fold
Zanamivir
No change
Emergence of oseltamivir resistance
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 resistance
– From untreated patients - low oseltamivir usage in Europe
Oseltamivir prescriptions in 2007
No. of prescriptions (millions)

6
5
Germany: 49 %
France: 38 %
Greece: 1 %
Finland: 3 %
Belgium: 7 %
Austria: 2 %
4
3
2
Other countries with
negligible use
1
0
Japan
Source : IMS Rx data
USA
Europe
Emergence of oseltamivir resistance

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 resistance
– From untreated patients - low oseltamivir usage in Europe
– Subsequent testing revealed spread of mutant throughout Europe
Emergence of oseltamivir resistance
Meijer et. al., EID, 15 (4), 2009
Emergence of oseltamivir resistance

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 resistance
– From untreated patients - low oseltamivir usage in Europe
– Subsequent testing revealed spread of mutant throughout Europe
– Variable resistance seen in different countries
Emergence of oseltamivir resistance
Meijer et. al., EID, 15 (4), 2009
1%
Compared with < 1%
resistance in previous
seasons across all countries!!
47 %
67 %
Emergence of oseltamivir resistance

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 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
– Oseltamivir resistant viruses being detected
16%
25%
?
16%
25%
WHO CC Melbourne received H1 viruses from
these countries during 2008 season
% Resistance in A(H1N1) viruses in
2008
6%
32%
(n=34)
0%
(n=5)
(n=22)
44%
(n=34)
91%
(n=11)
30%
(n=10)
100%
(n=7)
100%
(n=26)
86%
(n=111)
75%
(n=4)
Emergence of H274Y mutant viruses in different countries.
Australia
Macau
Oseltamivir sensitive virus
Malaysia
Oseltamivir resistant H274Y virus
New Caledonia
New Zealand
Philippines
Singapore
South Africa
Taiwan
Thailand
Oct
Nov Dec
2007
Jan
Feb Mar
Apr May Jun
2008
Jul
Aug Sep
Oct
Emergence of H274Y mutant viruses in different countries.
Predominantly H274Y mutants
Australia
Macau
Malaysia
New Caledonia
New Zealand
Philippines
Singapore
South Africa
Taiwan
Thailand
Oct
Nov Dec
2007
Jan
Feb Mar
Apr May Jun
2008
Jul Aug Sep Oct
Predominantly H274Y mutants
Speed of H274Y global spread
100
90
= prevalence
of oseltamivirresistant A(H1N1)
H274Y mutants in
Oceania, South
East Asia and
South Africa
20
24
28
32
36
40
From 0Meijertoet. al.,
100%
in one year!
EID, 15 (4), 2009
44
48
52
..and now in 2009…
Virtually 100% of seasonal A(H1N1) viruses are
oseltamivir resistant
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)
Regional publication –
WHO CC and NIC collaboration
It was great
opportunity for an NIC /
CC collaboration and
co-publication –
27 authors!!!
H274Y or H275Y ?
N1 amino acid sequence
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
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 ?
N1 amino acid sequence
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
Equivalent Histidine residue
N2 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
H274Y or H275Y ?



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
Important that:
– State which numbering system you are using
• eg H274Y (based on N2 numbering)
– Ensure that you are looking at the correct
residue!!!
Overview

Neuraminidase Inhibitors
– Emergence of oseltamivir resistance in A(H1N1) in
2008
– Resistance monitoring of pandemic H1N1 2009
viruses

Adamantanes
– Current levels of resistance in seasonal and
pandemic viruses

Summary
Pandemic H1N1 2009
•As of 31 July, 162,000 cases confirmed
•Unprecedented volumes of oseltamivir (and
zanamivir) used to treat infected patients and
prophylax contacts
•Only eight reported cases of oseltamivir resistance
• Denmark, 4 x Japan, Canada, Hong Kong,
Singapore
•All resistant viruses contained the H274Y
mutation
Oseltamivir resistant cases
•Only eight reported cases of oseltamivir resistance
• Denmark, 4 x Japan, Canada, Singapore, Hong Kong
Patients under
oseltamivir
prophylaxis
Patient
received full
oseltamivir
dose
Already infected?
Suboptimal dose?
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
Current resistance testing
at WHO CC, Melbourne
Cultured
isolates
Clinical specimens
(that have not been
cultured)
NA enzyme
inhibition assay
(Fluorescencebased)
Pyrosequencing
or conventional
sequencing
NA enzyme inhibition assay

Neuraminidase enzyme inhibition assay (fluoresencebased) testing of cultured isolates
April
May
June
July
Aug
TOTAL
New Zealand
4
0
22
1
0
27
Australia
0
15
69
27
0
111
Philippines
0
1
0
0
0
1
Singapore
0
4
7
0
0
11
Fiji
0
0
3
0
0
3
TOTAL
4
20
101
28
0
153
None of the isolates tested have demonstrated resistance
to either oseltamivir or zanamivir
Genetic analysis


Conventional sequencing
Pyrosequencing
– Mutation detection
•
H274Y
– 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
Monitoring for H274Y mutation

Advantages
– Most likely resistance mutation to arise in N1 under
oseltamivir pressure
– Quick, most labs have PCR / sequencing expertise

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!
– New strain unsure of which other resistance
mutations may occur (eg N294S in H5N1)
Overview

Neuraminidase Inhibitors
– Emergence of oseltamivir resistance in A(H1N1) in
2008
– Resistance monitoring of pandemic H1N1 2009
viruses

Adamantanes
– Current levels of resistance in seasonal and
pandemic viruses

Summary
Influenza antiviral drugs
Adamantanes
Inhibit M2 channel protein
Used since 1967
Amantadine and rimantadine
(Symmetrel ™ and Flumadine ™)
Influenza antiviral drugs
Adamantanes
Inhibit M2 channel protein
Used since 1967
Amantadine and rimantadine
(Symmetrel ™ and Flumadine ™)
Rapidly select for resistance
Effective only for influenza A
Adamantane resistance
• Overall – Australasia and South East Asia
100
A(H3N2)
% adamanatne resistant
90
80
70
60
50
40
30
20
10
0
2005
2006
2007
Sample date (Year)
2008
2009
Adamantane resistance
• Overall – Australasia and South East Asia
100
A(H3N2)
% adamanatne resistant
90
A(H1N1)
pandemic
2009
80
70
60
50
40
30
A(H1N1) seasonal
20
10
0
2005
2006
2007
Sample date (Year)
2008
2009
Overview

Neuraminidase Inhibitors
– Emergence of oseltamivir resistance in A(H1N1) in
2008
– Resistance monitoring of pandemic H1N1 2009
viruses

Adamantanes
– Current levels of resistance in seasonal and
pandemic viruses

Summary
Summary

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)
– Will the seasonal A(H1N1) continue to circulate?
Summary

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)
– Will the seasonal A(H1N1) continue to circulate?
Influenza specimens analysed from Victoria, Australia, by VIDRL
20 July
27 July
3 August
10 August
A(H1N1) pandemic
120
74
75
35
A(H3N2)
7
9
3
0
A(H1N1)
1
0
2
0
Influenza B
0
0
0
0
Data kindly provided by Dr Chris Birch, VIDRL, Melbourne
Summary

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)
– Will the seasonal A(H1N1) continue to circulate?

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

Continued monitoring of pandemic A(H1N1) viruses is essential,
particularly in patients under treatment and their contacts
WPRO / SEARO Antiviral Workshop





‘Hands-on’ laboratory based workshop
Melbourne WHO CC, November 9-13th 2009
Phenotypic assays
– Fluorescence-based
– Chemiluminescence-based
Genetic assays
– Real-time PCR
Information to get testing established
– SOPs, Sourcing antivirals, control viruses
Acknowledgements
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