Antiviral-resistant B viruses with a novel mutation detected by antiviral resistance surveillance in Japan
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1 6thMeeting of NICs in the WPR and SEAR May, 2012 Hanoi VN Antiviral-resistant B viruses with a novel mutation detected by antiviral resistance surveillance in Japan Masaki Imai WHO Collaborating Center for References and Researchon Influenza, National Institute of Infectious Diseases, Tokyo, Japan 1
2 Overview The level and spread of NA-inhibitor resistance in circulating strains in the 2010/11 and 2011/12 seasons in Japan NA inhibitor-resistant B viruses possessing a novel NA mutation detected in Japan 2
3 Overview The level of NA-inhibitor resistance in circulating strains in the 2010/11 and 2011/12 seasons in Japan NA-inhibitor-resistant B viruses possessing a novel mutation detected in Japan 3
4 Neuraminidase (NA) inhibitors for influenza in Japan NA inhibitors Brand name Route Oseltamivir Tamiflu Oral Zanamivir Relenza Inhalation Peramivir Rapiacta Intravenous Laninamivir Inavir Inhalation 4
5 NA inhibitor resistance monitoring in Japan 500 sentinels for infectious agent surveillance Collection of clinical specimens Local Public Health Institutes Virus isolation NIID Approximately 5-10% of total isolates Antigenic analysis of HA; Hemagglutination inhibition (HI) tests Genetic analysis of HA, NA, and M genes Detection of antiviral resistant viruses 5
6 NA inhibitor resistance monitoring in Japan For A/H1N1 viruses: Local Public Health institutes Genotypic; Screening of the H275Y mutation by TagMan RT- PCR. NIID Phenotypic; Confirmation of the screened viruses with H275Y mutation by NA inhibition assays. 5-10% of isolates selected randomly is tested. For A/H3N2 and B viruses: NIID Phenotypic; Detection of resistant viruses by NA inhibition assays (5-10% of isolates selected randomly) 6
7 Detection of 275Y and 275H/Y by TaqMan RT-PCR Fluorescence (FAM) 275Y (resistant) 275H +275Y Fluorescence (VIC) 275H (sensitive) 7
8 NA inhibitor susceptibility assays Fluorescentbased assay NA-Fluor assay kit MUNANA substrate Chemiluminescentbased assay NA-XTD assay kit NA-Star assay kit Low-priced 8
9 Isolation/Detection of influenza viruses in the season in Japan B 1124 (10%) A/H (33%) A/H1pdm (57%) 9
10 Detection of NA inhibitor-resistant viruses in the season in Japan A(H1N1)pdm09 78 / 3844 (2.0%) H275Y A(H3N2) 1 / 134 (0.7%) R292K B 0 / 146 (0%) 10
11 Isolation/Detection of influenza viruses in the season in Japan A/H1pdm09 13 (<0.1%) 11
12 Detection of NA inhibitor-resistant viruses in the season in Japan A(H1N1)pdm09 0 / 4 (0%) A(H3N2) 0 / 181 (0%) B 0 / 102 (0%) 12
13 Proportion of A(H1N1) pdm09 viruses with H275Y mutation is increasing in Japan 2008/09 0.5% (10 / 2,168) 2009/10 1.1% (69 / 6,005) 2010/11 2.0% (78 / 3,844) Clinical background of A(H1N1)pdm09 viruses with H275Y mutation in Japan Treated prophylaxed no known exposure to drug 2008/ / /11 13
14 Overview The level of NA-inhibitor resistance in circulating strains in the 2010/11 and 2011/12 seasons in Japan NA inhibitor-resistant B viruses possessing novel NA mutations detected in Japan 14
15 Detection of NA inhibitor-resistant viruses in the season in Japan A(H1N1)pdm09 78 / 3844 (2.0%) H275Y A(H3N2) 1 / 134 (0.7%) R292K B 0 / 146 (0%) 15
16 NA inhibitor-resistant B viruses were detected in Kochi prefecture in 2011 Kochi prefecture 16
17 Why did not NIID officially report the cases in Kochi prefecture? 17
18 Why did not NIID officially report the cases in Kochi prefecture? Kochi s lab; A total of 21 influenza B viruses were isolated using MDCK cells between January and June of NIID; 21 B viruses were tested in an NA inhibition assay to determine sensitivity to the NAIs (Oseltamivir/Peramivir/Zanamivir/Laninamivir). 18
19 Why did not NIID officially report the cases in Kochi prefecture? Kochi s lab; A total of 21 influenza B viruses were isolated using MDCK cells between January and June of NIID; 21 B viruses were tested in an NA inhibition assay to determine sensitivity to the NAIs (Oseltamivir/Peramivir/Zanamivir/Laninamivir). Of the 21 viruses, 4 exhibited increased IC50 to NA inhibitors 19
20 Why did not NIID officially report the cases in Kochi prefecture? We could not recover the resistant B viruses from the same original clinical specimens on MDCK cells at NIID. In addition, mutations in NA associated with NAIs resistance were not detected in the clinical specimens by pyrosequencing. 20
21 Why did NIID not officially report the cases in Kochi prefecture? We could not recover the resistant B viruses from the same original clinical specimens on MDCK cells at NIID. In addition, mutations in NA associated with NAIs resistance were not detected in the clinical specimens by pyrosequencing. The resistant B viruses were present in very low proportions in the clinical specimens and preferentially propagated only in MDCK cells at the local lab 21
22 B/KOCHI/41/2011; under investigation B/KOCHI/59/2011 B/KOCHI/60/2011; under investigation B/KOCHI/61/2011; under investigation 22
23 NA inhibitor susceptibilities of viruses isolated in MDCK cells at the local lab or NIID Virus B/KOCHI/59/2011 Virus isolation By Local lab IC50(nM)* Oseltamivir Peramivir Zanamivir Laninamivir B/KOCHI/59/2011 NIID B/Perth/211/01-197E (resistant) B/Perth/211/01-197D (sensitive)
24 NA inhibitor susceptibilities of viruses isolated in MDCK cells at the local lab or NIID Virus Virus isolation By IC50(nM)* Oseltamivir Peramivir Zanamivir Laninamivir B/KOCHI/59/2011 Local lab B/KOCHI/59/2011 NIID B/Perth/211/01-197E (resistant) B/Perth/211/01-197D (sensitive) 6.0- fold 7.5- fold fold fold
25 NA inhibitor susceptibilities of viruses isolated in MDCK cells at the local lab or NIID Virus Virus isolation By IC50(nM)* Oseltamivir Peramivir Zanamivir Laninamivir B/KOCHI/59/2011 Local lab B/KOCHI/59/2011 B/Perth/211/01-197E (resistant) B/Perth/211/01-197D (sensitive) NIID
26 NA inhibitor susceptibilities of viruses isolated in MDCK cells at the local lab or NIID Virus Virus isolation By IC50(nM)* Oseltamivir Peramivir Zanamivir Laninamivir B/KOCHI/59/2011 Local lab B/KOCHI/59/2011 NIID B/Perth/211/01-197E (resistant) B/Perth/211/01-197D (sensitive)
27 Amino acid substitution in NA of viruses isolated in the local lab or NIID Virus B/KOCHI/59/2011 B/KOCHI/59/2011 B/Perth/211/01 (resistant) B/Perth/211/01 (sensitive) Virus isolation By Local lab NIID Amino acid substitution in NA No mutations in NA that have been reported to confer resistance. D197E D197 27
28 Amino acid substitution in NA of viruses isolated in the local lab or NIID Virus Virus isolation By Amino acid substitution in NA B/KOCHI/59/2011 Local lab G140R>>>G B/KOCHI/59/2011 B/Perth/211/01 (resistant) B/Perth/211/01 (sensitive) NIID D197E D197 28
29 Amino acid substitution in NA of viruses isolated in the local lab or NIID Virus Virus isolation By Amino acid substitution in NA B/KOCHI/59/2011 Local lab G140R>>>G B/KOCHI/59/2011 NIID G140 B/Perth/211/01 (resistant) B/Perth/211/01 (sensitive) D197E D197 29
30 G140R mutation in NA is responsible for the resistance NA inhibitor susceptibilities of plaque-purified viruses Virus PP-B/KOCHI/59/11 #6 PP-B/KOCHI/59/11 #13 B/Perth/211/01 (resistant) B/Perth/211/01 (sensitive) Amino acid substitution in NA IC50(nM)* Oseltamivir Peramivir Zanamivir Laninamivir G G140R D197E D
31 G140R mutation in NA is responsible for the resistance NA inhibitor susceptibilities of plaque-purified viruses Virus PP-B/KOCHI/59/11 #6 PP-B/KOCHI/59/11 #13 B/Perth/211/01 (resistant) B/Perth/211/01 (sensitive) Amino acid substitution in NA IC50(nM)* Oseltamivir Peramivir Zanamivir Laninamivir G G140R fold fold fold fold D197E D
32 Localization of amino acid changes identified in this study on the 3D structure of the monomer of FluB NA Peramivir Top Side View View Mutations known to reduce sensitivity to NA inhibitors are shown in purple 32
33 Summary The resistant B virus, B/KOCHI/59/2011, isolated in MDCK cells at the local lab exhibited increased IC50 to NA inhibitors. The resistant B virus possessed a mutation (G140R) not previously associated with NAIs resistance. G140 is far from the NA active site and the framework. The resistant B virus possessing the G140R mutation was not recovered on MDCK cells at the NIID. The G140R mutation was not detected in the clinical specimens by pyrosequencing. 33
34 Conclusion: The resistant B virus with the G140R mutation was present in very low proportions and preferentially propagated only in MDCK cells at the local lab Comments: Proper maintenance of MDCK cell cultures is important for precision surveillance and monitoring for the emergence of antiviral resistance 34
35 Acknowledgement Influenza Virus Surveillance Group of Japan Staff of Laboratory of Influenza Virus Surveillance at WHO Collaborating Centers for Reference and Research on Influenza, Tokyo at NIID 35
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