Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (4): 816-824.doi: 10.3864/j.issn.0578-1752.2022.04.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Amino Acid of 225 in the HA Protein Affects the Pathogenicities of H1N1 Subtype Swine Influenza Viruses

YANG ShiMan1(),XU ChengZhi1(),XU BangFeng1,WU YunPu2,JIA YunHui1,QIAO ChuanLing1(),CHEN HuaLan1   

  1. 1Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/State Key Laboratory of Veterinary Biotechnology, Harbin 150069
    2Laboratory Animal Center, Liaoning University of Traditional Chinese Medicine, Shenyang 110847
  • Received:2020-12-31 Accepted:2021-03-30 Online:2022-02-16 Published:2022-02-23
  • Contact: ChuanLing QIAO E-mail:ysm196033@163.com;xucz1994@163.com;qiaochuanling@caas.cn

RichHTML

31

PDF

120

Abstract:

【Objective】 The pathogenicities of influenza viruses are determined by multiple viral genes. The results of our previous study indicated that hemagglutinin (HA) gene substitutions of the two genetically similar H1N1 swine influenza viruses altered their pathogenicities in mice. This study aimed to further identify the key amino acids affecting viral pathogenicity. 【Method】 After analyzing the amino acid differences of HA protein between the two H1N1 viruses, the reassortant viruses bearing the single amino acid mutations were constructed using the site-directed mutagenesis primers, and their EID50 values were determined. To determine the growth of the parental, reassortant and mutant viruses in vitro, MDCK cells and A549 cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.001 and 0.1, respectively. The BALB/c mice was further intranasally (i.n.) inoculated with 106 EID50 of each virus, and three mice were euthanized at 3 days post-infection (dpi).The organs, including brain, nasal turbinate, lung, kidney and spleen, were collected from the mice and titrated in eggs to evaluate the viral replication abilities in vivo. The MLD50 values of the indicated viruses were determined by inoculating i.n. groups of five mice with 101-106 EID50 of viruses. The body weight was measured daily for 14 dpi, and the mice that lost more than 25% of their original weight were euthanized for humane reasons. 【Result】 The HA proteins of the ZD71 and SY130 viruses differed at four amino acids at positions 4, 138, 144, and 225 (H3 numbering). Four reassortants were rescued, followed by whole-genome sequencing to ensure the absence of unwanted mutations. The viral replication abilities of the reassortant viruses (rZD71-HA/G225E and rSY130-HA/E225G) were significantly affected in MDCK, as well as in A549 cells, when G225E and E225G substitutions were introduced into the rZD71 and rSY130 virus, respectively. In contrast, the mutations of the other three amino acids had little effect on viral replication in vitro. Further mouse infection experiments also demonstrated that amino acid substitutions at site 225 of HA protein significantly affected the viral pathogenicities in mice. In particular, the substitution G225E increased the pathogenicity of rZD71-HA/G225E virus, with the MLD50 value of rZD71-HA/G225E virus decreasing from 4.32 log10EID50 to 3.0 log10EID50, compared with that of rZD71 virus. And the virus replicated well not only in the nasal turbinate and lung, but also in the spleen and kidney. 【Conclusion】 A single amino acid at position 225 in the HA protein significantly affects the viral replication capacity and virulence of these two H1N1 swine influenza viruses. It is suggested that close monitoring for this residue should be paid in the future virological surveillance, so as to provide a scientific basis for better prevention and control of animal influenza, and even human influenza pandemic.

Key words: swine influenza virus, HA protein, amino acid, pathogenicity

Table 1

The different amino acids between the ZD71 and SY130 viruses"

病毒
Viruses		 氨基酸差异位点 Amino acid difference sites
4 138 144 225
ZD71 V A T G
SY130 A S A E

Table 2

Determination of EID50 of the site-directed mutant viruses"

拯救病毒
Rescued viruses		 氨基酸差异位点
Amino acid difference sites		 log10EID50
(mL)
4 138 144 225
rZD71 V A T G 8.38
rZD71-HA A S A E 8.68
rZD71-HA/V4A A A T G 8.83
rZD71-HA/A138S V S T G 9.17
rZD71-HA/T144A V A A G 8.83
rZD71-HA/G225E V A T E 8.63
rSY130 A S A E 8.68
rSY130-HA V A T G 8.17
rSY130-HA/A4V V S A E 8.50
rSY130-HA/S138A A A A E 7.83
rSY130-HA/A144T A S T E 8.50
rSY130-HA/E225G A S A G 8.38

Fig. 1

Growth kinetics of the viruses in MDCK cells (A, C) and A549 cells (B, D)"

Fig. 2

Viral replication titers in the organs of mice infected with viruses"

Fig. 3

Determination of MLD50 of the parental viruses, single-gene reassortants, and single-site mutants"

Fig. 4

Analysis of amino acids at position 225 in the HA protein of H1N1 subtype SIV “X”denotes amino acid D, K, or N"

[1] GAMBLIN S J, SKEHEL J J. Influenza hemagglutinin and neuraminidase membrane glycoproteins. The Journal of Biological Chemistry, 2010, 285(37):28403-28409. doi: 10.1074/jbc.R110.129809.
doi: 10.1074/jbc.R110.129809
[2] WILLE M, HOLMES E C. The ecology and evolution of influenza viruses. Cold Spring Harbor Perspectives in Medicine, 2020, 10(7):a038489. doi: 10.1101/cshperspect.a038489.
doi: 10.1101/cshperspect.a038489
[3] MA W J. Swine influenza virus: current status and challenge. Virus Research, 2020, 288:198118. doi: 10.1016/j.virusres.2020.198118.
doi: 10.1016/j.virusres.2020.198118
[4] CHEN Y, ZHANG J, QIAO C L, YANG H L, ZHANG Y, XIN X G, CHEN H L. Co-circulation of pandemic 2009 H1N1, classical swine H1N1 and avian-like swine H1N1 influenza viruses in pigs in China. Infection, Genetics and Evolution, 2013, 13:331-338. doi: 10.1016/j.meegid.2012.09.021.
doi: 10.1016/j.meegid.2012.09.021
[5] YANG H L, CHEN Y, QIAO C L, HE X J, ZHOU H, SUN Y, YIN H, MENG S S, LIU L P, ZHANG Q Y, KONG H H, GU C Y, LI C J, BU Z G, KAWAOKA Y, CHEN H L. Prevalence, genetics, and transmissibility in ferrets of Eurasian avian-like H1N1 swine influenza viruses. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(2):392-397. doi: 10.1073/pnas.1522643113.
doi: 10.1073/pnas.1522643113
[6] SUN H L, XIAO Y H, LIU J Y, WANG D Y, LI F T, WANG C X, LI C, ZHU J D, SONG J W, SUN H R, JIANG Z M, LIU L T, ZHANG X, WEI K, HOU D J, PU J, SUN Y P, TONG Q, BI Y H, CHANG K C, LIU S D, GAO G F, LIU J H. Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection. PNAS, 2020, 117(29):17204-17210. doi: 10.1073/pnas.1921186117.
doi: 10.1073/pnas.1921186117
[7] CHEN Y, TROVÃO N S, WANG G J, ZHAO W F, HE P, ZHOU H B, MO Y N, WEI Z Z, OUYANG K, HUANG W J, GARCÍA-SASTRE A, NELSON M I. Emergence and evolution of novel reassortant influenza A viruses in canines in Southern China. mBio, 2018, 9(3):e00909-18. doi: 10.1128/mBio.00909-18.
doi: 10.1128/mBio.00909-18
[8] LIU J H, LI Z H, CUI Y L, YANG H Y, SHAN H, ZHANG C M. Emergence of an Eurasian avian-like swine influenza A (H1N1) virus from mink in China. Veterinary Microbiology, 2020, 240:108509. doi: 10.1016/j.vetmic.2019.108509.
doi: 10.1016/j.vetmic.2019.108509
[9] ZHU W F, ZHANG H, XIANG X Y, ZHONG L L, YANG L, GUO J F, XIE Y R, LI F C, DENG Z H, FENG H, HUANG Y W, HU S X, XU X, ZOU X H, LI X D, BAI T, CHEN Y K, LI Z, LI J H, SHU Y L. Reassortant Eurasian avian-like influenza A(H1N1) virus from a severely ill child, Hunan Province, China, 2015. Emerging Infectious Diseases, 2016, 22(11):1930-1936. doi: 10.3201/eid2211.160181.
doi: 10.3201/eid2211.160181
[10] YANG H L, QIAO C L, TANG X, CHEN Y, XIN X G, CHEN H L. Human infection from avian-like influenza A (H1N1) viruses in pigs, China. Emerging Infectious Diseases, 2012, 18(7):1144-1146. doi: 10.3201/eid1807.120009.
doi: 10.3201/eid1807.120009
[11] RUSSELL C J. Acid-induced membrane fusion by the hemagglutinin protein and its role in influenza virus biology. Current Topics in Microbiology and Immunology, 2014, 385:93-116. doi: 10.1007/82_2014_393.
doi: 10.1007/82_2014_393
[12] STEINHAUER D A. Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology, 1999, 258(1):1-20. doi: 10.1006/viro.1999.9716.
doi: 10.1006/viro.1999.9716
[13] MATROSOVICH M, STECH J, KLENK H D. Influenza receptors, polymerase and host range. Revue Scientifique et Technique (International Office of Epizootics), 2009, 28(1):203-217. doi: 10.20506/rst.28.1.1870.
doi: 10.20506/rst.28.1.1870
[14] MATROSOVICH M, TUZIKOV A, BOVIN N, GAMBARYAN A, KLIMOV A, CASTRUCCI M R, DONATELLI I, KAWAOKA Y. Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. Journal of Virology, 2000, 74(18):8502-8512. doi: 10.1128/jvi.74.18.8502-8512.2000.
doi: 10.1128/jvi.74.18.8502-8512.2000
[15] KANNAN S, KOLANDAIVEL P. Computational studies of pandemic 1918 and 2009 H1N1 hemagglutinins bound to avian and human receptor analogs. Journal of Biomolecular Structure and Dynamics, 2016, 34(2):272-289. doi: 10.1080/07391102.2015.1027737.
doi: 10.1080/07391102.2015.1027737
[16] DAS P, LI J Y, ROYYURU A K, ZHOU R H. Free energy simulations reveal a double mutant avian H5N1 virus hemagglutinin with altered receptor binding specificity. Journal of Computational Chemistry, 2009, 30(11):1654-1663. doi: 10.1002/jcc.21274.
doi: 10.1002/jcc.21274
[17] STEVENS J, BLIXT O, GLASER L, TAUBENBERGER J K, PALESE P, PAULSON J C, WILSON I A. Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. Journal of Molecular Biology, 2006, 355(5):1143-1155. doi: 10.1016/j.jmb.2005.11.002.
doi: 10.1016/j.jmb.2005.11.002
[18] TUMPEY T M, MAINES T R, VAN HOEVEN N, GLASER L, SOLÓRZANO A, PAPPAS C, COX N J, SWAYNE D E, PALESE P, KATZ J M, GARCÍA-SASTRE A. A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science, 2007, 315(5812):655-659. doi: 10.1126/science.1136212.
doi: 10.1126/science.1136212
[19] WANG Z, YANG H L, CHEN Y, TAO S Y, LIU L L, KONG H H, MA S J, MENG F, SUZUKI Y, QIAO C L, CHEN H L. A single-amino-acid substitution at position 225 in hemagglutinin alters the transmissibility of Eurasian avian-like H1N1 swine influenza virus in Guinea pigs. Journal of Virology, 2017, 91(21):e00800-17. doi: 10.1128/JVI.00800-17.
doi: 10.1128/JVI.00800-17
[20] XU C Z, XU B F, WU Y P, YANG S M, JIA Y H, LIANG W H, YANG D W, HE L K, ZHU W F, CHEN Y, YANG H L, YU B L, WANG D Y, QIAO C L. A single amino acid at position 431 of the PB2 protein determines the virulence of H1N1 swine influenza viruses in mice. Journal of Virology, 2020, 94(8):e01930-19. doi: 10.1128/JVI.01930-19.
doi: 10.1128/JVI.01930-19
[21] ZHANG Y, ZHANG Q Y, GAO Y W, HE X J, KONG H H, JIANG Y P, GUAN Y T, XIA X Z, SHU Y L, KAWAOKA Y, BU Z G, CHEN H L. Key molecular factors in hemagglutinin and PB2 contribute to efficient transmission of the 2009 H1N1 pandemic influenza virus. Journal of Virology, 2012, 86(18):9666-9674. doi: 10.1128/JVI.00958-12.
doi: 10.1128/JVI.00958-12
[22] STEEL J, LOWEN A C, MUBAREKA S, PALESE P. Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathogens, 2009, 5(1):e1000252. doi: 10.1371/journal.ppat.1000252.
doi: 10.1371/journal.ppat.1000252
[23] LEE J, HENNINGSON J, MA J J, DUFF M, LANG Y K, LI Y H, LI Y H, NAGY A, SUNWOO S, BAWA B, YANG J M, BAI D P, RICHT J A, MA W J. Effects of PB1-F2 on the pathogenicity of H1N1 swine influenza virus in mice and pigs. The Journal of General Virology, 2017, 98(1):31-42. doi: 10.1099/jgv.0.000695.
doi: 10.1099/jgv.0.000695
[24] SUN Y P, HU Z, ZHANG X X, CHEN M Y, WANG Z, XU G L, BI Y H, TONG Q, WANG M Y, SUN H L, PU J, IQBAL M, LIU J H. An R195K mutation in the PA-X protein increases the virulence and transmission of influenza A virus in mammalian hosts. Journal of Virology, 2020, 94(11):e01817-e01819. doi: 10.1128/JVI.01817-19.
doi: 10.1128/JVI.01817-19
[25] ABED Y, PIZZORNO A, HAMELIN M E, LEUNG A, JOUBERT P, COUTURE C, KOBASA D, BOIVIN G. The 2009 pandemic H1N1 D222G hemagglutinin mutation alters receptor specificity and increases virulence in mice but not in ferrets. The Journal of Infectious Diseases, 2011, 204(7):1008-1016. doi: 10.1093/infdis/jir483.
doi: 10.1093/infdis/jir483
[26] OTTE A, SAUTER M, DAXER M A, MCHARDY A C, KLINGEL K, GABRIEL G. Adaptive mutations that occurred during circulation in humans of H1N1 influenza virus in the 2009 pandemic enhance virulence in mice. Journal of Virology, 2015, 89(14):7329-7337. doi: 10.1128/JVI.00665-15.
doi: 10.1128/JVI.00665-15
[27] JACKSON D, HOSSAIN M J, HICKMAN D, PEREZ D R, LAMB R A. A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(11):4381-4386. doi: 10.1073/pnas.0800482105.
doi: 10.1073/pnas.0800482105
[28] RAMOS I, FERNANDEZ-SESMA A. Cell receptors for influenza a viruses and the innate immune response. Frontiers in Microbiology, 2012, 3:117. doi: 10.3389/fmicb.2012.00117.
doi: 10.3389/fmicb.2012.00117
[29] LIU Q F, QIAO C L, MARJUKI H, BAWA B, MA J Q, GUILLOSSOU S, WEBBY R J, RICHT J A, MA W J. Combination of PB2 271A and SR polymorphism at positions 590/591 is critical for viral replication and virulence of swine influenza virus in cultured cells and in vivo. Journal of Virology, 2012, 86(2):1233-1237. doi: 10.1128/JVI.05699-11.
doi: 10.1128/JVI.05699-11
[30] SUPHAPHIPHAT P, FRANTI M, HEKELE A, LILJA A, SPENCER T, SETTEMBRE E, PALMER G, CROTTA S, TUCCINO A B, KEINER B, TRUSHEIM H, BALABANIS K, SACKAL M, ROTHFEDER M, MANDL C W, DORMITZER P R, MASON P W. Mutations at positions 186 and 194 in the HA gene of the 2009 H1N1 pandemic influenza virus improve replication in cell culture and eggs. Virology Journal, 2010, 7:157. doi: 10.1186/1743-422X-7-157.
doi: 10.1186/1743-422X-7-157
[1] DONG Yu, WU Qian, FENG Xuan, ZHENG YinYing, CUI BaiMing. A Novel Plasmid pEA60 of Erwinia amylovora Enhances the Pathogenicity of Strains by Regulating the Synthesis of Virulence Factors [J]. Scientia Agricultura Sinica, 2026, 59(5): 996-1007.
[2] CONG QiQi, ZHANG JingYi, MENG XiangLong, DAI PengBo, LI Bo, HU TongLe, WANG ShuTong, CAO KeQiang, WANG YaNan. Identification of Hypovirus in Apple Ring Rot Fungus Botryosphaeria dothidea and Detection of Virus-Carrying Status in China [J]. Scientia Agricultura Sinica, 2025, 58(3): 478-492.
[3] TONG ZhaoYang, LIU WenHua, ZHANG GuoXin, DONG ChunYan, ZHANG YanXia, XU XiaoWei, HE Dong, LIU HeChun, LI Yang, WANG FengTao, FENG Jing, YAO XiaoBo, LIU MeiJin, LIN RuiMing. The Relationship Between Occurrence of Hulless Barley Ear Rot and Population Migration of Grass Mite (Siteroptes spp.) [J]. Scientia Agricultura Sinica, 2025, 58(3): 493-506.
[4] YANG WenJuan, GAO JiaCheng, WANG YanTing, LI Yan, GUO Ming, WANG JunCheng, MENG YaXiong, WANG HuaJun, SI ErJing. Function of Effector Pg00778 Regulation on the Pathogenicity of Pyrenophora graminea to Barley [J]. Scientia Agricultura Sinica, 2025, 58(15): 3020-3035.
[5] YU YunYan, MA GaoXing, DUAN YaNing, TAO Qi, LI XinYi, HU QiuHui, MA Ning. The Potential of Alternative Proteins from Edible Fungi Based on Amino Acid and Physicochemical Characterization [J]. Scientia Agricultura Sinica, 2025, 58(15): 3097-3117.
[6] WAN YunFei, YANG YuYing, ZHANG NaiXin, XU MengMeng, YU QinHao, QIAO ChuanLing, CHEN HuaLan. Identification and Functional Analysis of Adaptive Amino Acid Mutations in the Eurasian Avian-Like H1N1 Swine Influenza Virus [J]. Scientia Agricultura Sinica, 2025, 58(10): 2035-2044.
[7] DONG ZaiFang, DING TengTeng, SHAN YiXuan, LI HongLian, CHEN LinLin, XING XiaoPing. Autophagy-Related Gene FpAtg3 Involves in Growth and Pathogenicity of Fusarium pseudograminearum [J]. Scientia Agricultura Sinica, 2024, 57(6): 1080-1090.
[8] ZHANG AiHong, YANG Fei, ZHAO YuanYe, ZHAO YiHan, DI DianPing, MIAO HongQin. Pathogenicity and Epidemic Risk of Barley Yellow Striate Mosaic Virus [J]. Scientia Agricultura Sinica, 2024, 57(23): 4686-4697.
[9] ZHAO WenShuo, ZHANG JinLong, YAO ZhaoRan, SONG YuQi, LÜ Shun, LIU YingXue, YUAN CongCong, SUN YuHang. Effects of Aflatoxin B1 on Influenza Virus Replication, Organ Damages and Intestinal Microbiota Disorder of Swine [J]. Scientia Agricultura Sinica, 2024, 57(20): 4145-4160.
[10] WANG Yuan, DU MengDan, LI ZhengGang, SHE XiaoMan, YU Lin, LAN GuoBing, DING ShanWen, HE ZiFu, TANG YaFei. Identification of Pathogen Causing Tomato White Tip and Curl Leaf Disease and Its Pathogenicity in Guangdong Province [J]. Scientia Agricultura Sinica, 2024, 57(12): 2350-2363.
[11] ZHANG Jian, ZHAO BinSen, FENG Hao, HUANG LiLi. Function and Mechanism Analysis of Vm-milRN7 Regulating the Pathogenicity of Valsa mali [J]. Scientia Agricultura Sinica, 2024, 57(10): 1930-1942.
[12] XING YuTong, TENG YongKang, WU TianFan, LIU YuanYuan, CHEN Yuan, CHEN Yuan, CHEN DeHua, ZHANG Xiang. Mepiquat Chloride Increases the Cry1Ac Protein Content Through Regulating Carbon and Amino Acid Metabolism of Bt Cotton Under High Temperature and Drought Stress [J]. Scientia Agricultura Sinica, 2023, 56(8): 1471-1483.
[13] GAO XiaoXiao, TU LiQin, YANG Liu, LIU YaNan, GAO DanNa, SUN Feng, LI Shuo, ZHANG SongBai, JI YingHua. Construction of an Infectious Clone of Tobacco Mild Green Mosaic Virus Isolate Infecting Pepper from Jiangsu Based on Genomic Clone [J]. Scientia Agricultura Sinica, 2023, 56(8): 1494-1502.
[14] GONG AnDong, LEI YinYu, WU NanNan, LIU JingRong, SONG MengGe, ZHANG YiMei, YANG Guang, YANG Peng. The Effect of 3-Oxyacyl ACP Reductase Gene FgOAR1 on the Growth, Development and Pathogenicity of Fusarium graminearum [J]. Scientia Agricultura Sinica, 2023, 56(24): 4854-4865.
[15] SONG ZhiZhong, WANG JianPing, SHI ShengPeng, CAO JingWen, LIU WanHao, XU WeiHua, XIAO HuiLin, TANG MeiLing. Identification and Cloning of Ferritin Family Genes in Grape and Response to Compound Amino Acid-Iron Spraying During Different Fruit Developmental Stages [J]. Scientia Agricultura Sinica, 2023, 56(18): 3629-3641.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd