Summary of neuraminidase amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors (NAI)*

Summary of neuraminidase amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors (NAI)* Susceptibility assessed by NA inhibition assays Source of Type/Subtype Amino acid N2 Comments substitution a numbering a (IC 50 fold change vs. wild type [NAI susceptible virus]) b viruses/ Reference Oseltamivir Zanamivir Peramivir Laninamivir Selection with c A(H1N1)pdm09 E119G 119 NI (3) HRI (832) RI (51)? d RG (52) Some IC 50 s were given for mixed virus populations; to be assessed using recombinant NA Only I427T affects IC 50?- to be assessed using recombinant NA E119V 119 RI (60) HRI (571) RI (25)? RG (52) D199G 198 RI (17) NI (6) NI (2)? RG (52) I223K 222 RI (12-39) NI (5-6) NI (1-4)? Sur (43) I223R 222 RI (28-45) RI (10-12)?? Clin/No; Clin/Ose/Zan; RG (17, 51, 63) I223V 222 NI (6) NI (2) NI (2)? RG (52) S247N 246 NI (4-8) NI (2-5) NI (1)? Sur (29) H275Y 274 HRI (221-1637) NI (1-6) RI/HRI (50-751) NI (1-2) Clin/Ose; Sur; In vitro (5, 12, 22, 32, 39, 45, 46, 48, 50, 60) N295S 294 HRI (208) NI (3) RI (12)? RG (52) D199N+H275Y 198+274 HRI (318-744) NI (2-3) HRI (108)? Clin/Ose (20, 50) I223K+H275Y 222+274 HRI (>10822) RI (11) HRI (5343) RI (11) Sur; In vitro (46) I223R+H275Y 222+274 HRI (>9100) RI (27-22) HRI (>7500) RI (17) Clin/Ose; RG (43, 46, 51) I223V+H275Y 222+274 HRI (1733) NI (2) HRI (1331)? Clin/Ose; RG (10, 52) S247N+H275Y 246+274 HRI (5880) NI (5) HRI (334)? Sur (29) Q313R+I427T 313+427 RI (10-43) NI/RI (3-20) NI (4)? Sur (22, 25) A(H1N1) E119V 119 RI/HRI (15/1727) HRI (136/2144) HRI (5050)? RG (1, 11) Host cell- selected in both studies; not found in Q136K 136 NI (1) RI/HRI (36-327) RI (75-80)? Sur (27, 49) clinical specimens This result is virus specific Y155H 155 HRI (123) HRI (555)?? Sur (41) I223M 222 NI (8) NI (1) NI (1)? RG; In vitro/zan (26) I223V 222 NI (3) NI (2) NI (1)? RG; In vitro/zan (26) S247G 246 NI (5) NI (1)?? Sur (55) H275Y 274 HRI (321-2597) NI (1-2) HRI (111-1095)? Sur; RG (11, 22, 40, 55) R293K 292 NI (1) RI (18?? RG (11) Last updated 21 October 2016 Page 1 of 9

Type/Subtype Comments Both changes were host cellselected Q136K was host cell-selected D151E/N was host cell-selected D151G was host cell-selected Q136K+D151N were host cellselected Susceptibility assessed by NA inhibition assays Source of Amino acid N2 substitution a numbering a (IC 50 fold change vs. wild type [NAI susceptible virus]) b viruses/ Reference Oseltamivir Zanamivir Peramivir Laninamivir Selection with c N295S 294 RI/HRI (40/197) NI (1/5) RI (12)? RG (1, 11) I117V+E119V 117+119 NI (7) HRI (391)?? RG (11) I117M+E119V 117+119 RI (20) HRI (181)?? RG (11) I117V+H275Y 117+274 HRI (683) NI (1)?? RG (11) I117M+H275Y 117+274 HRI (395) NI (1)?? RG (11) I117V+N295S 117+294 RI (60) NI (1)?? RG (11) I117M+N295S 117+294 RI (52) NI (4)?? RG (11) E119V+ H275Y 119+274 RI (21) HRI (132)?? RG (11) Q136K+D151E 136+151 NI (1) RI (25) RI (18)? Sur (49) Q136K+H275Y 136+274 HRI (198) RI (15) HRI (1805)? Sur (49) D151E/N+H275Y 151+274 HRI (231-799) NI (1-3) RI/HRI (80-718)? Sur (49) D151G+H275Y 151+274 HRI (1189) RI (14) HRI (1161)? Sur (49) I223M+H275Y 222+274 HRI (1943) NI (1) HRI (400)? RG; In vitro/zan (26) I223V+H275Y 222+274 HRI (584/971) NI (1/2) HRI (893)? RG; In vitro/zan (11, 26) I223V+N295S 222+294 RI (97) NI (2)?? RG (11) R293K+H275Y 292+274 HRI (207) NI (1)?? RG (11) N295S+H275Y 294+274 HRI (108) NI (1)?? RG (11) S334N+H275Y 336+274 HRI (415) NI (1)?? RG (11) I117V+N295S+H275Y 117+294+274 HRI (336) RI (12)?? RG (11) I117M+N295S+H275Y 117+294+274 HRI (121) RI (11)?? RG (11) Q136K+D151N+H275Y 136+151+274 HRI (356) NI (4) HRI (23 00)? Sur (49) I223V+N295S+H275Y 222+294+274 HRI (300) RI (13)?? RG (11) A(H5N1) V96A 116 RI (11-18) RI (10-63) NI (4)? Sur (6, 31) I97V 117 NI (5) NI (4)?? RG; Sur (33, 37) E99A 119 RI (35) HRI (1254)?? RG (33) E99G 119 NI (3-4) HRI (1345-1485) RI/HRI (12-164)? RG; In vitro/zan (26) Q116L 136 RI (26) HRI (350)?? In vivo/zan (30) V129A 149 NI (4) NI (8)?? Sur (42) D179G 198 RI (32) RI (44) NI (4)? RG; In vitro/zan (26) I203M 222 RI (36) NI (1) NI (1)? RG; In vitro/zan (26) I203V 222 NI (7) NI (1) NI (1)? RG; In vitro/zan (26) Last updated 21 October 2016 Page 2 of 9

Type/Subtype Comments Susceptibility assessed by NA inhibition assays Source of Amino acid N2 substitution a numbering a (IC 50 fold change vs. wild type [NAI susceptible virus]) b viruses/ Oseltamivir Zanamivir Peramivir Laninamivir Selection with c Reference S227N 246 RI (24) NI (2)?? Sur (6) H255Y 274 HRI (911-2502) NI (1-3) HRI (108-533) NI (6) Sur; Clin/Ose; RG; In vitro/zan (26, 33, 38, 44) N275S 294 RI/HRI (12-138) NI/RI (2-27) NI/RI/HRI (3-130)? Clin/Ose; RG (14, 33, 38) I97V+I294V 117+314 RI (16) NI (1) NI (1)? Sur (31) I203M+H255Y 222+274 HRI (8024) NI (3) HRI (3340)? RG; In vitro/zan (26) I203V+H255Y 222+274 HRI (1925) NI (2) HRI (2106)? RG; In vitro/zan (26) K130N+I203L+S227N 150+222+246 RI (77) NI (1)?? Sur (6) A(H3N2) E119D 119 NI (2) RI (32) NI (2)? RG (67) E119I 119 HRI (208) RI (17) NI (3)? Clin/Ose; In vitro (47) E119V 119 RI/HRI (18-2057) NI (1-7) NI (1-3) NI (3-4) Sur; Clin/Ose; In vitro; RG Host cell-selected, not found in clinical specimens T148I was host cell-selected (1, 34, 40, 47, 56, 61, 62, 67) Q136K 136 NI (1-7) RI/HRI (30-132)?? Sur (13, 48) D151E 151 RI (11) NI (2)?? RG (66) D151V/D 151 NI (8) HRI (164)?? Sur (55) I222L 222 NI (9) NI (2)?? RG (53) R224K 224 HRI (>4000) RI (>50)?? RG (66) Del 245-248 e 245-248 HRI (157-175) NI (3) NI (1)? Clin/Ose (2) E276D 276 RI (15) HRI (160)?? RG (66) Clin/Ose; In (1, 8, 36, 55, 62, R292K 292 HRI (>10000) NI/RI/HRI (3-134) RI/HRI (14-719)? vitro/zan; RG 65-67) N294S 294 HRI (300-1879) NI (8) NI (1)? Clin/Ose; RG (1, 36) R371K 371 RI (45) RI (15)?? RG (66) E119V+T148I 119+148 HRI (>6000) HRI (>800) HRI (>110) HRI (>250) Sur; In vitro; RG (61) E119V+I222L 119+222 HRI (1571) RI (5)?? RG (53) E119V+I222V 119+222 HRI (293-2286) NI (2) NI (7)? Clin/Ose (4, 56) A(H3N2)v S245N 245 NI (1) NI (1) NI (1) NI (1) Sur; RG (58) S247P 247 RI (42) HRI (266) NI (2) NI (9) Sur; RG (58) S245N+S247P 245+247 RI (25-40) HI/HRI (66-235) NI (1-2) NI (7-9) Sur; RG (48, 58) A(H7N9) R289K 292 HRI (>4600) RI (11-64) HRI (405-997) RI (15-33) In vitro; RG (23, 57) B E105K 110 NI (4-8) RI (15-42) HRI (213-436) NI/RI (3-12) Clin/No; RG (19) Last updated 21 October 2016 Page 3 of 9

Type/Subtype Comments CDC: IC 50 s for a mixed virus population reported Susceptibility assessed by NA inhibition assays Source of Amino acid N2 substitution a numbering a (IC 50 fold change vs. wild type [NAI susceptible virus]) b viruses/ Reference Oseltamivir Zanamivir Peramivir Laninamivir Selection with c E117A 119 RI/HRI (3171) RI/HRI (12538) HRI (13780)? Sur; RG (7, 54) E117A or D 119 HRI (>300) HRI (>560) HRI (>1598)? RG (35) E117G 119 RI (31) RI/HRI (33-560) HRI (>1598)? In vitro; RG (3, 35) E117V 119 HRI (300) NI (2) HRI (531)? RG (35) Q138R 140 NI (1) RI (15) RI (41) RI (7) Sur; In vitro (18) P139S 141 RI (10) RI (25) HRI (322) RI (6) Sur; In vitro (18) G140R 142 RI (9) RI (30) HRI (321) RI (9) Sur; In vitro (18) R150K 152 HRI (100-252) NI/RI/HRI (5-1000) HRI (214-400)? Clin; Clin/Zan; (21, 35, 40, 59, RG; Sur 66) D197E 198 RI (12-26) NI (3-7) RI (16-18)? Clin/No; Sur; RG (7, 28, 41) D197N 198 NI/RI (4-10) NI/RI (3-17) NI (5)? Clin/No; Clin/Ose; Sur (24, 34, 40, 55) D197Y 198 RI/HRI (15/57) RI (14) HRI (168)? Sur; RG (7, 16) A200A/T 201 RI (5-8) RI (5-7)?? Sur (48) I221L 222 HRI (70-121) RI (7-13)?? Clin/Ose (15) I221T 222 NI/RI (5-8) NI (2-4) RI (15-43) NI (<3) Clin/No; Sur; RG (24, 39, 41, 64){Burnham, 2014 #6313 I221V/I 222 NI (3-6) NI (1) NI (4-8)? Sur (59) A245T 246 RI (24) RI (39) RI (5) NI (3) Sur (39) H273Y 274 NI/RI (2-12) NI (1) RI/HRI (15-110)? Sur; RG (7, 50, 55) R292K 292 HRI (>300) RI (29) HRI (502)? RG (35) N294S 294 RI (17-61) NI (1-4) RI (31)? Clin/No; RG (7, 9) K360E 358-359 f NI (2) NI (2) HRI (165) NI (<3) Sur (39) R374K 371 HRI (101/407) RI/HRI (29/145) HRI (352)? Sur; RG (7, 55) A395E 390 RI (5) NI (1) RI (5) NI (<3) Sur (39) G407S 402 NI (4) NI (7)?? Clin/Ose; Sur (24) D432G 429 NI (1) NI (1) RI (41) NI (<3) Sur (39) G140R+N144K 142+146 NI (6) RI (10) HRI (257)? Sur (50) Y142H+G145R 144+147 RI (5) NI (4) HRI (487) NI (<3) Sur (39) a Numbering is based on an alignment of neuraminidases from: A/Brisbane/59/2007 [H1N1], A/California/7/2009 [H1N1pdm09], A/turkey/Turkey/1/2005 [H5N1], A/Singapore/1/57 [H2N2], A/Perth/16/2009 [H3N2, A/Hong Kong/1074/97 [H9N2], A/Anhui/1/2013 [H7N9], A/Jiangxi-Donghu/346/2013 [H10N8], B/Brisbane/60/2008 [B/Vic lineage], B/Bangladesh/3333/2007 [B/Yam lineage]. Last updated 21 October 2016 Page 4 of 9

b Assessed by NA inhibition assays chemiluminescent (NA-Star), fluorescent (MUNANA), and/or colorimetric (fetuin). NAI phenotype is shown according to the referenced studies: NI, normal inhibition; RI, reduced inhibition; HRI, highly reduced inhibition as defined in - World Health Organization. Meetings of the WHO working group on surveillance of influenza antiviral susceptibility Geneva, November 2011 and June 2012. Wkly Epidemiol Rec. 2012 Sep 28;87(39):369-74. c Source: Sur, surveillance studies; Clin, clinical detection; RG, reverse genetics; In vitro, cell-culture studies; In vivo, animal studies. Drug use: No, no NAI used; Ose, oseltamivir; Zan, zanamivir. d? indicates that the NAI(s) indicated were not studied. e Del indicated deletion of the three amino acids indicated. f Precise N2 numbering cannot be given as the influenza B NA carries an insertion in the alignment compared to the H3N2 NA. * Substitutions table developed by the WHO GISRS antiviral susceptibility expert working group (AVWG). 2014. Last updated 21 October 2016 Page 5 of 9

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