Elsevier

Antiviral Research

Volume 153, May 2018, Pages 95-100
Antiviral Research

Identification of Indonesian clade 2.1 highly pathogenic influenza A(H5N1) viruses with N294S and S246N neuraminidase substitutions which further reduce oseltamivir susceptibility

https://doi.org/10.1016/j.antiviral.2018.03.007Get rights and content

Highlights

  • Susceptibilities to the NAIs of 96 HPAI clade 2.1 (H5N1) viruses from Indonesia were tested in an enzyme assay.

  • Box Plot analysis identified 4 viruses as extreme outliers to oseltamivir with IC50 values ranging from 46 to 62 nM.

  • Two of the NAs had an N294S substitution, one had an S246N substitution.

  • There was minimal effect on sensitivity to zanamivir or peramivir for any of the isolates, including the mutants.

Abstract

We have tested the in vitro susceptibility to the neuraminidase (NA) inhibitors of 96 highly pathogenic clade 2.1 A(H5N1) viruses from Indonesia, isolated between 2008 and 2011. HPAI virus samples obtained through the Influenza Virus Monitoring (IVM) surveillance program in Indonesia were tested for susceptibility to oseltamivir and zanamivir. The NAs of four viruses were identified as extreme outliers to oseltamivir, based on statistical analysis by box plots, with IC50 values ranging from 46 to 62 nM. The NAs of two of these viruses from Sumatra and Aceh, had an N294S substitution, while one virus from Sulawesi had an S246N NA substitution. The NAs of all four viruses showed a specific loss of slow binding to oseltamivir in an IC50 kinetics assay. As observed in our previous surveillance, there was only a minimal effect on the sensitivity to zanamivir or peramivir for these mutants or any of the other isolates tested. The continued circulation of subtype H5N1 viruses in avian species poses an on-going zoonotic threat. The fact that we continue to identify avian isolates with naturally occurring mutations conferring reduced oseltamivir susceptibility remains a concern, given oseltamivir will be a key antiviral in the event of a new pandemic emerging.

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

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