Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Isolated from Poultry, Vietnam, 2009–2011 - Vol. 19 No. 12 - December 2013 - Emerging Infectious Disease journal - CDC

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Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Isolated from Poultry, Vietnam, 2009–2011 - Vol. 19 No. 12 - December 2013 - Emerging Infectious Disease journal - CDC

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Volume 19, Number 12—December 2013

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Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Isolated from Poultry, Vietnam, 2009–2011

Article Contents

• Materials and Methods
• Results
• Discussion
• Conclusion
• Acknowledgment
• References
• Figure 1
• Figure 2
• Figure 3
• Table 1
• Table 2
• Table 3
• Table 4
• Technical Appendix 1  [25 KB]
• Technical Appendix 2  [768 KB - 3 pages]
• Suggested Citation

Ha T. Nguyen, Tung Nguyen, Vasiliy P. Mishin, Katrina Sleeman, Amanda Balish, Joyce Jones, Adrian Creanga, Henju Marjuki, Timothy M. Uyeki, Dang H. Nguyen, Diep T. Nguyen, Hoa T. Do, Alexander I. Klimov, Charles T. Davis, and Larisa V. Gubareva 

Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (H.T. Nguyen, V.P. Mishin, K. Sleeman, A. Balish, J. Jones, H. Marjuki, T.M. Uyeki, A.I. Klimov, C.T. Davis, L.V. Gubareva); Battelle Memorial Institute, Atlanta (H.T. Nguyen, A. Creanga); National Center for Veterinary Diagnostics, Hanoi, Vietnam (T. Nguyen, D.H. Nguyen, D.T. Nguyen); Atlanta Research and Education Foundation, Atlanta (J. Jones)

Suggested citation for this article

Abstract

We assessed drug susceptibilities of 125 avian influenza A(H5N1) viruses isolated from poultry in Vietnam during 2009–2011. Of 25 clade 1.1 viruses, all possessed a marker of resistance to M2 blockers amantadine and rimantadine; 24 were inhibited by neuraminidase inhibitors. One clade 1.1 virus contained the R430W neuraminidase gene and reduced inhibition by oseltamivir, zanamivir, and laninamivir 12-, 73-, and 29-fold, respectively. Three of 30 clade 2.3.4 viruses contained a I223T mutation and showed 7-fold reduced inhibition by oseltamivir. One of 70 clade 2.3.2.1 viruses had the H275Y marker of oseltamivir resistance and exhibited highly reduced inhibition by oseltamivir and peramivir; antiviral agents DAS181 and favipiravir inhibited H275Y mutant virus replication in MDCK-SIAT1 cells. Replicative fitness of the H275Y mutant virus was comparable to that of wildtype virus. These findings highlight the role of drug susceptibility monitoring of H5N1 subtype viruses circulating among birds to inform antiviral stockpiling decisions for pandemic preparedness.

Sporadic transmission of highly pathogenic avian influenza (HPAI) A(H5N1) viruses from birds to humans has been documented since 1997 (1), and these viruses continue to cause severe illness and death in humans. Their wide geographic spread and rapid evolution have raised concerns over emergence of a novel, virulent virus that could efficiently transmit among humans, leading to a pandemic. Vietnam is among the countries experiencing the highest number of human fatalities caused by zoonotic H5N1 subtype infections. Since the introduction of HPAI (H5N1) viruses into poultry in Vietnam during 2003 (1,2), there have been dynamic changes in their genetic and antigenic properties. Clade 1 viruses predominated in Vietnam before 2007, and were the most commonly detected H5N1 subtype group in the Mekong Delta region through 2010 (3). However, in northern Vietnam provinces, clade 2.3.4 viruses became the predominant group during 2007–2010. Since 2010, viruses of clade 2.3.2.1 have been detected in poultry from both regions (3). Since 2009, multiple subgroups of 2.3.2.1 rapidly emerged and have circulated among domestic poultry in Asia, including several provinces of Vietnam (4).
Genetic and antigenic divergence of HPAI (H5N1) viruses among poultry challenges development of effective vaccines for poultry and to pandemic preparedness and development of antiviral drugs for humans. Assessment of drug susceptibility has become an integral part of subtype H5N1 virus surveillance. To assist laboratories worldwide in their surveillance and pandemic preparedness efforts, the Influenza Division of the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, along with other partners, developed the H5N1 Genetic Changes Inventory that includes established and potential markers of drug resistance (5). Resistance to matrix 2 (M2) protein blockers amantadine and rimantadine, caused by mutations in the M2 protein, is detected commonly in clade 1.1 (S31N) and clade 2.1.3 (V27A) H5N1 virus subtypes and sporadically in other groups (6,7). Oseltamivir, an orally administered neuraminidase (NA) inhibitor, is the most prescribed medication for the treatment of persons with influenza virus infections. Emergence of resistance to NA inhibitors among H5N1 virus subtypes, especially oseltamivir resistance among H5N1 subtypes caused by the H275Y mutation, is a constant threat (8). Assessment of susceptibility to NA inhibitors is hampered by several factors: insufficient knowledge of molecular markers of resistance, lack of harmonized approaches for testing and data analysis and, most critically, lack of established laboratory correlates of clinically relevant resistance. Taking into account these and other limitations, the current method for monitoring susceptibility to NA inhibitors is a critical element needed to evaluate pandemic risk.
In this study, we assessed drug susceptibility profiles of HPAI A(H5N1) viruses isolated from poultry specimens collected in Vietnam during 2009–2011. The antiviral drugs tested included FDA-approved medications and investigational antiviral agents. We report the detection of an oseltamivir-resistant virus with H275Y mutation from the expanding clade 2.3.2.1.

Conclusion

Our findings demonstrate the critical role of ongoing monitoring of antiviral drug susceptibility in HPAI (H5N1) viruses sampled from poultry on informing antiviral stockpiling decisions for pandemic preparedness. Because 15 countries have reported human cases of HPAI (H5N1) virus infection to date, these findings also emphasize the need to enhance the armamentarium of available anti-influenza drugs worldwide for treatment of subtype H5N1-infected patients, including agents with diverse mechanisms of action, which could enable combination treatment (37), and host-directed antiviral therapy, and which may be less vulnerable to resistance.

Dr Ha T. Nguyen is a Battelle research scientist on the Molecular Epidemiology Team of the Influenza Division, Centers for Disease Control and Prevention, in Atlanta, Georgia. Her research interests include influenza viruses and mechanisms of resistance to antiviral agents.

Acknowledgment

We thank our collaborators in the National Center for Veterinary Diagnostics, Hanoi, Vietnam, for their valuable contributions to this study. We also thank Ronald B. Moss and Yousuke Furuta for kindly providing investigational anti-influenza drugs DAS181 and favipiravir, respectively, and Anton Chesnokov for his excellent technical assistance.

References

1. Wan XF, Nguyen T, Davis CT, Smith CB, Zhao ZM, Carrel M, Evolution of highly pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and 2007. PLoS ONE. 2008;3:e3462. DOIPubMed
2. Dung Nguyen T, Nguyen TV, Vijaykrishna D, Webster RG, Guan Y, Malik Peiris JS, Multiple sublineages of influenza A virus (H5N1), Vietnam, 2005–2007. Emerg Infect Dis. 2008;14:632–6. DOIPubMed
3. Nguyen T, Rivailler P, Davis CT. Hoa do T, Balish A, Dang NH, et al. Evolution of highly pathogenic avian influenza (H5N1) virus populations in Vietnam between 2007 and 2010. Virology. 2012;432:405–16.
4. World Health Organization. Antigenic and genetic characteristics of zoonotic influenza viruses and development of candidate vaccine viruses for pandemic preparedness. Influenza, Vaccines, Vaccine Viruses. February 2013 [cited 2013Aug 6]. http://www.who.int/influenza/vaccines/virus/characteristics_virus_vaccines/en/
5. Centers for Disease Control and Prevention. H5N1 Genetic changes inventory. [cited 2013 Aug 6]. http://www.cdc.gov/flu/avianflu/h5n1/inventory.htm
6. Cheung CL, Rayner JM, Smith GJ, Wang P, Naipospos TS, Zhang J, Distribution of amantadine-resistant H5N1 avian influenza variants in Asia. J Infect Dis. 2006;193:1626–9. DOIPubMed
7. Hill AW, Guralnick RP, Wilson MJ, Habib F, Janies D. Evolution of drug resistance in multiple distinct lineages of H5N1 avian influenza. Infect Genet Evol. 2009;9:169–78. DOIPubMed
8. Moscona A. Oseltamivir resistance—disabling our influenza defenses. N Engl J Med. 2005;353:2633–6. DOIPubMed
9. World Health Organization Global Influenza Surveillance Network. Manual for the laboratory diagnosis and virological surveillance of influenza. 2011 [cited 2013 Aug 6]. http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf 
10. World Health Organization/ World Organisation for Animal Health/ Food and Agriculture Organization (WHO/OIE/FAO) H5N1 Evolution Working Group. Continued evolution of highly pathogenic avian influenza A (H5N1): updated nomenclature. Influenza Other Respi Viruses. 2012;6:1–5 .
11. Nguyen HT, Fry AM, Gubareva LV. Neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods. Antivir Ther. 2012;17:159–73. DOIPubMed
12. Deyde VM, Nguyen T, Bright RA, Balish A, Shu B, Lindstrom S, Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method. Antimicrob Agents Chemother. 2009;53:1039–47. DOIPubMed
13. Nguyen HT, Sheu TG, Mishin VP, Klimov AI, Gubareva LV. Assessment of pandemic and seasonal influenza A (H1N1) virus susceptibility to neuraminidase inhibitors in three enzyme activity inhibition assays. Antimicrob Agents Chemother. 2010;54:3671–7. DOIPubMed
14. Okomo-Adhiambo M, Sleeman K, Ballenger K, Nguyen HT, Mishin VP, Sheu TG, Neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective. Viruses. 2010;2:2269–89. DOIPubMed
15. Gubareva LV, Trujillo AA, Okomo-Adhiambo M, Mishin VP, Deyde VM, Sleeman K, Comprehensive assessment of 2009 pandemic influenza A (H1N1) virus drug susceptibility in vitro. Antivir Ther. 2010;15:1151–9. DOIPubMed
16. Triana-Baltzer GB, Gubareva LV, Klimov AI, Wurtman DF, Moss RB, Hedlund M, Inhibition of neuraminidase inhibitor-resistant influenza virus by DAS181, a novel sialidase fusion protein. PLoS ONE. 2009;4:e7838. DOIPubMed
17. Klimov A, Balish A, Veguilla V, Sun H, Schiffer J, Lu X, Influenza virus titration, antigenic characterization, and serological methods for antibody detection. Methods Mol Biol. 2012;865:25–51. DOIPubMed
18. Naughtin M, Dyason JC, Mardy S, Sorn S, von Itzstein M, Buchy P. Neuraminidase inhibitor sensitivity and receptor-binding specificity of Cambodian clade 1 highly pathogenic H5N1 influenza virus. Antimicrob Agents Chemother. 2011;55:2004–10. DOIPubMed
19. World Health Organization. Meetings of the WHO working group on surveillance of influenza antiviral susceptibility– Geneva, November 2011 and June 2012. Wkly Epidemiol Rec. 2012;87:369–74 .PubMed
20. Yen HL, Ilyushina NA, Salomon R, Hoffmann E, Webster RG, Govorkova EA. Neuraminidase inhibitor-resistant recombinant A/Vietnam/1203/04 (H5N1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo. J Virol. 2007;81:12418–26. DOIPubMed
21. de Jong MD, Tran TT, Truong HK, Vo MH, Smith GJ, Nguyen VC, Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med. 2005;353:2667–72 . DOIPubMed
22. Le QM, Kiso M, Someya K, Sakai YT, Nguyen TH, Nguyen KH, Avian flu: isolation of drug-resistant H5N1 virus. Nature. 2005;437:1108. DOIPubMed
23. McKimm-Breschkin JL, Selleck PW, Usman TB, Johnson MA. Reduced sensitivity of influenza A (H5N1) to oseltamivir. Emerg Infect Dis. 2007;13:1354–7 .PubMed
24. Stoner TD, Krauss S, DuBois M, Negovetich J, Stallknecht DE, Senne DA, Antiviral susceptibility of avian and swine influenza virus of the N1 neuraminidase subtype. J Virol. 2010;84:9800–9. DOIPubMed
25. Kiso M, Ozawa M, Le MT, Imai H, Takahashi K, Kakugawa S, Effect of an asparagine-to-serine mutation at position 294 in neuraminidase on the pathogenicity of highly pathogenic H5N1 influenza A virus. J Virol. 2011;85:4667–72. DOIPubMed
26. Collins PJ, Haire LF, Lin YP, Liu J, Russell RJ, Walker PA, Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature. 2008;453:1258–61. DOIPubMed
27. Ilyushina NA, Seiler JP, Rehg JE, Webster RG, Govorkova EA. Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets. PLoS Pathog. 2010;6:e1000933. DOIPubMed
28. Earhart KC, Elsayed NM, Saad MD, Gubareva LV, Nayel A, Deyde VM, Oseltamivir resistance mutation N294S in human influenza A(H5N1) virus in Egypt. J Infect Public Health. 2009;2:74–80. DOIPubMed
29. Amaro RE, Swift RV, Votapka L, Li WW, Walker RC, Bush RM. Mechanism of 150-cavity formation in influenza neuraminidase. Nat Commun. 2011;2:388.
30. Hurt AC, Holien JK, Barr IG. In vitro generation of neuraminidase inhibitor resistance in A(H5N1) influenza viruses. Antimicrob Agents Chemother. 2009;53:4433–40. DOIPubMed
31. Nguyen HT, Fry AM, Loveless PA, Klimov AI, Gubareva LV. Recovery of a multidrug-resistant strain of pandemic influenza A 2009 (H1N1) virus carrying a dual H275Y/I223R mutation from a child after prolonged treatment with oseltamivir. Clin Infect Dis. 2010;51:983–4. DOIPubMed
32. Le MT, Wertheim HF, Nguyen HD, Taylor W, Hoang PV, Vuong CD, Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam. PLoS ONE. 2008;3:e3339. DOIPubMed
33. Chen H, Smith GJ, Li KS, Wang J, Fan XH, Rayner JM, Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control. Proc Natl Acad Sci U S A. 2006;103:2845–50. DOIPubMed
34. Boltz DA, Douangngeun B, Phommachanh P, Sinthasak S, Mondry R, Obert C, Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People’s Democratic Republic. J Gen Virol. 2010;91:949–59. DOIPubMed
35. Wathen MW, Barro M, Bright RA. Antivirals in seasonal and pandemic influenza-future perspectives. Influenza Other Respitory Viruses. 2013;7:76–80.
36. Chan-Tack KM, Gao A, Himaya AC, Thompson EG, Singer ME, Uyeki TM, Clinical experience with intravenous zanamivir under an emergency investigational new drug program in the United States. J Infect Dis. 2013;207:196–8. DOIPubMed
37. White NJ, Webster RG, Govorkova EA, Uyeki TM. What is the optimal therapy for patients with H5N1 influenza? PLoS Med. 2009;6:e1000091. DOIPubMed

Figures

• Figure 1. . Description of 125 highly pathogenic avian influenza A(H5N1) viruses collected from poultry in Vietnam during 2009–2011 and tested during this study.
• Figure 2. . Distribution of log-transformed 50% inhibitory concentration (IC₅₀) values for oseltamivir, zanamivir, and peramivir: Box-and-whisker plot analysis of all tested highly pathogenic avian influenza A(H5N1) viruses (n = 125)...
• Figure 3. . Replicative capacity of the oseltamivir-resistant highly pathogenic avian influenza A(H5N1) virus possessing the H275Y substitution and the wild type virus in (A) MDCK and MDCK-SIAT1 cell lines and...

Tables

• Table 1. Clade-specific analysis of drug susceptibility of highly pathogenic avian Influenza A(H5N1) viruses in the neuraminidase inhibition assay
• Table 2. Characterization of statistical outliers identified in neuraminidase inhibition assay
• Table 3. Reduction of virus yield in MDCK-SIAT1 cells in the presence of antiviral agent DAS181
• Table 4. Reduction of virus yield in MDCK-SIAT1 cells in the presence of favipiravir

Technical Appendices

• Technical Appendix 1. . Drug susceptibility and accession data for highly pathogenic avian influenza A(H5N1) viruses.    [25 KB]
• Technical Appendix 2. . Analysis of highly pathogenic avian influenza A(H5N1) viruses.    [768 KB - 3 pages]

Suggested citation for this article: Nguyen HT, Nguyen T, Mishin VP, Sleeman K, Balish A, Jones J, et al. Antiviral susceptibility of highly pathogenic avian influenza A(H5N1) viruses isolated from poultry, Vietnam, 2009–2011. Emerg Infect Dis. 2013 Dec [date cited]. http://dx.doi.org/10.3201/eid1912.130705

DOI: 10.3201/eid1912.130705