Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (10): 2035-2044.doi: 10.3864/j.issn.0578-1752.2025.10.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Identification and Functional Analysis of Adaptive Amino Acid Mutations in the Eurasian Avian-Like H1N1 Swine Influenza Virus

WAN YunFei(), YANG YuYing, ZHANG NaiXin, XU MengMeng, YU QinHao, QIAO ChuanLing(), CHEN HuaLan   

  1. Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/State Key Laboratory for Animal Disease Control and Prevention, Harbin 150069
  • Received:2025-01-09 Accepted:2025-03-21 Online:2025-05-16 Published:2025-05-21
  • Contact: QIAO ChuanLing

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Abstract:

【Objective】 Influenza A virus (IAV) can cross species barriers through adaptive mutations, and infect a variety of mammals, including humans, posing a continual threat to global public health. Based on cell passage culture, the adaptive amino acid mutations of influenza virus were identified and their impacts on viral biological characteristics of were further investigated, which could provide a theoretical guidance for the prevention and control of influenza 【Method】 A/swine/Zhejiang/199/2013 (H1N1) (ZJ199) virus was passaged in MDCK cells, with the adaptive amino acid mutations occurred in the virus after three passages, which was named ZJ199-P3. The replication capacity and infectivity of the ZJ199 and ZJ199-P3 viruses were compared both in vitro and in vivo. A reverse genetics system for ZJ199 was established, and recombinant viruses containing single-site mutation were rescued using rZJ199 as a backbone. The replication capacities of the parental virus and the single-site mutant recombinant viruses were determined in vitro to identify key amino acid sites affecting viral replication. The impacts of the adaptive mutations on viral biological activity were further investigated by measuring viral polymerase activity, neuraminidase activity, and the activity of ISRE and NF-κB reporter genes. 【Result】 Comparative analysis of the amino acid sequences between the ZJ199 and ZJ199-P3 viruses revealed three mutations in the ZJ199-P3 virus, including A234T in the NP protein, S21G in the NA protein, and M100I in the NS2 protein, respectively. Replication capacity assays demonstrated that the ZJ199-P3 virus exhibited reduced replication abilities both in vitro and in vivo, compared with the ZJ199 virus. Using reverse genetics system, recombinant virus rZJ199 and its single-point mutant viruses including rZJ199-NA-S21G, rZJ199-NP-A234T, and rZJ199-NS2-M100I were rescued. In vitro replication analysis showed that the NP-A234T mutation significantly impaired viral replication capacity compared with the rZJ199 virus, whereas the NA-S21G and NS2-M100I mutations had no apparent effect on viral replication. Further studies demonstrated that the NP-A234T mutation reduced viral polymerase activity, the NA-S21G mutation decreased neuraminidase enzymatic activity, and the NS2-M100I mutation weakened the inhibitory effects on ISRE and NF-κB reporter gene activities. 【Conclusion】 Through serial passaging in cells, three adaptive mutations were identified in the EA H1N1 SIV. The A234T mutation in the NP reduced viral polymerase activity, resulting in diminished viral replication in vitro. The S21G mutation in the NA protein and the M100I mutation in the NS2 protein both impacted the virus's biological activity. These findings underscored the importance of enhanced surveillance of influenza viruses to provide critical scientific evidence for the development of effective influenza prevention and control strategies.

Key words: H1N1, swine influenza virus, replication, adaptive mutation

Table 1

The different amino acid positions between ZJ199 and ZJ199-P3 viruses"

基因（位点）
Gene (Position)		 ZJ199 ZJ199-P3
NP (234) A T
NA (21) S G
NS2 (100) M I

Fig. 1

Replication kinetics of virus in MDCK and A549 cells **: P<0.01; ***: P<0.001; ****: P<0.0001"

Fig. 2

Infectivity of ZJ199 and ZJ199-P3 viruses in mice ****: P<0.0001 A: Weight change; B: Survival; C: Viral titer"

Fig. 3

Replication kinetics of recombinant virus in vitro A: MDCK cells; B: MDCK cells; C: 549 cells *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001"

Fig. 4

The biological activity of ZJ199 and ZJ199-P3 viruses *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001 A: Polymerase activity; B: Neuraminidase activity; C: ISRE reports gene activity; D: NF-κB reports gene activity"

Table 2

Distribution of amino acids in NP, NA and NS2 protein of influenza virus in different hosts"

宿主Host 基因Gene 位点Position 氨基酸残基百分比Percentage of amino acid residues (%)
猪
Swine		 NP (4956)
NA (8220)
NS2 (5191)		 234
21
100		 A (99.80)
S (72.30)
M (99.02)		 S (0.16)
N (27.60)
I (0.87)		 P (0.04)
G (0.04)
L (0.11)
人
Human		 NP (9047)
NA (6332)
NS2 (8618)		 234
21
100		 A (99.90)
N (99.80)
M (99.87)		 S (0.02)
S (0.17)
I (0.13)		 T (0.02)
G (0.01)
\
禽
Avian		 NP (653)
NA (690)
NS2 (674)		 234
21
100		 A (100)
S (97.50)
M (61.70)		 \
N (2.33)
L (38.10)		 \
G (0.14)
\
[1]
MENG F, CHEN Y, SONG Z C, ZHONG Q, ZHANG Y J, QIAO C L, YAN C, KONG H H, LIU L L, LI C J, YANG H L, CHEN H L. Continued evolution of the Eurasian avian-like H1N1 swine influenza viruses in China. Science China Life Sciences, 2023, 66(2): 269-282.
[2]
ZHAO Y Z, HAN L B, SANG H T, LIU S D, YANG P P, HOU Y M, XIAO Y H. Swine influenza viruses isolated from 2019 to 2022 in Shandong Province, China, exemplify the dominant genotype. Genes, 2024, 15(7): 849.
[3]
CUI X X, MA J H, PANG Z F, CHI L Z, MAI C S, LIU H L, LIAO M, SUN H L. The evolution, pathogenicity and transmissibility of quadruple reassortant H1N2 swine influenza virus in China: A potential threat to public health. Virologica Sinica, 2024, 39(2): 205-217.

doi: 10.1016/j.virs.2024.02.002 pmid: 38346538
[4]
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, et al. Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(29): 17204-17210.
[5]
LI C, MELIOPOULOS V, RENDAHL A, SCHULTZ-CHERRY S, TORREMORELL M. Naturally occurring influenza reassortment in pigs facilitates the emergence of intrahost virus subpopulations with distinct genotypes and replicative fitness. mBio, 2025, 16(1): e01924-24.
[6]
STORMS S M, LEONARDI-CATTOLICA A, PREZIOSO T, VARGA C, WANG L Y, LOWE J. Influenza A virus shedding and reinfection during the post-weaning period in swine: Longitudinal study of two nurseries. Frontiers in Veterinary Science, 2024, 11: 1482225.
[7]
CHOI E J, LEE Y J, LEE J M, KIM Y J, CHOI J H, AHN B, KIM K, HAN M G. The effect of mutations derived from mouse-adapted H3N2 seasonal influenza A virus to pathogenicity and host adaptation. PLoS ONE, 2020, 15(1): e0227516.
[8]
URBANIAK K, KOWALCZYK A, POMORSKA-MÓL M, KWIT K, MARKOWSKA-DANIEL I. Effect of serial in vivo passages on the adaptation of H1N1 avian influenza virus to pigs. Journal of Veterinary Research, 2022, 66(1): 9-19.
[9]
ZHU M, HE J Q, ZENG H, WANG P P, ZHU Y H, SUN F Y, HUANG X, XU Y, HUANG C Q, CHEN J C, et al. Synergistic HA and NS mutations enhanced the virulence of a mouse-adapted H1N1 influenza A virus. Veterinary Microbiology, 2023, 276: 109615.
[10]
LIU L L, WANG F J, WU Y, MI W Y, ZHANG Y P, CHEN L, WANG D X, DENG G H, SHI J Z, CHEN H L, KONG H H. The V223I substitution in hemagglutinin reduces the binding affinity to human-type receptors while enhancing the thermal stability of the H3N2 canine influenza virus. Frontiers in Microbiology, 2024, 15: 1442163.
[11]
LIU K T, GUO Y Q, ZHENG H F, JI Z X, CAI M, GAO R Y, ZHANG P H, LIU X W, XU X L, WANG X Q, LIU X F. Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K. Virologica Sinica, 2023, 38(1): 47-55.
[12]
GILBERTSON B, SUBBARAO K. Mammalian infections with highly pathogenic avian influenza viruses renew concerns of pandemic potential. Journal of Experimental Medicine, 2023, 220(8): e20230447.
[13]
CHAN M, LEUNG A, HISANAGA T, PICKERING B, GRIFFIN B D, VENDRAMELLI R, TAILOR N, WONG G, BI Y H, BABIUK S, BERHANE Y, KOBASA D. H7N9 influenza virus containing a polybasic HA cleavage site requires minimal host adaptation to obtain a highly pathogenic disease phenotype in mice. Viruses, 2020, 12(1): 65.
[14]
MENG F, YANG H L, QU Z Y, CHEN Y, ZHANG Y J, ZHANG Y P, LIU L L, ZENG X Y, LI C J, KAWAOKA Y, CHEN H L. A Eurasian avian-like H1N1 swine influenza reassortant virus became pathogenic and highly transmissible due to mutations in its PA gene. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119(34): e2203919119.
[15]
ZHU W F, FENG Z M, CHEN Y K, YANG L, LIU J, LI X Y, LIU S L, ZHOU L J, WEI H J, GAO R B, WANG D Y, SHU Y L. Mammalian-adaptive mutation NP-Q357K in Eurasian H1N1 Swine Influenza viruses determines the virulence phenotype in mice. Emerging Microbes & Infections, 2019, 8(1): 989-999.
[16]
CARDENAS M, SEIBERT B, COWAN B, CACERES C J, GAY L C, CARGNIN FACCIN F, PEREZ D R, BAKER A L, ANDERSON T K, RAJAO D S. Modulation of human-to-swine influenza a virus adaptation by the neuraminidase low-affinity calcium-binding pocket. Communications Biology, 2024, 7: 1230.

doi: 10.1038/s42003-024-06928-6 pmid: 39354058
[17]
CAI M K, ZHONG R T, QIN C X, YU Z Q, WEN X Y, XIAN J S, CHEN Y J, CAI Y, YI H Y, GONG L, ZHANG G H. The R251K substitution in viral protein PB2 increases viral replication and pathogenicity of Eurasian avian-like H1N1 swine influenza viruses. Viruses, 2020, 12(1): 52.
[18]
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, et al. A single amino acid at position 431 of the PB 2 protein determines the virulence of H1N1 swine influenza viruses in mice. Journal of Virology, 2020, 94(8): e01930-19.
[19]
ZHAO Z Z, YI C Y, ZHAO L Z, WANG S Y, ZHOU L S, HU Y, ZOU W, CHEN H C, JIN M L. PB2-588I enhances 2009 H1N1 pandemic influenza virus virulence by increasing viral replication and exacerbating PB2 inhibition of beta interferon expression. Journal of Virology, 2014, 88(4): 2260-2267.
[20]
LUO W Y, ZHANG J, LIANG L B, WANG G W, LI Q B, ZHU P Y, ZHOU Y, LI J P, ZHAO Y H, SUN N, et al. Correction: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication. PLoS Pathogens, 2024, 20(2): e1012035.
[21]
WANG X Y, JIANG L, WANG G W, SHI W J, HU Y Z, WANG B, ZENG X Y, TIAN G B, DENG G H, SHI J Z, et al. Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importin α7 is counteracted by TBK1-p62 axis-mediated autophagy. Cellular & Molecular Immunology, 2022, 19(10): 1168-1184.
[22]
PFLUG A, LUKARSKA M, RESA-INFANTE P, REICH S, CUSACK S. Structural insights into RNA synthesis by the influenza virus transcription-replication machine. Virus Research, 2017, 234: 103-117.

doi: S0168-1702(16)30782-1 pmid: 28115197
[23]
ARRAGAIN B, KRISCHUNS T, PELOSSE M, DRNCOVA P, BLACKLEDGE M, NAFFAKH N, CUSACK S. Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase. Nature Communications, 2024, 15: 6910.

doi: 10.1038/s41467-024-51007-3 pmid: 39160148
[24]
AREA E, MARTÍN-BENITO J, GASTAMINZA P, TORREIRA E, VALPUESTA J M, CARRASCOSA J L, ORTÍN J. 3D structure of the influenza virus polymerase complex:Localization of subunit domains. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(1): 308-313.
[25]
ENGELHARDT O G, FODOR E. Functional association between viral and cellular transcription during influenza virus infection. Reviews in Medical Virology, 2006, 16(5): 329-345.

doi: 10.1002/rmv.512 pmid: 16933365
[26]
YANG J, LIU S W, DU L Y, JIANG S B. A new role of neuraminidase (NA) in the influenza virus life cycle: implication for developing NA inhibitors with novel mechanism of action. Reviews in Medical Virology, 2016, 26(4): 242-250.

doi: 10.1002/rmv.1879 pmid: 27061123
[27]
SCHEIBNER D, SALAHELDIN A H, BAGATO O, ZAECK L M, MOSTAFA A, BLOHM U, MÜLLER C, EWEAS A F, FRANZKE K, KARGER A, et al. Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses. PLoS Pathogens, 2023, 19(2): e1011135.
[28]
HUANG X Y, YIN G H, ZHOU B, CAI Y Q, HU J N, HUANG J W, CHEN Z L, LIU Q T, FENG X L. KRT 10 plays an important role in the release of viral genome from endosomes during H9N2 subtype AIV replication in HeLa cells. Veterinary Microbiology, 2023, 284: 109824.
[29]
ZHANG B, LIU M X, HUANG J X, ZENG Q Y, ZHU Q Y, XU S, CHEN H L. H1N1 influenza A virus protein NS2 inhibits innate immune response by targeting IRF7. Viruses, 2022, 14(11): 2411.
[30]
HU Y, LIU X K, ZHANG A D, ZHOU H B, LIU Z D, CHEN H C, JIN M L. CHD 3 facilitates vRNP nuclear export by interacting with NES1 of influenza A virus NS2. Cellular and Molecular Life Sciences, 2015, 72(5): 971-982.
[31]
ZHANG L, SHAO Y K, WANG Y Y, YANG Q X, GUO J M, GAO G F, DENG T. Twenty natural amino acid substitution screening at the last residue 121 of influenza A virus NS2 protein reveals the critical role of NS2 in promoting virus genome replication by coordinating with viral polymerase. Journal of Virology, 2024, 98(1): e01166-23.
[32]
KANG M, WANG L F, SUN B W, WAN W B, JI X, BAELE G, BI Y H, SUCHARD M A, LAI A, ZHANG M, et al. Zoonotic infections by avian influenza virus: changing global epidemiology, investigation, and control. The Lancet Infectious Diseases, 2024, 24(8): e522-e531.
[33]
NAKAMURA K, HARADA Y, TAKAHASHI H, TRUSHEIM H, BERNHARD R, HAMAMOTO I, HIRATA-SAITO A, OGANE T, MIZUTA K, KONOMI N, et al. Systematic evaluation of suspension MDCK cells, adherent MDCK cells, and LLC-MK 2 cells for preparing influenza vaccine seed virus. Vaccine, 2019, 37(43): 6526-6534.
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