Variant swine H3N2 and novel reassortant H3N1 viruses with A(H1N1)pdm09-like segments, Republic of Korea

Spread of North American-like TRSw and European swine influenza viruses in Asia?

H1 ph1n1 2009 virus Human infection Sw-like H1 virus TRSw H1N2 virus in Korea TRSw H1N1 virus in Korea Human seasonal H1N1 virus 2 A/South Carolina/1/18 A/BrevigMission/1/18 A/Puerto Rico/8/34 A/Wisconsin/301/76 A/New Jersey/8/76 A/Sw/Nebraska/1/92 A/Sw/Ontario/11112/04 A/Sw/Ontario/23866/04 A/Sw/Ontario/53518/03 A/Maryland/12/91 A/Sw/North Carolina/46203/03 A/Sw/Korea/CY02/02* A/Sw/Colorado/17871/01* A/Sw/Minnesota/45319/99* A/Sw/Missouri/1877/01* A/Sw/Korea/CAS08/05 A/Sw/Korea/CAN01/04 A/Iowa/01/06 A/Sw/Iowa/00239/04 A/Iowa/CEID23/05 A/Sw/Minnesota/34893/01* A/California/04/09 A/Sw/Korea/CJ1/09 A/Sw/Korea/SCJ1/09 A/Sw/Norway/3206/09 A/Hong Kong/2369/09 A/Sw/Kansas/13481-S/00* A/Sw/Arkansas/12491/00* A/Sw/Nebraska/18339/01* A/Sw/Hong Kong/915/04* A/Sw/Hong Kong/1562/05* A/Sw/Hong Kong/NS623/02* A/Sw/Hong Kong/NS30/04* A/Sw/Hong Kong/1578/03* A/Sw/Guangxi/17/05* A/Sw/Hong Kong/823/06* A/Wisconsin/10/98 A/Sw/Hong Kong/78/03* A/Sw/Indiana/9K035/99* A/Sw/Illinois/085A/01* A/Wisconsin/87/05 A/Sw/Ohio/C62006/06 A/Ohio/2/07 A/Ohio/1/07 A/Sw/Ohio/24366/07 A/Sw/Minnesota/16419/01* A/Sw/Korea/1130/09* A/Sw/Korea/S15/06* A/Sw/Korea/JL01/05* A/Sw/Korea/Asan04/06* A/Sw/Korea/HS2/04* A/Sw/Korea/JL04/05* A/Sw/Korea/JL02/05* A/Sw/Korea/CY08/07* A/Sw/Korea/1204/09* Sw/Korea/PZ4/06* A/Sw/Korea/PZ11/06* A/Sw/Korea/PZ14/06* A/Michigan/9/07 A/Brisbane/59/07 A/Sw/Illinois/00685/05 A/Sw/Minnesota/07002083/07 A/Sw/Iowa/02039/08* A/Sw/Texas/008648/08* A/Sw/Ontario/52156/03* A/Sw/Illinois/07003243/07* A/Sw/Minnesota/SG-00239/07* α-h1 cluster β-h1 cluster ɣ -H1 cluster δ -H1 cluster Human Seasonal-like Classical Swine-like A/Sw/Korea/1130/09 A/Sw/Korea/1204/09

H3 2 70 73 A/Sw/Changhua/72-10/03 A/Sw/Chiai/77-10/01 A/Sw/ON/41848/97 A/Nagasaki/97/95 A/Finland/339/95 99 A/Sw/NC/35922/98 A/Sw/Korea/CAS09/06 A/Sw/Korea/CAS07/05 A/Sw/Korea/CAN04/05 A/Sw/TX/4199-2/98 A/Sw/NC/16497/99 A/Sw/IA/569/99 A/Sw/NE/209/98 A/Sw/MN/593/99 A/Sw/IA/533/99 A/New York/622/96 A/New York/647/95 A/Sw/Korea/CY04/07 A/Sw/Korea/CY05/07 A/Sw/Korea/CY09/07 A/Wuhan/359/95 A/New York/521/98 A/Sw/Korea/PZ72-1/06 A/Sydney/5/97 A/Memphis/98 99 A/Sw/CO/23619/99 93 99 A/Paris/896/97 A/Texas/9/96 85 98 96 A/Panama/2007/99 A/Sw/Korea/CAS05/04 A/Canterbury/01/05 A/Brisbane/3/05 A/Wisconsin/67/05 A/Aichi/133/05 A/Sw/Korea/CY07/07 A/Sw/Korea/CY10/07 A/Sw/IL/21587/99 A/Sw/WI/14094/99 A/Sw/OK/18717/99 A/Sw/OK/18089/99 A/Sw/Korea/JNS06/04 A/Sw/NC/29974/03 A/Sw/MN/22057/02 A/Tk/MN/674-2/03 A/Tk/NC/12344/03 Sero-group I (US H3N2) A/Sw/Korea/CY05/07 Sero-group II (US H3N2) A/Sw/Korea/CY07/07 Sero-group III (US H3N2)

Copyright 2013. Chungbuk National University. All rights reserved. Swine influenza viruses in Korea Spanish Flu (H1N1) Hong Kong Flu (H3N2) Asian Flu (H2N2) HuSw H3N2 CSw H1N1 TRSw H1N2 TRSw H3N2 TRSw H1N1 A(H1N1)pdm09???? 18 57 68 96 98 01 02 03 04 2005 06 07 08 09 11 12 13 1900 2000 2010 2 Av H11N6 H7N2 Av H9N2 RA H3N1 TRSw H3N2 Av H5N2 RA H1N1 RA H1N2 RA H1N2 Appearance of strain with changes in pre-existing ones; violet entries designate isolation of virus strains atypical in swine.???

Genotypes of identified reassortant H3 swine influenza viruses. Isolate Subtype No. of virus Genotype HA NA PB2 PB1 PA NP M NS Stock titers (TCID 50 /ml) MLD 50 Sw/Korea/CY02-07/12 H3N1 1 H3 ph1n1 ph1n1 TRSw ph1n1 TRSw ph1n1 TRSw 6.30E+07 > 6.0 Sw/Korea/CY02-08/12 H3N1 1 H3 ph1n1 ph1n1 TRSw ph1n1 ph1n1 ph1n1 ph1n1 6.30E+07 > 6.0 Sw/Korea/CY03-12/12 H3N1 1 H3 ph1n1 ph1n1 ph1n1 ph1n1 ph1n1 ph1n1 ph1n1 1.00E+08 > 6.0 Sw/Korea/CY03-13/12 H3N1 1 H3 ph1n1 ph1n1 TRSw ph1n1 ph1n1 ph1n1 TRSw 4.00E+07 > 6.0 Sw/Korea/CY02-10/12 H3N2 8 H3 N2 TRSw TRSw TRSw TRSw ph1n1 TRSw 2.00E+06 > 6.0 Shaded segments indicate TRSw H3N2 virus origin. The MLD50 (expressed as log 10 TCID 50 ) was determined by inoculating groups of 10 mice with 10 5 TCID50 of virus in 30 µl of sterile PBS. H1N1p TRS H3N2 CY02-07 CY02-08 CY03-12 CY03-13 CY02-10

Virus titers (log 10 TCID 50 /0.1 ml) Growth properties in cell lines 8 MDCK 8 HTBE 8 A549 7 7 7 6 6 6 5 5 5 4 3 4 3 * 4 3 2 2 2 1 1 1 0 0 0 CY02-07 CY02-08 CY03-12 CY03-13 CY02-10

Virus titers (log 10 TCID 50 /g) Survival rate (%) Virulence in mice 7 6 5 4 3 2 1 0 Day post-inoculation Day post-inoculation CY02-07 CY02-08 CY03-12 CY03-13 CY02-10

Experimental set-up in ferrets Intranasal and intra-tracheal inoculation with 10 5.5 TCID 50 /ml of respective viruses.

Virus titers (log 10 TCID 50 /0.1 ml) Virus titers (log 10 TCID 50 /0.1 ml) Ferret to ferret transmission Sw/Korea/CY02-07/12 (rh3n1p) Sw/Korea/CY02-08/12 (rh3n1p) Sw/Korea/CY03-12/12 (rh3n1p) CY02-07 CY02-08 CY03-12 Days post-infection/exposure Days post-infection/exposure Days post-infection/exposure Sw/Korea/CY03-13/12 (rh3n1p) CY03-13 Sw/Korea/CY02-10/12 (rh3n2p) CY02-10 Days post-infection/exposure Days post-infection/exposure

Virus titers (log 10 TCID 50 /g) Viral titers in tissues CY02-07 CY02-08 CY03-12 CY03-13 CY02-10

Evaluation of clinical disease in experimentally inoculated ferrets. Virus exposure No. with sneezin g No. with weight l oss (%)* No. with fev er ( o C) No. with virus detec tion ( log 10 TCID 50 /ml) No. with serocon version Sw/Korea/CY02-07/12 Inoculated 2/2 2/2 (8.5) 2/2 (2) 2/2 (6.4) 2/2 (2560) RD contact 2/2 2/2 (5.7) 2/2 (1.2) 2/2 (5.5) 2/2 (2560) Sw/Korea/CY02-08/12 Inoculated 2/2 2/2 (7.3 2/2 (1.6) 2/2 (5.9) CY03-12 2/2 (2560) RD contact 0/2 2/2 (1.8) 2/2 (2.2) 0/2 2/2 (320) Sw/Korea/CY03-12/12 Inoculated 1/2 2/2 (6.2) 2/2 (1.7) 2/2 (5.4) 2/2 (1280) RD contact 0/2 0/2 1/2(0.8) 0/2 0/2 Sw/Korea/CY03-13/12 Inoculated 2/2 2/2 (7.9) 2/2 (1.9) 2/2 (5.8) 2/2 (2560) RD contact 2/2 2/2 2/2 (1) 1/2 (5.3) 2/2 (1920) Sw/Korea/CY02-10/12 Inoculated 2/2 2/2 (6.8) 2/2 (1.2) 2/2 (4.2) 2/2 (640) RD contact 2/2 2/2 (2.1) 2/2 (1) 2/2 (4.8) 2/2 (480) *, mean maximum weight loss;, mean maximum temperature increase;, mean peak titer;, geometric mean HI titer.

Immunologic cross-reactivity Ferret antiserum Viruses Subtype Sw/Korea/CY02-07/12 Sw/Korea/CY02-08/12 Sw/Korea/CY03-12/12 Sw/Korea/CY03-13/12 Sw/Korea/CY02-10/12 Perth/16/0 9 California/04/2009 H1N1 <20 <20 <20 <20 <20 <20 Sw/Korea/CY02-07/12 H3N1 2560 2560 2560 2560 640 <20 Sw/Korea/CY02-08/12 H3N1 2560 2560 2560 2560 640 <20 Sw/Korea/CY03-12/12 H3N1 2560 2560 2560 2560 640 <20 Sw/Korea/CY03-13/12 H3N1 2560 2560 2560 2560 640 <20 Sw/Korea/CY02-10/12 H3N2 640 640 640 640 1280 <20 Brisbane/10/07 H3N2 <20 <20 <20 <20 <20 40 Perth/16/09 H3N2 <20 <20 <20 <20 <20 1280 Sw/Korea/CAN04/05 * H3N2 40 80 40 40 20 <20 Sw/Korea/CAS05/04 H3N2 160 160 80 80 160 <20 Sw/Korea/CY07/07 H3N2 80 80 40 40 40 <20 Human seasonal H3N2 influenza A virus; *, prototype cluster I TRSw;, prototype cluste II TRSw;, prototype cluster III TRSw.

Copyright 2013. Chungbuk National University. All rights Are reassortant H3 swine viruses still circulating in Korea? CY0423-12/13(H1N2) Surface genes: H1 Eurasian avian-like swine N2: Korean-like swine Internal genes: H3N2pM-like

Novel TRSW H1N2 in Korea Title: Characterization of the novel reassortant H1N2 swine influenza virus in South Korea and assessment of its transmissibility in ferrets Table 1. Nucleotide homology (%) between a novel H1N2 SIV (A/swine/Korea/CY0423-12/2013) and reference strain obtained from GenBank using BLAST search. SEGMENT A(H1N1)pdm09 Eurasian H1N1 Korean-like H1N2 H3N2pM-like California/07/09 Korea/SCJ01/09 SW/HongKong/199/09 SW/Jiangsu/40/11 SW/Korea/PZ4/06 SW/Korea/VDS2/09 SW/Korea/CY03-11/12 SW/Ohio/12TOSU293/12 SW/Korea/CY03-19/12 SW/Korea/D79/11 HA 70.9 71.1 97.3 97.2 70.8 70.9 70.3 39 38.4 38 NA 37.4 37.7 37.5 37.8 97.2 96.1 95.1 89.4 90.3 90.6 PB2 93.5 93.2 83.1 83.2 94.8 94.3 93.9 98 99.5 99.4 PB1 93.4 93.3 85.2 84.3 94.5 93.9 93.4 99.2 99.4 99.3 PA 93.4 93.4 84.2 83.7 94.6 93.3 93.2 99.1 99.4 99.4 NP 94.6 94.1 80.7 81.1 96.6 95.5 95.6 99 99.4 99.2 M 98.8 98.6 94.1 94.1 86 85.2 85.3 99.1 99.47 99.2 NS 94 93.6 78.6 78.2 96.8 96.7 95.6 99 99.4 99.3 Note: Highlighted in bold are the highest nucleotide homologies of reference strains with the corresponding gene of A/swine/Korea/CY0423-12/2013. *Abbreviations of viruses used as reference strains: California/07/2009, A/California/07/2009; Korea/SCJ01/2009, A/Korea/SCJ01/2009; SW/HongKong/199/2009, A/swine/HongKong/199/2009; SW/Jiangsu/40/2011, A/swine/Jiangsu/40/2011; SW/Korea/PZ4/06, A/swine/Korea/PZ4/06; SW/Korea/VDS2/2009, A/swine/Korea/VDS2/2009; SW/Korea/CY03-11/2012, A/swine/Korea/CY03-11/2012; SW/Ohio/12TOSU293/2012, A/swine/Ohio/12TOSU293/2012; SW/Korea/CY03-19/2012, A/swine/Korea/CY03-19/2012; and SW/Korea/KSB/2012, A/swine/Korea/KSB/2012.

Unique SIV Genetic Combination

H1 PHYLOGENETIC ANALYSIS A/swine/Korea/SCJ01/2009 (H1N1) 99 A/Shenzhen/24/2009 (H1N1) 80 A/Australia/59/2009 (H1N1) 9 Supported results obtained from the A/swine/Illinois/SG1141/2003 (H1N1) A/swine/North Carolina/8161/2002 (H1N1) A/swine/Korea/1130/2009 (H1N2) 82 94 A/swine/Korea/CY03-11/2012 (H1N2) A/swine/Korea/JL02/2005 (H1N2) BLAST homology A/swine/Korea/PZ4/2006 analysis (H1N2) 91 94 A/BrevigMission/1/1918 (H1N1) A/California/04/2009 (H1N1) A/swine/California/T9001707/1991 (H1N1) 70 A/swine/Korea/CY02/2002 (H1N2) A/swine/Korea/CAN01/2004 (H1N1) 99 A/swine/Indiana/00427/2004 (H1N2) A/swine/Wisconsin/8/1980 (H1N1) 72 A/swine/Ontario/23866/04 (H1N1) A/mallard/Netherlands/10-Cam/1999 (H1N1) A/mallard/Bavaria/185-8/2008 (H1N1) 96 A/duck/Shimane/188/1999 (H1N1) A/duck/Zhejiang/0224-6/2011 (H1N2) A/mallard/Marquenterre/Z237/1983 (H1N1) 96 A/swine/France/WVL3/1984 (H1N1) A/swine/Arnsberg/6554/1979 (H1N1) A/swine/Belgium/WVL2/1983 (H1N1) 98 A/swine/Netherlands/12/1985 (H1N1) A/swine/England/WVL14/1996 (H1N1) A/swine/England/453/2006 (H1N1) 95 A/swine/England/17788/2000 (H1N1) A/swine/Italy/22530/2002 (H1N2) A/swine/Cotes d'armor/1455/99 (H1N1) A/swine/Fujian/204/2007 (H1N1) A/swine/Shandong/692/2008 (H1N1) A/swine/Jiangsu/zg3/2010 (H1N1) 95 A/swine/Jiangsu/40/2011 (H1N1) 93 A/swine/Shaanxi/s2/2012 (H1N1) A/swine/Korea/CY0423-18/2013 (H1N2) A/swine/Korea/CY0423-16/2013 (H1N2) A/swine/Korea/CY0423-33/2013 (H1N2) A/swine/Korea/CY0423-10/2013 (H1N2) A/swine/Korea/CY0423-15/2013 (H1N2) A/swine/Korea/CY0423-14/2013 (H1N2) A/swine/Korea/CY0423-12./2013 (H1N2) A/swine/Korea/CY0423-37/2013 (H1N2) A/swine/Korea/CY0423-6/2013 (H1N2) 0.02 A/swine/Korea/CY0423-2/2013 (H1N2) A/swine/Korea/CY0423-25/2013 (H1N2) A/swine/Korea/CY0423-39/2013 (H1N2) A(H1N1)pdm0 North American (Classical) swine Eurasian avian Eurasian avian-like swine

N2 64 94 95 A/Lyon/2279/1995 (H3N2) A/VictOria/75/1995 (H3N2) A/South Africa/1147/96 (H3N2) A/Buenos Aires/4559/96 (H3N2) 91 99 A/swine/Mexico/Mex50/2010 (H3N2) A/swine/Korea/CY09/2007 (H3N2) A/swine/Hong Kong/1435/2009 (H1N2) 84 A/swine/Hong Kong/NS252/2009 (H1N2) A/swine/Hong Kong/1110/2006 (H1N2) 97 A/swine/Hong Kong/1111/2004 (H1N2) A/swine/Hong Kong/1578/2003 (H1N2) 72 A/swine/Korea/CY10/2007 (H3N2) A/swine/Korea/JNS06/2004 (H3N2) A/swine/Texas/4199-2/1998 (H3N2) A/swine/Ohio/891/01 (H1N2) A/swine/Indiana/00427/2004 (H1N2) A/swine/Minnesota/5763/2003 (H1N2) 93 A/swine/Korea/S5/2005 (H1N2) A/Swine/Korea/CY02/02 (H1N2) 69 94 90 A/Fujian/411/2002 (H3N2) 61 A/Perth/16/2009 (H3N2) 97 A/Brisbane/10/2007 (H3N2) 92 83 A/swine/Korea/CAS09/2006 (H3N2) A/swine/Korea/CAS09/2006 (H3N2) A/swine/Korea/CAS07/2005 (H3N2) 99 79 A/Singapore/1/1957 (H2N2) A/Hong Kong/MA/1968 (H3N2) 68 91 A/Pennsylvania/04/2012 (H3N2) A/West Virginia/17/2012 (H3N2) A/Maine/05/2011 (H3N2) A/Minnesota/10/2010 (H3N2) A/Wisconsin/67/2005 (H3N2) A/Hiroshima/52/2005 (H3N2) A/swine/Korea/PL01/2012 (H3N2) A/swine/Ohio/A01349615/2013 (H3N2) A/swine/Ohio/12TOSU293/2012 (H3N2) 97 A/swine/Indiana/A00968373/2012 (H3N2) A/Indiana/10/2011 (H3N2) A/swine/Korea/D79/2011 (H3N2)_ 81 A/swine/Korea/CY03-14/2012 (H3N2) 90 A/swine/Korea/CY02-10/2012 (H3N2) A/swine/Korea/KSB/2012 (H3N2) 77 Korea/VDS1/2010 (H1N2) huh3n2 A/swine/Korea/Asan04/2006 (H1N2) A/swine/Korea/Hongsong2/2004 (H1N2) A/swine/Korea/CY03-11/2012 (H1N2) A/swine/Korea/CY12-03/2011 (H1N2) 92 A/swine/Korea/VDS2/2009 (H1N2) A/swine/Korea/PZ4/2006 (H1N2) A/swine/Korea/CY08/2007 (H1N2) 89 A/swine/Korea/JL02/2005 (H1N2) A/swine/Korea/4940/2010 (H1N2) A/swine/Korea/4383/2009 (H1N2) A/swine/Korea/4372/2009 (H1N2) 82 A/swine/Korea/CY0423-39/2013 (H1N2) A/swine/Korea/CY0423-16/2013 (H1N2) A/swine/Korea/CY0423-37/2013 (H1N2) A/swine/Korea/CY0423-33/2013 (H1N2) A/swine/Korea/CY0423-2/2013 (H1N2) A/swine/Korea/CY0423-14/2013 (H1N2) A/swine/Korea/CY0423-15/2013 (H1N2) A/swine/Korea/CY0423-40/2013 (H1N2) A/swine/Korea/CY0423-28/2013 (H1N2) 0.01 A/swine/Korea/CY0423-25/2013 (H1N2) A/swine/Korea/CY0423-12/2013 (H1N2) A/swine/Korea/CY0423-6/2013 (H1N2) H3N2pM/H3N2v North American swine

EA avian-like swine H1 viruses have never been reported in Korea Carried out serological surveillance of pig sera from Chungcheong, Cheonbuk and Gyeonsang regions of South Korea n=371 in 2012 and n=1185 in 2013 Total: 1556

SEROPREVALENCE Seropositivity Number (%) of sera positive Subtotal 2012 2013 None 338 (91.1) 669 (56.5) 7 (64.7) A(H1N1)pdm09 24 (6.5) 126 (10.6) 150 (9.6) Korean H1N2 5 (1.3) 47 (4) 52 (3.3) EA avian-like swine H1 0 131 (11.1) 131 (8.4) A(H1N1)pdm09, Korean H1N2 4 (1.1) 23 (1.9) 27 (1.7) A(H1N1)pdm09, EA avian-like swine H1 0 157 (13.2) 157 (10.1) Korean H1N2, EA avian-like swine H1 0 6 (0.5) 6 (0.4) A(H1N1)pdm09, Korean H1N2, EA avian-like s wine H1 0 26 (2.2) 26 (1.7) Total sera positive 33 (8.9) 516 (43.5) 549 (35.3) Total sera tested 371 1185 1556

Number (%) of sera positive Seropositivity 2012 2013 Subtotal None 141 (38) 1035 (87) 1176 (76) H3N2pM 109 (29) 115 (10) 224 (14) CY05/07 16 (4) 7 (1) 23 (1) CY07/07 11 (3) 11 (1) 22 (1) H3N2pM, CY05/07 17 (5) 0 17 (1) H3N2pM, CY07/07 12 (3) 4 (0.3) 16 (1) CY05/07, CY07/07 4 (1) 0 4 (0.3) H3N2pM, CY05/07, CY07/07 61 16) 13 (1) 74 (5) Total sera positive 230 (62) 150 (13) 380 (24) Total sera tested 371 1185 1556 Abbreviations of H3N2 viruses tested: H3N2pM: A/swine/Korea/SwLu0306-4/2012 CY05/07: A/swine/Korea/CY05/2007 CY07/07: A/swine/Korea/CY07/2007

Log10 TCID50/ml CELL GROWTH KINETICS 8 Cell Growth Kinetics ** CY03-11/2012 CY0423-12/2013 6 4 2 MDCK NHBE hours post infection (hpi)

Log10 TCID50/ml Log10 TCID50/ml Nasal turbinates 6 5 4 CY03-11/2012 CY0423-12/2013 3 2 1 1 3 5 7 9 day post infection (dpi) 6 Mice Lungs CY03-11/2012 CY0423-12/2013 5 4 3 2 1 1 3 5 7 9 day post infection (dpi)

HISTOPATHOLOGY A. CY03-11/2012 400X B. CY423-12/2013 400X

PATHOGENICITY AND TRANSMISSIBILITY IN FERRETS 6.0 log 10 TCID 50 /ml

Timeline of the genotype of viruses identified in surveillance program in China Zhu et al., 2013. Curr Top Microbiol Immunol

2001 2002 2004 TR H1N2 Genotypes of swine influenza viruses detected in South Korea TR H1N1 TR H3N2 A(H1N1) pdm09 TR H1N2 reassortant H3N2pM H11N6 H7N2 Transient variants and atypical viruses Avian TR H1N2 TR H3N2 TR H1N1 Classical Swine H1 A(H1N1)pdm09 H3N2pM EA avian-like swine H1 2005 2006 H3N1 2007 2008 H5N2 H5N2 H1N1 2009 H5N2 H1N2 H1N2 H1N2 2010 H1N2 H1N2 2011 2012 H3N1 H3N1 H3N1 H3N1 2013 H1N2 2014

Concluding remarks - Overall findings underscore the possibility that influenza viruses in swine populations can become a threat to animal and human health. - Reviving concerns over the capacity of pigs to potentially create future pandemic influenza viruses, continuous and efficient surveillance among swine herds worldwide remains vital and beneficial.