Characterization of Hemagglutinin Gene Fragment of H9N2 Avian Influenza Virus Isolated from Environmental Live Bird Market in the Greater Jakarta Area

N. R. Maharani, H. Susetya, M. H. Wibowo


Virus H9N2 has become the most common subtype of low pathogenic avian influenza (LPAI) in poultry and shows the ability to infect humans. One of the important factors triggering the virus ​​pandemic is the live bird market (LBM). The virus acts as an internal gene donor in the other subtypes, such as H5N1, H5N2, H7N9, and H10N8, in poultry and humans. This study was conducted to detect the presence of the H9N2 virus and the molecular characteristics of the hemagglutinin gene fragment at the cleavage site, receptor binding site, antigenic site, and glycosylation site positions of the H9N2 subtype AI virus isolated from the LBM environment. This study used Disease Investigation Center Subang isolates from environmental samples of LBM in the Greater Jakarta area (DKI Jakarta, Bogor, Depok, Tangerang, and Bekasi) in 2019. Based on molecular detection using RT-PCR and RT-qPCR, it was found that avian influenza subtype H9N2 was detected. This indicates that LBM has the potential to be the source of the spread of the ​​H9N2 virus. The pattern of the amino acid cleavage site PSRSSR↓GLF indicates that the research sample belongs to the low pathogenic AI. The substitution of amino acids in the receptor binding site and antigenic site increases the specificity of non-human host recognition. The potential glycosylation site, with the NCS motif found in amino acid position 295-297, close to the receptor-binding site. Based on genetic analysis and phylogenetic topography, the virus is included in the CVI (China, Vietnam, Indonesia) lineage and subclade H9.4.2.5.


Abe, Y., E. Takashita, K. Sugawara, Y. Matsuzaki, Y. Muraki, & S. Hongo. 2020. Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemegglutinin. ASM Journal 78:9605-9611.
Adu, B. G., M. S. B. Sembiring, O. H. Sibarani, I. G. N. K. Mahardika, I. B. K. Suardana, I. G. A. A. Suartini, & T. S. Nindhia. 2020. Seroprevalensi virus Avian Influenza H9N2 pada ayam kampung (Gallus domesticus) di Pasar Beringkit, Kabupaten Badung, Bali. Jurnal Veteriner 21:258-366.
Azizpour, A., H. Goudarzi, S. Charkhkar, R. Momayez, & M. H. Hablolvarid. 2014. Experimental study on tissue tropism and dissemination of H9N2 avian influenza virus and Ornithobacterium rhinotracheale co-infection in SPF chickens. J. Anim. Plant Sci. 24:1655–1662.
Baron, J., T. Carolin, M. Deborah, S. Eva, M. Daniela, H. Maya, S. Folker, S. Torsten, G. Yi, G. Wolfgang, K. Hans-Dieter, & B. Eva. 2012. Matriptase, HAT, and PRSS2 activate the hemagglutinin of H9N2 influenza A viruses. J. Virol. 87:1811–1820.
Belser, J. A., X. Sun, N. Brock, C. Pappas, J. A. Pulit-Penaloza, H. Zeng, Y. Jang, J. Jones, P. J. Carney, J. Chang, N. V. Long, N. T. Diep, S. Thor, H. Di, G. Yang, P. W. Cook, H. M. Creager, D. Wang, J. McFarland, P. V. Dong, D. E. Wentworth, T. M. Tumpey, J. R. Barnes, J. Stevens, C. T. Davis, & T. R. Maines. 2020. Genetically and antigenically divergent influenza A (H9N2) viruses exhibit differential replication and transmission phenotypes in mammalian models. J. Virol. 94:e00451-20.
Biswas, P. K., M. Giasuddin, P. Chowdhury, H. Barua, N. C. Debnath, & M. Yamage. 2018. Incidence of contamination of live bird markets in Bangladesh with influenza A virus and subtypes H5, H7 and H9. Transbound. Emerg. Dis. 65:687-695.
Blaurock, C., D. Scheibner, M. Landmann, M. Vallbracht, R. Ulrich, E. B. Friebertshauser, T. C. Mettenleiter, & E. M. Abdelwhab. 2020. Non-basic amino acids in the hemagglutinin proteolytic cleavage site of a European H9N2 avian influenza virus modulate virulence in turkeys.  Sci. Rep. 10: 21226.
Burke, D. F. & D. J. Smith. 2014. A recommended numbering scheme for influenza A HA subtypes. PLoS ONE 9: e112302.
Carnaccini, S. & D. R. Perez. 2020. H9 influenza viruses: an emerging challenge. Cold Spring Harb Perspect Med. 10:1-11.
Cheng, K. L., J. Wu, W. L. Shen, A. Y. L. Wong, Q. Guo, J. Yu, X. Zhuang, W. Su, T. Song, M. Peiris, H. L. Yen, & E. H. Y. Lau. 2020. Avian influenza virus detection rates in poultry and environment at live poultry markets, Guangdong, China. Emerg. Infect. Dis. 26: 591-595.
Cui, L., D. Liu, W. Shi, J. Pan, X. Qi, X. Li, X. Guo, M. Zhou, W. Li, J. Li, J. Haywood, H. Xiao, X. Yu, X. Pu, Y. Wu, H. Yu, K. Zhao, Y. Zhu, B. Wu, T. Jin, Z. Shi, F. Tang, F. Zhu, Q. Sun, L. Wu, R. Yang, J. Yan, F. Lei, B. Zhu, W. Liu, J. Ma, H. Wang, & G. F. Gao. 2014. Dynamic reassortments and genetic heterogeneity of the human-infecting influenza A (H7N9) virus. Nat. Commun. 5:3142.
Dharmayanti, N. L. P. I., R. Hartawan, Pudjiatmoko, H. Wibawa, Hardiman, A. Balish, R. Donis, C. T. Davis, & G. Samaan. 2014. Genetic characterization of Clade Avian Influenza A(H5N1) Viruses, Indonesia, 2012. Emerg. Infect. Dis. 20:671-674.
Dharmayanti, N. L. P. I., D. A. Hewajuli, A. Ratnawati, & R. Hartawan. 2020. Genetic diversity of H5N1 viruses in live bird markets, Indonesia. J. Vet. Sci. 21:e56.
Dirjen PKH, Kementerian Pertanian. 2018. Deteksi Virus Avian Influenza dengan Teknik Real-Time Reverse Transcription Polymerase Chain Reaction (rRT-PCR). FAO SOP book, Balai Besar Veteriner Wates.
Fereidouni, S. R., E. Starick, C. Grund, A. Globig, T. C. Mettenleiter, M. Beer, & T. Harder. 2009. Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses. Vet. Microbiol. 135:253-60.
Guan, Y., K. F. Shortridge, S. Krauss, & R. G. Webster. 1999. Molecular characterization of H9N2 influenza viruses: Were they the donors of the “internal” genes of H5N1 viruses in Hong Kong? J. Virol. 96: 9363-9367.
Hassan, M. M., M. A. Hoque, B. Ujvari, & M. Klaassen. 2018. Live bird markets in Bangladesh as a potentially important source for Avian Influenza Virus transmission. Prev. Vet. Med. 156:22–27.
Homme, P. J. & B. C. Easterday. 1970. Avian influenza virus infections. I: characteristics of influenza A/turkey/Wisconsin/1966 virus. Avian Dis. 14: 66–74.
Huang, Y., X. Li, H. Zhang, B. Chen, Y. Jiang, L. Yang, & L. Gao. 2015. Human infection with an avian influenza A (H9N2) virus in the middle region of China. J. Med. Virol. 87:1641–1648.
Ito, T., J. S. S. Nelson, C. S. Keln, L. G. Baum, S. Krauss, M. R. Lastrucci, I. Donatelli, H. Kida, J. C. Paulson, R. G. Webster, & Y. Kawaoka. 2000. Molecular basis for the generation in pigs of Influenza A viruses with pandemic potential. J. Virol. 79:7367-7373.
Jiahao, Z., M. Kaixiong, L. Bo, C. Yiqun, Q. Ziwen, X. Jinchao, H. Jinyu, H. Chen, H. Yifan, L. Huanan, L. Dingxiang, L. Ming, & Q. Wenbao. 2021. A risk marker of tribasic hemagglutinin cleavage site in influenza A (H9N2) virus. Commun. Biol. 4:71.
Jiang, W., S. Liu, G. Hou, J. Li, Q. Zhuang, S. Wang, P. Zhang, & J. Chen. 2012. Chinese and global distribution of H9 subtype Avian Influenza viruses. PLoS ONE 7:e52671.
Jonas, M., A. Sahesti, T. Murwijati, C. L. Lestariningsih, I. Irine, C. S. Ayesda, & G. N. Mahardika. 2018. Identification of avian influenza virus subtype H9N2 in chicken farms in Indonesia. Prev. Vet. Med. 159:99–105.
Kang, J., J. Wen-Ming, L. Shuo, C. Ji-Ming, C. Jie, H. Guang-Yu, L. Jin-Ping, & H. Bao-Xu. 2010. Characterization of the hemagglutinin gene of subtype H9 avian influenza viruses isolated in 2007–2009 in China. J. Virol. Methods 163:186-189.
Kumar, S., G. Stecher, & K. Tamura. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33:1870-1874.
Lestari, H. Wibawa, E. P. Lubis, R. Dharmawan, R. A. Rahayu, H. Mulyawan, K. Charoenkul, C. Nasamran, B. Poermadjaja, & A. Amonsin. 2019. Co-circulation and characterization of HPAI-H5N1 and LPAI-H9N2 recovered from a duck farm, Yogyakarta, Indonesia. Transbound Emerg. Dis. 67:994–1007.
Liu, D., W. Shi, & G. F. Gao. 2014. Poultry carrying H9N2 act as incubators for novel human avian influenza viruses. The Lancet. 384:869.
Liu, S., K. Ji, J. Chen, D. Tai, W. Jiang, G. Hou, J. Chen, J. Li, & B. Huang. 2009. Panorama phylogenetic diversity and distribution of type A influenza virus. PLoS ONE 4:e5022.
Marinova-Petkova, A., S. Karthik, M. F. Mohammed, J. E. Lisa, H. M. Kamrul, A. Sharmin, T. Jasmine, W. David, S. Patrick, F. John, M. Pamela, K. Scott, J. W. Richard, & R. G. Webster. 2016. The continuing evolution of H5N1 and H9N2 influenza viruses in Bangladesh. Avian Dis. 60:108–117.
Matrosovich, M., A. Tuzikov, N. Bovin, A. Gambaryan, A. Klimov, M. R. Castrucci, I. Donatelli, & Y. Kawaoka. 2000. Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J. Virol. 7:8502-8512.
Min, K. & K. J. Hyung. 2017. Genetics and biological property analysis of Korea lineage of influenza A H9N2 viruses. Vet. Microbiol. 204:96-103.
Moosakhani, F., A. H. Shoshtari, S. A. Pourbakhsh, Keyvanfar, & H. A. Ghorbani. 2010. Phylogenetic analysis of the hemagglutinin genes of 12 H9N2 influenza viruses isolated from chickens in Iran from 2003 to 2005. Avian Dis. 54:870-874.
Mosleh, N., H. Dadras, K. Asasi, M. J. Taebipour, S. S. Tohidifar, & G. Farjanikish. 2017. Evaluation of the timing of the Escherichia coli co-infection on pathogenecity of H9N2 avian influenza virus in broiler chickens. Iran J. Vet. Res. 18:86–91.
Muflihanah, E. Andesfha, H. Wibawa, F. C. Zenal, F. Hendrawati, Siswani, Wahyuni, D. Kartini, I. Rahayuningtyas, S. Hadi, S. Mukartini, B. Poermadjaja, & F. S. T. Rasa. 2017. Kasus pertama low pathogenic Avian Influenza Subtipe H9N2 pada peternakan ayam petelur di Kabupaten Sidrap, Sulawesi Selatan Indonesia. Buletin InfoVet 16:1-13.
Naguib, M. M., R. Ulrich, E. Kasbohm, C. L. P. Eng, D. Hoffmann, C. Grund, M. Beer, & T. C. Harder. 2017. Natural reassortants of potentially zoonotic avian influenza viruses H5N1 and H9N2 from Egypt display distinct pathogenic phenotypes in experimentally infected chickens and ferrets. J. Virol. 91: e01300.
Novianti, A. N., K. Rahardjo, R. R. Prasetya, A. M. Nastri, J. R. Dewantari, A. P. Rahardjo, A. T. S. Estoepangestie, Y. K. Shimizu, E. D. Poetranto, G. Soegiarto, Y. Mori, & K. Shimizu. 2019. Whole-Genome sequence of an avian influenza A/H9N2 virus isolated from an apparently healthy chicken at a live-poultry market in Indonesia. Microbiol. Resour. Announc. 8:e01671-18.
Nugroho, C. M. H., Soejoedono, R. D. Poetri, & O. Nadia. 2018. Karakterisasi Molekuler Gen Hemaglutinin Virus Avian Influenza Subtipe H9N2 yang Diisolasi dari Ayam Layer di Pulau Jawa. Scientific Repository. IPB University. 2014. Influenza A Cleavage Sites. [8 Juli 2020].
Parvin, R., A. A. Shehata, K. Heenemann, K. Gac, A. Rueckner, M. Y. Halami, & T. M. Vahlenkamp. 2015. Differential replication properties among H9N2 avian influenza viruses of Eurasian origin. Vet. Res. 46:75.
Parvin, R., J. Schinkoethe, C. Grund, R. Ulrich, F. Bönte, K. P. Behr, M. Voss, M. A. Samad, K. E. Hassan, C. Luttermann, M. Beer, & T. Harder. 2020. Comparison of pathogenicity of subtype H9 avian influenza wild‑type viruses from a wide geographic origin expressing mono‑, di‑, or tri‑basic hemagglutinin cleavage sites. Vet. Res. 51:48.
Peacock, T. H. P., J. James, J. E. Sealy, & M. Iqbal. 2019. A global perspective on H9N2 avian influenza virus. Viruses 11: E620.
Pusch, E. A. & D. L. Suarez. 2018. The multifaceted zoonotic risk of H9N2 avian influenza. Vet. Sci. 82: 1-18.
Rachmawati, P. D., T. S. Adikara, H. Plumeriastuti, R. Ernawati, J. Rahmahani, D. Handijatno, & C. M. H. Nugroho. 2020. Analisis filogenetik gen hemaglutinin dan neuraminidase avian influenza H9N2 asal ayam petelur di Jawa Timur. Jurnal Veteriner 21:216-226.
Ratnawati, A. & N. L. P. I. Dharmayanti. 2015. Deteksi virus avian influenza subtipe H5N1 di beberapa pasar unggas hidup dalam wilayah Provinsi Jawa Barat sekitarnya. Jurnal Kedokteran Hewan 9:14-19.
Reed, C., D. Bruden, K. K. Byrd, V. Veguilla, M. Bruce, D. Hurlburt, D. Wang, C. Holiday, K. Hancock, J. R. Ortiz, J. Klejka, J. M. Katz, & T. M. Uyeki. 2014. Characterizing wild bird contact and seropositivity to highly pathogenic avian influenza A (H5N1) virus in Alaskan residents. Influenza and Other Respiratory Viruses 8:516–523.
Rogers, G. N., J. C. Paulson, R. S. Daniels, J. J. Skehel, I. A. Wilson, & D. C Wiley. 1983. Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature 304:76-78.
Sayeed, Md. A., C. Smallwood, T. Imama, R. Mahmud, R. B. Hasan, M. Hasan, M. S. Anwerb, Md. H. Rashid, & Md. A Hoquea. 2017. Assessment of hygienic conditions of live bird markets on avianinfluenza in Chittagong metro, Bangladesh. Prev. Vet. Med. 142: 7–15.
Sealy, J. E., T. Yaqub, T. P. Peacock, P. Chang, B. Ermetal, A. Clements, J. R. Sadeyen, A. Mehboob, H. Shelton, J. E. Bryant, R. S. Daniels, J. W. McCauley, & M. Iqbal. 2019. Association of increased receptor-binding avidity of influenza A(H9N2) viruses with escape from antibody-based immunity and enhanced zoonotic potential. Emerg. Infect Dis. 25:63–72.
Shen, H. Q., Z. Q. Yan, F. G. Zeng, C. T. Liao, Q. F. Zhou, J. P. Qin, Q. M. Xie, Y. Z. Bi, & F. Chen. 2015. Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013. J. Vet. Sci. 16: 317-324.
Sriwilaijaroen, N. & Y. Suzuki. 2012. Review: Molecular basis of the structure and function of H1 hemagglutinin of influenza virus. Proc. Jpn. Acad. Ser. B. 88: 226-249.
Stech, O., J. Veits, S. Weber, D. Deckers, D. Schroer, T. W. Vahlenkamp, A. Breithaupt, J. Teifke, T. C. Mettenleiter, & J. Stech. 2009. Acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain. J. Virol. 83:5864–5868.
Sung-su, Y., L. Dong-Hun, J. Jei-Hyun, M. J. Pantin-Jackwood, S. Chang-seon, & D. E. Swayne. 2020. Live bird markets as evolutionary epicentres of H9N2 low pathogenicity avian influenza viruses in Korea. Emerg. Microbes Infect. 9:616-627.
Tate, M. D., E. R. Job, Y. M. Deng, V. Gunalan, S. Maurer-Stroh, & P. C. Reading. 2014. Playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection. Viruses 6: 1294–1316.
Teng, Q., D. Xu, W. Shen, Q. Liu, G. Rong, X. Li, & Z. Li. 2016. A single mutation at position 190 in hemagglutinin enhances binding affinity for human type sialic acid receptor and replication of H9N2 avian influenza virus in mice. J. Virol. 90:9806–9825.
Tse, L.V. & G. R. Whittaker. 2015. Modification of the hemagglutinin cleavage site allows indirect activation of avian influenza virus H9N2 by bacterial Staphylokinase. Virol. 482:1–8.
Turner, J. C. M., M. M. Feeroz, M. K. Hasan, S. Akhtar, D. Walker, P. Seiler, S. Barman, J. Franks, L. Jones-Engel, P. McKenzie, S. Krauss, R. J. Webby, G. Kayali, & R. G. Webster. 2017. Insight into live bird markets of Bangladesh: an overview of the dynamics of transmission of H5N1 and H9N2 avian influenza viruses. Emerg. Microbes Infect. 6:1-8.
Van der Kolk, J. H. 2019. Role for migratory domestic poultry and/ or wild birds in the global spread of avian influenza?. Veterinary Quarterly 39:161-167.
Wibowo, M. H., H. Susetya, T. Untari, K. Putri, C. R. Tabbu, & W. Asmara. 2006. Molecular study on the pathogenicity of avian influenza virus. Indones. J. Biotechnol. 11:901-907.
Wibowo, M. H., E. A. Srihanto, K. Putri, W. Asmara, & C. R. Tabbu. 2013. The development of pathogenicity of avian influenza virus isolated from Indonesia. Indones. J. Biotechnol. 18:2, 133-143.
Wu, A., C. Su, D. Wang, Y. Peng, M. Liu, S. Hua, T. Li, G. F. Gao, H. Tang, J. Chen, X. Liu, Y. Shu, D. Peng, & T. Jiang. 2013. Sequential reassortments underlie diverse influenza H7N9 genotypes in China. Cell Host Microbe 14:446–452.
Xia, J., J-Q. Cui, X. He, Y-Y. Liu, K-C. Yao, S-J. Cao, X-F. Han, & Y. Huang. 2017. Genetic and antigenic evolution of H9N2 subtype avian influenza virus in domestic chickens in southwestern China, 2013-2016. PLoS ONE 12: e0171564.
Yanheng, W., L. Jinsi, Y. Shuhuan, X. Ying, W. Man, C. Xueqin, Z. Yayang, L. Le, & S. Wuyang. 2018. The molecular characteristics of avian influenza viruses (H9N2) derived from air samples in live poultry markets. Infect. Genet. Evol. 60: 191-196.
Yuan, J., Y. Dong, R. Hongguang, Y. Zhiqiu, H. Zhisong, H. Mingda, L. Beiping, Z. Wei, Y. Junjie, & L. Long. 2014. Phylogeograohy of avian influenza A H9N2 in Cina.Vet. Res. Biomed. Central. 15:1110.
Yuxin, Z., L. Song, Z. Yufa, S. Wengang, T. Yujing, P. Quanhai, & M. Zengmin. 2015. Phylogenetic analysis of hemagglutinin genes of H9N2 avian influenza viruses isolated from chickens in Shandong, China, between 1998 and 2013. BioMed Res. Int. 2015: 1-6.
Zhong, L., X. Wang, Q. Li, D. Liu, H. Chen, M. Zhao, X. Gu, L. He, X. Liu, M. Gu, D. Peng, & X. Liu. 2014. Molecular mechanism of the airborne transmissibility of H9N2 avian influenza A viruses in chickens. J. Virol. 88:9568-9578.
Zhou, X., Y. Li, Y. Wang, J. Edwards, F. Guo, A. C. A. Clements, B. Huang, & R. J. S. Magalhaes. 2015. The role of live poultry movement and live bird market biosecurity in the epidemiology of influenza A (H7N9): A cross-sectional observational study in four eastern China provinces. J. Infect. xx:1-10.
Zhu, Y. C., B., Zhang, Z. H. Sun, X. J. Wang, X. H. Fan, L. X. Gao, Y. Liang, X. Y. Chen, & Z. F. Zhang. 2018. Replication and pathology of duck influenza virus subtype H9N2 in Chukar. Biomed Environ. Sci. 31:306–310.


N. R. Maharani
H. Susetya
M. H. Wibowo (Primary Contact)
MaharaniN. R., SusetyaH., & WibowoM. H. (2022). Characterization of Hemagglutinin Gene Fragment of H9N2 Avian Influenza Virus Isolated from Environmental Live Bird Market in the Greater Jakarta Area. Tropical Animal Science Journal, 45(2), 141-153.

Article Details