Identification of Indonesian clade 2.1 highly pathogenic influenza A(H5N1) viruses with N294S and S246N neuraminidase substitutions which further reduce oseltamivir susceptibility
Introduction
Avian influenza A(H5N1) viruses continue to circulate among commercial, domestic and wild birds in Asia, the Middle East and Africa (OIE, 2017, WHO, 2017). Up until 2009 Indonesia had the highest numbers of human cases and a high mortality rate. However, there has been a significant decrease in the numbers of human infections in Indonesia, especially since 2013, with only 2 cases and 2 deaths reported in 2015, none in 2016 and one in 2017 (WHO, 2017). In contrast, Egypt saw a dramatic upsurge in human infections and deaths since 2013, with 136 cases and 39 deaths in 2015, (WHO, 2015), although only 10 cases and 3 deaths were reported for 2016 and 3 cases and one death in 2017 (WHO, 2017).
The neuraminidase inhibitor (NAI), oseltamivir (Tamiflu) remains the drug of choice for the treatment of infected humans. However, resistance is known to arise not only because of oseltamivir treatment but also through random mutations. The latter has been seen in human seasonal influenza viruses (Hurt et al., 2009a, Meijer et al., 2009, Takashita et al., 2015) and in highly pathogenic avian influenza (HPAI) A(H5N1) viruses isolated from infected poultry (McKimm-Breschkin et al., 2007, McKimm-Breschkin et al., 2013a, Nguyen et al., 2013). Due to differences in the chemical structures of the NAIs, resistance to oseltamivir may not necessarily confer resistance to the other two licensed NAIs, zanamivir and peramivir (McKimm-Breschkin, 2013). Hence these may be suitable as an alternative therapy. As HPAI (H5N1) viruses remain endemic in many parts of the world, they will not be easily eradicated. Since human (H5N1) virus infections arise from contact with infected poultry the virus is fortunately not yet transmissible between humans, however it remains a potential pandemic threat. Hence, it is important to monitor the emergence and circulation of resistance in avian influenza isolates from poultry and other avian species, in order for the appropriate therapy to be used if the viruses become transmissible between humans.
We have previously shown that the NAs from clade 2.1 viruses from Indonesia have a reduced sensitivity to oseltamivir compared to clade 1 viruses from Vietnam (McKimm-Breschkin et al., 2007). This is primarily due to an H252Y substitution in the NA. We also subsequently identified other substitutions which further reduce sensitivity including I222M/V/T and I117V (N2 numbering) (McKimm-Breschkin et al., 2013a). Clade 1 viruses with an S246G also demonstrate reduced oseltamivir sensitivity (McKimm-Breschkin et al., 2007, McKimm-Breschkin et al., 2013a). Through the Influenza Virus Monitoring (IVM) surveillance program in Indonesia which primarily aims to monitor antigenic drift of the (H5N1) virus hemagglutinin (Hartaningsih et al., 2015), we further tested samples of (H5N1) viruses obtained from 2008 to 2011 from various regions of Indonesia for sensitivity to the NAIs. We have screened samples for sensitivity to oseltamivir and zanamivir in a fluorescence based enzyme inhibition assay as previously described (McKimm-Breschkin et al., 2013a). We also determined the kinetics of drug binding for those isolates showing altered sensitivity against both these drugs and peramivir (Barrett et al., 2011). Of 96 samples screened, based on box plot statistical analysis, four viruses were considered to be extreme outliers to oseltamivir, with IC50 values in the range of 46–62 nM (IC50 = concentration of drug to inhibit enzyme activity by 50%). Substitutions were different to those reported in our previous surveillance. Although the oseltamivir resistant viruses demonstrated altered kinetics of drug binding for zanamivir and peramivir, none of the viruses were extreme outliers to these drugs, with all IC50 values less than 10 nM.
Section snippets
Viruses
HPAI (H5N1) viruses isolated primarily from chickens were supplied to the CSIRO Australian Animal Health Laboratory (AAHL) as part of the Indonesian IVM program (Hartaningsih et al., 2015). Viruses were amplified in specific pathogen free eggs under BSL3 conditions at AAHL. Virus cultures were inactivated by gamma irradiation prior to use in the fluorescence based NA inhibition assays.
Chemicals and inhibitors
Zanamivir and peramivir were kindly provided by GlaxoSmithKline (Stevenage, UK). Oseltamivir carboxylate was
NAI susceptibility
A total of 96 clade 2.1 (H5N1) virus samples were tested in this study (Table 1 and Supplementary Table 2). Thirty-nine viruses were from the western Indonesian provinces of Sumatra and Aceh that were associated with HPAI outbreaks in 2008–2009. Thirty-two virus samples from 2009 were from the eastern province of South Sulawesi, and two 2008 viruses were from West Sulawesi. Finally, 23 viruses were from mostly central Indonesian provinces in Java, Bali and Kalimantan collected from 2010 to
Discussion
The WHO laboratories and National Influenza Centers are now more actively involved in surveillance and testing the sensitivity of human influenza isolates to the NAIs (Hurt et al., 2016). However, there remains limited surveillance of the NAI sensitivity of circulating avian influenza isolates, especially the HPAI (H5N1) and A(H7N9) viruses. Yet these remain as potential pandemic threats, and the NAIs remain the only globally approved influenza-specific antiviral.
We have previously demonstrated
Declarations of interest
None.
Author's contribution
Conceived and designed the experiments: JMB. Performed the experiments: JMB, SB, FW, PS, KRD. Analyzed the data: JMB, SB, FW, KRD. Contributed reagents/materials/analysis tools: P, MA, NH, JM. Wrote the paper: JMB, FW.
Acknowledgements
We would like to thank the veterinary and diagnostic scientists of the regional Disease Investigation Centers (DIC) in Indonesia, and the Diagnosis, Surveillance and Response group of the CSIRO Australian Animal Health Laboratory (AAHL) for collection of samples, diagnosis and culture of viruses and DNA sequencing. The support and encouragement of the Director of Animal Health, Directorate General of Livestock and Animal Health Services, Ministry of Agriculture, Indonesia is gratefully
References (41)
- et al.
Solid phase assay for comparing reactivation rates of neuraminidases of influenza wild type and resistant mutants after inhibitor removal
Antivir. Res.
(2014) - et al.
Oseltamivir resistance mutation N294S in human influenza A(H5N1) virus in Egypt
J. Infect. Public Health
(2009) - et al.
Surveillance at the molecular level: developing an integrated network for detecting variation in avian influenza viruses in Indonesia
Prev. Vet. Med.
(2015) - et al.
Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2014-2015
Antivir. Res.
(2016) - et al.
Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in oceania, South East Asia and South Africa
Antivir. Res.
(2009) - et al.
Assessing the development of oseltamivir and zanamivir resistance in A(H5N1) influenza viruses using a ferret model
Antivir. Res.
(2010) - et al.
Resistant influenza A viruses in children treated with oseltamivir: descriptive study
Lancet
(2004) - et al.
Host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in A(H1N1) viruses
Antivir. Res.
(2010) - et al.
Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl- alpha-D-N-acetylneuraminate) substrate
Anal. Biochem.
(1979) - et al.
Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2013-2014
Antivir. Res.
(2015)
Real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses
PLoS One
Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People's Democratic Republic
J. Gen. Virol.
Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome
Lancet
H7N9 Situation Update
Human infection with a novel avian-origin influenza A (H7N9) virus
N. Engl. J. Med.
Competitive fitness of oseltamivir-sensitive and -resistant highly pathogenic H5N1 influenza viruses in a ferret model
J. Virol.
BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT
Nucleic Acids Symp. Ser.
Zanamivir-resistant influenza viruses with a novel neuraminidase mutation
J. Virol.
Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets
PLoS Pathog.
Continuing threat of influenza (H5N1) virus circulation in Egypt
Emerg. Infect. Dis.
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2022, Comprehensive PharmacologyA novel I117T substitution in neuraminidase of highly pathogenic avian influenza H5N1 virus conferring reduced susceptibility to oseltamivir and zanamivir
2019, Veterinary MicrobiologyCitation Excerpt :The new influenza antiviral drug Baloxavir marboxil (trade name Xofluza) which is a PA protein inhibitor was approved in 2018 in Japan and the US for the treatment of influenza (Ng, 2019). As drug resistant variants may exist naturally or may emerge due to selection, resistance to these drugs due to amino acid changes in the viral NA has been studied extensively (Gubareva et al., 1997; Oh and Hurt, 2014; Takashita et al., 2015; McKimm-Breschkin et al., 2018). Several subtype-specific mutations in framework or catalytic residues of NA, such as V116A, I117V, E119A/G, Q136L/K, V149A, D198G, I222M/V, S246N, H274Y, N294S (N2 numbering) that confer resistance to these drugs have been described previously.