monitoring and characterizations of antiviral drug resistant...

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


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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


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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


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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


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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


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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


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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


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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


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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


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Influenza Virus Research Center(established at April 2009)

Influenza Virus Research Center(established at April 2009)


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monitoring and characterizations of antiviral drug resistant...

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