Therapeutic Trial of an Endothelin Receptor Agonist for the Highly Pathogenic Avian Influenza A/H5N1 Virus Infection in Chicks


The rapid spread of the highly pathogenic A/H5N1 avian influenza virus among domestic birds and its transmission to humans has induced world-wide fears of a new influenza pandemic. A/H5N1 has infected over 300 people since 1997, and has shown a mortality rate of over 50%. The high mortality in human cases is thought to be enhanced by the excessive secretion of various endogenous factors, including cytokines and interleukins, stimulated by viral infections. Chickens infected with A/H5N1 viruses experience sudden death without showing severe clinical symptoms or inflammation. However, severe hemorrhage and congestion are seen in various tissues in sporadic chicken cases of A/H5N1-infections, especially in the pulmonary tissues, thus indicating that there is ischemia due to vascular abnormalities. Our previous studies have focused on the expression pattern of endothelin-1, which modulates the vascular tone via endothelin receptors. An Indonesian sporadic strain of A/H5N1 virus was intranasally administered to 10-day-old chicks, and the expression of endothelin was examined in the infected birds. All birds died within five days of inoculation, and had moderate inflammation accompanied by severe hemorrhage and congestion in the lungs. Immunohistochemical studies showed enhanced expression of endothelin-1 in the infected lungs. In addition, the real-time PCR analyses revealed that endothelin-1 and endothelin receptor A mRNA were significantly elevated in the birds with A/H5N1 infections. Subsequently, H5N1-infected birds were inoculated with bosentan hydrate, a competitive antagonist of endothelin receptors. Interestingly, the mortality rate of the infected birds was dramatically decreased in a dose-dependent manner by the administration of bosentan hydrate. The pathological lesions, including congestion and hemorrhage in the pulmonary tissues, were clearly inhibited. These findings are promising, and suggest that endothelin receptor antagonists are a potential treatment for the highly pathogenic avian flu.

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Adachi, K. , Soejoedono, R. , Handharyani, E. , Inai, M. and Tsukamoto, Y. (2014) Therapeutic Trial of an Endothelin Receptor Agonist for the Highly Pathogenic Avian Influenza A/H5N1 Virus Infection in Chicks. Health, 6, 2553-2561. doi: 10.4236/health.2014.619294.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Alexander, D.J. and Brown, I.H. (2000) Recent Zoonoses Caused by Influenza A Viruses. Revue scientifique et technique, 19, 197-225.
[2] Spickler, A.R., Trampel, D.W. and Roth, J.A. (2008) The Onset of Virus Shedding and Clinical Signs in Chickens Infected with High-Pathogenicity and Low-Pathogenicity Avian Influenza Viruses. Avian Pathology, 37, 555-557.
[3] Normile, D. (2005) Infectious Diseases. North Korea Collaborates to Fight Bird Flu. Science, 308, 175.
[4] Leroux-Roels, I., Borkowski, A., Vanwolleghem, T., Dreame, M., Clement, F., Hons, E., Devaster, J.M. and Leroux-Roeis, G. (2007) Antigen Sparing and Cross-Reactive Immunity with an Adjuvanted rH5N1 Prototype Pandemic Influenza Vaccine: A Randomized Controlled Trial. The Lancet, 370, 580-589.
[5] Peiris, J.S.M., Yu, W.C., Leung, C.W., Cheung, C.Y., Ng, W.F., Nicholls, J.M., Ng, T.K., Chan, K.H., Lai, S.T., Lim, W.L., Yuen, K.Y. and Guan, Y. (2004) Re-Emergence of Fatal Human Influenza A Subtype H5N1 Disease. The Lancet, 363, 617-619.
[6] Beigel, J.H., Farrar, J., Han, A.M., Hayden, F.G., Hyer, R., de Jong M.D., Lochindarat, S., Nguyen, T.K., Nguyen, T.H., Tran, T.H., Nicoll, A., Touch, S. and Yuen, K.Y. (2005) Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A/H5: Avian Influenza A (H5N1) Infection in Humans. The New England Journal of Medicine, 353, 1374-1385.
[7] Poland, G.A. (2006) Vaccines against Avian Influenza—A Race against Time. The New England Journal of Medicine, 354, 1411-1413.
[8] Ungchusak, K., Auewarakul, P., Dowell, S.F., Kitphati, R., Auwanit, W., Puthavathana, P., Uiprasertkul, M., Boonnak, K., Pittayawonganon, C., Cox, N.J., Zaki, S.R., Thawatsupha, P., Chittaganpitch, M., Khontong, R., Simmerman, J.M. and Chunsutthiwat, S. (2005) Probable Person-to-Person Transmission of Avian Influenza A (H5N1). The New England Journal of Medicine, 352, 333-340.
[9] Chan, M.C., Cheung, C.Y., Chui, W.H., Tsao, S.W., Nicholls, J.M., Chan, Y.O., Chan, R.W., Long, H.T., Poonm, L.L., Guan, Y. and Peiris, J.S. (2005) Proinflammatory Cytokine Responses Induced by Influenza A (H5N1) Viruses in Primary Human Alveolar and Bronchial Epithelial Cells. Respiratory Research, 6, 135.
[10] Shinya, K., Ebina, M., Yamada, S., Ono, M., Kasai, N. and Kawaoka, Y. (2006) Avian Flu: Influenza Virus Receptors in the Human Airway. Nature, 440, 435-436.
[11] Yamada, S., Suzuki, Y., Suzuki, T., Le, M.Q., Nidom, C.A., Sakai-Tagawa, Y., Muramoto, Y., Ito, M., Kiso, M., Horimoto, T., Shinya, K., Sawada, T., Kiso, M., Usui, T., Murata, T., Lin, Y., Ha, A., Haire, L.F., Stevens, D.J., Russell, R.J., Gamblin, S.J., Skehel, J.J. and Kawaoka, Y. (2006) Haemagglutinin Mutations Responsible for the Binding of H5N1 Influenza A Viruses to Human-Type Receptors. Nature, 444, 378-382.
[12] Cheung, C.Y., Poon, L.L., Lau, A.S., Luk, W., Lau, Y.L., Shortridge, K.F., Gordon, S., Guan, Y. and Peiris, J.S. (2002) Induction of Proinframmatory Cytokines in Human Macrophage by Influenza A (H5N1) Viruses: A Mechanism for the Unusual Severity of Human Disease? The Lancet, 360, 1831-1837.
[13] de Jong, M.D. (2006) Fetal Outcome of Human Influenza A (H5N1) Is Associated with High Viral Load and Hypercytokinemia. Nature Medicine, 12, 1203-1207.
[14] Salomon, R., Hoffmann, E. and Webster, R.G. (2007) Inhibition of the Cytokine Response Does Not Protect against Lethal H5N1 Influenza Infection. Proceedings of the National Academy of Sciences of the United States of America, 104, 12479-12481.
[15] Szretter, K.J., Gangappa, S., Lu, X., Smith, C.W., Shieh, J., Zaki, S.R., Sambhara, S., Tumpey, T.M. and Katz, J.M. (2007) Role of Hostcytokine Responses in the Pathogenisis of Avian H5N1 Viruses in Mice. Journal of Virology, 81, 2736-2744.
[16] Inagami, T., Naruse, M. and Hoover, R. (1995) Endothelium as an Endocrine Organ. Annual Review of Psychology, 57, 171-189.
[17] Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. and Masakai, T. (1989) The Human Endothelin Family: Three Structurally and Pharmacologically Distinct Isopeptides Predicted by Three Separate Genes. Proceedings of the National Academy of Sciences of the United States of America, 86, 2863-2867.
[18] Black, S.M., Kumar, S., Wiseman, D., Ravi, K., Wedgwood, S., Ryzhov, V. and Fineman, J.R. (2007) Pediatric Pulmonary Hypertension: Roles of Endothelin-1 and Nitric Oxide. Clinical Hemorheology and Microcirculation, 37, 111-120.
[19] Shimoda, L.A., Sham, J.S., Liu, Q. and Sylvester, J.T. (2002) Acute and Chronic Hypoxic Pulmonary Vasoconstriction: A Central Role for Endothelin-1. Respiratory Physiology & Neurobiology, 132, 93-106.
[20] Weigand, L., Sylvester, J.T. and Shimoda, L.A. (2006) Mechanisms of Endothelin-1-Induced Contraction in Pulmonary Arteries from Chronically Hypoxic Rats. American Journal of Physiology, 290, L284-L290.
[21] Sakurai, M., Yanagisawa, Y., Takuwa, H., Miyazaki, S., Kimura, K., Goto, Y. and Masaki, T. (1990) Cloning of a cDNA Encoding a Non-Isopeptides-Selective Subtype of the Endothelin Receptor. Nature, 348, 732-735.
[22] Carr, M.J., Spalding, L.J., Goldie, R.G. and Henry, P.J. (1998) Distribution of Immunoreactive Endothelin in the Lungs of Mice during Respiratory Viral Infection. European Respiratory Journal, 11, 79-85.
[23] Adachi, K., Handharyani, E., Sari, D.K., Takama, K., Fukuda, K., Endo, I., Yamamoto, R., Sawa, M., Tanaka, M., Konishi, I. and Tsukamoto, Y. (2008) Development of Neutralization Antibodies against Highly Pathogenic H5N1 Avian Influenza Virus Using Ostrich (Struthio camelus) Yolk. Molecular Medicine Reports, 1, 203-209.
[24] Kamiyama, Y., Adachi, K., Handharyani, E., Soejoedono, R.D., Kusano, T., Inai, M., Tsukamoto, M., Kashiwagi, S. and Tsukamoto, Y. (2011) Protection from Avian Influenza H5N1 Virus Infection with Antibody-Impregnated Filters. Virology Journal, 8, 54.
[25] Reed, L.J. and Muench, H. (1938) A Simple Method of Estimating Fifty Percent End-Points. American Journal of Hygiene, 27, 493-497.
[26] Clozel, M., Breu, V., Gray, G.A., Kalina, B., Loffler, B.M., Burri, K., Cassal, J.M., Hirth, G., Müller, M. and Neidhart, W. (1994) Pharmacological Characterization of Bosentan, a New Potent Orally Active Nonpeptide Endothelin Receptor Antagonist. Journal of Pharmacology and Experimental Therapeutics, 270, 228-235.
[27] Seo, B., Oemar, B.S., Siebenmann, R., von Segesser, L. and Lüscher, T.F. (1997) Endothelin Plays a Role in the Maintenance of Blood Pressure in Normotensive Guinea Pigs. Circulation, 89, 1203-1208.
[28] Gomez, A.P., Moreno, M.J., Iglesias, A., Coral, P.X. and Hernandez, A. (2007) Endothelin 1, Its Endothelin Type A Receptor, Connective Tissue Growth Factor, Platelet-Derived Growth Factor and Adrenomedullin Expression in Lungs of Pulmonary Hypertensive and Nonhypertensive Chickens. Poultry Science, 86, 909-916.
[29] Emori, T., Hirata, Y., Ohta, K., Shichiri, M. and Marumo, F. (1989) Secretory Mechanism of Immunoreactive Endothelin in Cultured Bovine Endothelial Cells. Biochemical and Biophysical Research Communications, 160, 93-100.
[30] Kurihara, H., Yoshizumi, M., Sugiyama, T., Takaku, F., Yanagisawa, M., Masaki, T., Hamaoki, M., Kato, H. and Yazaki, Y. (1989) Transforming Growth Factor-Beta Stimulates the Expression of Endothelin mRNA by Vascular Endothelial Cells. Biochemical and Biophysical Research Communications, 159, 1435-1440.
[31] Yoshizumi, M., Kurihara, H., Morita, T., Yamashita, T., Oh-hashi, Y., Sugiyama, T., Takaku, F., Yanagisawa, M., Masaki, T. and Yazaki, Y. (1990) Interleukin 1 Increases the Production of Endothelin-1 by Cultured Endothelial Cells. Biochemical and Biophysical Research Communications, 166, 324-329.
[32] Maemura, K., Kurihara, H., Morita, T., Oh-hashi, Y. and Yazaki, Y. (1992) Production of Endothelin-1 in Vascular Endothelial Cells Is Regulated by Factors Associated with Vascular Injury. Gerontology, 38, 29-35.
[33] Yoshizumi, M., Kurihara, H., Sugiyama, T., Takaku, F., Yanagisawa, M., Masaki, T. and Yazaki, Y. (1989) Hemodynamic Shear Stress Stimulates Endothelin Production by Cultured Endothelial Cells. Biochemical and Biophysical Research Communications, 161, 859-864.
[34] Bridges, C.B., Kuehnert, M.J. and Hall, C.B. (2003) Transmission of Influenza: Implications for Control in Health Care Settings. Clinical Infectious Diseases, 37, 1094-1101.

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