monitoring and characterizations of antiviral drug resistant...
TRANSCRIPT
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Takato Odagiri
National Influenza Center, WHO Collaborating Center for References
and Research of Influenza at National Institute of Infectious Diseases,
Tokyo, Japan
Monitoring and characterizations of antiviral drug resistant pandemic A/H1N1 influenza
viruses in Japan
WHO Meeting with NIC on Strengthening the Global Influenza Surveillance Network (GISN)30 November to 3 December 2010 Tunis, Tunisia
NAIs Drag Name Administrationroute Licensed date
Zanamivir Relenza inhaled (multi-dose) Dec, 2000
Oseltamivir Tamiflu oral (multi-dose) Feb, 2001
Peramivir Rapiacta intravenous (single-dose) Jan, 2010
Laninamivir Inavir inhaled (single-dose) Sept, 2010
Licensed NAI antiviral drugs in Japan
NH
NH2
HNH
HO
HNH3C
HH
H3C―OOH
OH
CO2H
HO
Laninamivir(active form)
NH
NH2
HNH
HO
HNH3C
HH
HOOH
OH
CO2H
HO
Zanamivir
OHHNH3CH OH
O
CH3H3C
H
OH2N
Oseltamivir(active form)
NH2
NH
HN
OH
OOH
HHN
O
Peramivir
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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2002 2003 2004 2005 2006 2007 2008 2009 2010
Supplied numbers of Oseltamivir in Japan
Data from Chugai Co.Ltd.
NA inhibitor resistance mutations generated and characterised in vitro
Neuraminidasemutation Subtype
NAI susceptibility(IC50 fold change)
Oseltamivir Zanamivir PeramivirFunctional residues
R292K H3N2 RH3N2 R (>1580) RS (2.5) RS (14)H3N2 R (>30 000) RS (13)B R (>300) RS (28) R (500)
D151E H3N2 RS (10) S (2)R152K H3N2 S (1) S (2)
B R (252) RS (4.7) R (214)R224K H3N2 R (>4000) R (>50)E276D H3N2 R (15) R (160)R371K H3N2 R (45) R (15)
Framework residuesH275Y H1N1 R (3000)
H1N1 R (247) S RH3N2 S S SH5N1 R (1672) S (2)
N294S H1N1 R(113)H5N1 R (21) RS(3)
E119V H3N2 RH3N2 R (200) SH3N2 R (18) S (0.8) S (0.7)B R (300) S (1.9) R (531)
E119D H3N2 S (1.6) R (32) S (2.2)B R (>300) R (>560) R (>1598)
E119G B RB RS (31) R (>560) R (1598)
E119A B R (>300) R (>560) R (1598)R = resistant; RS = reduced susceptibility; S = sensitive; RG = reverse genetics
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Surveillance Network to detect NAI resistants in JapanSurveillance Network to detect NAI resistants in Japan
Local Public Health Laboratories (60~70 sites)Virus isolation
Partial sequencing of NA gene(Randomly selected 5 viruses/week)
Confirmation of H275YConfirmation of H275YNAI susceptibility assayNAI susceptibility assay
NIIDNIID
Full length sequencing of NA gene
NITE
Viruses with H275Y Positive and 275H/Y mixture
Collection of clinical specimens(Approx. 10% of outpatients)
Sentinel hospitals (~500 sites)
Weekly information sharing by website for general in Japan
Information sharing with WHO
Primers, Sequence reagentsProtocol, Technical support
Chemiluminescent assay
• Complete Kit: Available– Applied Biosystems
• Substrate: NA‐star
fluorescent assay
• Complete Kit: Not available• Substrate: MUNANA– Sigma‐Aldrich
NA inhibitor susceptibility assays
NIID used
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Oseltamivir Peramivir Zanamivir Laninamivir
275H viruses 0.09±0.03 0.05±0.02 0.31±0.11 0.38±
275Y viruses 38.0±13.5 10.4±3.90 0.37±0.10 0.57±
A/H1N1pdmisolates
Mean IC50 value (nM) using NA Star
IC50 values of A/H1N1pdm viruses by NA inhibitor susceptibility assays
Sialic acid ZanamivirOseltamivir (active form)Peramivir
Modified from Shiraishi et al., Influenza 2002;3(3):201-207
Binding profiles of NAIs
Laninamivir (active form)
GlycerolCarboxyl
GuanidinoNA active site
HN
O
OH
HOOH
O
HOO
OH
OH
H2N HN
O
NH
HO
OHO
HOO
NH
O
H2N HN
O
NH
HOOH
O
HOO
NH
OH
OH
Pocket of hydrophobic
Hydrophobic
HN
O
NH2
HO
OO
H275Ymutation
NH2
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Total1.1% (76/6917)
All resistant viruses were sensitive to zanamivir
54 (71%)
11 (15%)
11(15%)
treated
untreated
prophylaxis
Cases of 275Y virus emergence
Detection of oseltamivir and peramivir resistant viruses with 275Y mutation
NAIs susceptibility assay of AH1N1pdm viruses
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Haemagglutination inhibition tests of A/H1N1pdm viruses using 0.5% TRBCs
Numbers of 275Y viruses detected/month
6,917 viruses analyzed
76 viruses with 275Y
May
Jun
July
Aug
Sept
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Jun
July
2009 2010
Viru
ses
anal
yzed
Num
ber o
f 275
Y vi
rus
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Development of TaqMan RT-PCR system to detect 275Y and 275H/Y
mixture viruses by collaboration with the principal members of local PHLs
Objectives
To simplify the first screening of 275Y viruses at local public health labs, TaqMan RT-PCR system was developed. By collaboration, two-way communication between local PHLs and NIID can be retained.
Benefits: Establish a relationship of mutual trust between local PHLs and NIID Bottom up of level of technical and scientific knowlege of staff in local Labs Smooth control of all local PHLs by NIID Prompt responsibility and feedback by local PHLs
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Yamagata pref
Fukuoka pref
Principal membersSupport member for principal Labs
Yokohama city
Toyama pref
Sakai city
Okinawa pref
Tokyo
Aichi pref(Chief of Local side)
PHL in Osaka pref
Yamaguchi pref
Principal members of local PHLs for collaboration with NIID
Hokkaido pref
(12 Labs)
(26 Labs)(8 Labs)(15 Labs)(10 Labs)
(12 Labs)
Yamagata pref
Fukuoka pref
Principal membersSupport member for principal Labs
Yokohama city
Toyama pref
Sakai city
Okinawa pref
Tokyo
Aichi pref(Chief of Local side)
PHL in Osaka pref
Yamaguchi pref
Principal members of local PHLs for collaboration with NIID
Hokkaido pref
(12 Labs)
(26 Labs)(8 Labs)(15 Labs)(10 Labs)
(12 Labs)
NIID
CorePHL
CorePHL
CorePHL
CorePHL
CorePHL
CorePHL
PHLs (12)in region
PHLs (26)in region
PHLs (8)in region
PHLs (15)in region
PHLs (10)in region
PHLs (12)in region
Collaboration for system development
Transfer of validated technology
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Primers
NA gene of A/H1N1pdm
690 872823
5’‐ATGTGCATGTGTAAATGGTTCTTGCTTTAC‐3’
Forward primer Reverse primer
5’‐ACACATGTGATTTCACTAGAATCAGG‐3’
Cultured virus: without RNA extraction, 90 min reaction timeClinical specimens: required RNA extraction
Development of TaqMan RT-PCR assay
274 275 276 277 278..TAT TAC TAT GAG GAA.... Y Y Y E E ..
..TAT CAC TAT GAG GAA..
.. Y H Y E E ..
FAM
VIC
5’-TACTATGAGGAAT-3’
5’-CACTATGAGGAAT-3’
MGB
MGBH275
Y275
Probes
Detection of 275Y and 275H/Y by TaqMan RT-PCR
a
b
bc
d
c
d
a: 3.16 x 10 1 TCID50/reactionb: 3.16 x 10 0 TCID50/reactionc: 3.16 x 10-1 TCID50/reactiond: 3.16 x 10-2 TCID50/reaction
a
Y275
H275
275H/Y mix
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Acknowledgement
Influenza Virus Surveillance Group (77 Local PHLs) in JapanInfluenza Virus Surveillance Group (77 Local PHLs) in Japan
Influenza Virus Research Center at NIIDInfluenza Virus Research Center at NIIDDr Emi Takashita, Dr Seiichiro Fujisaki, Dr Noriko Kishida,
Dr Hong Xu, Dr Mina Nakauchi, Dr Ikuyo Takayama, Dr Tsutomu Kageyama, Miho Ejima, Namhee Kim, Reiko Ito,
Teruko Doi, Hiromi Sugawara, Dr Masato Tashiro
Center for Pathogen Genomics at NIIDCenter for Pathogen Genomics at NIIDDr Masaru Yokoyama, Dr Kazumasa Motomura,
Aya Sato, Dr Hironori Sato
Infectious Disease Surveillance Center at NIIDInfectious Disease Surveillance Center at NIIDDr Kazuyo Yamashita
Bioresource Information Center at National Institute of TechnoloBioresource Information Center at National Institute of Technology gy and Evaluation (NITE)and Evaluation (NITE)
Dr Akio Oguchi, Dr Shuji Yamazaki, Dr Nobuyuki Fujita
Department of Pediatrics, Showa General Hospital, TokyoDepartment of Pediatrics, Showa General Hospital, TokyoDr Kunihiro Oba
Medical Corporation JinjiMedical Corporation Jinji--kai, Takahashi iin, Ibaraki, Japkai, Takahashi iin, Ibaraki, Japan an Dr Nami Konomi
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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Influenza Virus Research Center(established at April 2009)
Influenza Virus Research Center(established at April 2009)
WHO Global NIC Meeting, Hammamet, Tunisia 30 November 2010 - 3 December 2010
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