Highly pathogenic avian influenza (HPAI) H5N1 remains endemic in at least six countries including China, Egypt, Indonesia, Viet Nam, Bangladesh and parts of India, and continues to impact livelihoods and poultry farming in several other countries in South East Asia [1]. While not yet capable of human to human transmission, concerns regarding potential emergence of a pandemic virus remain as millions of poultry are infected annually. Sporadic transmission from poultry to humans continues; as of February 2013, the largest numbers of human cases reported to WHO were from Indonesia, Egypt, and Viet Nam (n = 192, 169, and 123 respectively) [2]. The case fatality rates vary significantly across these three countries, ranging from 36% (60/169) in Egypt to 83% (160/192) in Indonesia. This variation may reflect delays in initiating treatment including antiviral therapy, but may also represent some inherent variability in the drug susceptibility of different isolates.

Two drugs are licensed globally for the treatment and prevention of influenza, zanamivir (Relenza) administered by oral inhalation, delivering high doses to the upper respiratory tract, and oseltamivir (Tamiflu) which is taken orally and disseminates systemically. A third drug, peramivir, is licensed in Japan and South Korea for intravenous administration, but it is still undergoing clinical trials elsewhere. These drugs target the neuraminidase (NA) enzyme, a surface glycoprotein of the influenza virus, and are effective against all strains of influenza due to the high degree of conservation at the NA active site. Oseltamivir is the drug of choice for treatment of H5N1 infected patients due to concerns regarding potential systemic infection. Oseltamivir is also the primary drug stockpiled globally for potential pandemics. All these drugs, classed as neuraminidase inhibitors (NAIs), are designed based on 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) a transition state analogue of the sialic acid substrate. Zanamivir has a single modification of a C4-guanidinium group compared to DANA [3], while oseltamivir has both a C4-amino group, and a pentyl ether group replaces the glycerol side chain in DANA [4]. Peramivir has features of both inhibitors, with a C4-guanidinium group and a pentyl side chain [5]. Both oseltamivir and peramivir require structural rearrangements in the active site for high affinity binding. Residue E276 rotates to form a salt link to R224, creating the pocket which accommodates their hydrophobic side chains. Because of the structural changes required to accommodate the binding of these inhibitors, we had predicted resistance was more likely to arise against oseltamivir than zanamivir [6]. Widespread resistance to oseltamivir was demonstrated by the global spread of the oseltamivir resistant seasonal H1N1 influenza strain during 2007–9 [7]–[10].

Despite the millions of poultry infected with HPAI, routine surveillance for drug susceptibility has not been conducted on avian isolates with information primarily from small studies [11]–[15]. Analysis of NA gene sequences in public data bases has identified known NAI resistance mutations in HPAI H5N1 avian virus isolates. Sequence analysis revealed mixed populations of oseltamivir sensitive wild type viruses and some with the H274Y mutation conferring oseltamivir resistance from chickens, ducks and geese [16]. Virus with a N294S mutation was detected in ducks [17]. However, while sequencing approaches can detect mixtures of wild type and mutant populations, only known mutations can be detected [18]. In contrast, screening using the NA enzyme inhibition assay will detect phenotypic differences caused by any mutation, but the resistant population generally needs to be in excess over the wild type population to detect a shift in susceptibility [19]. The technique also requires level 3 biocontainment facilities for virus culture to produce sufficient material to be analyzed in the assay, which can be a barrier to large scale surveillance of drug susceptibility.

We previously evaluated a panel of clade 1 HPAI H5N1 viruses from Viet Nam and Cambodia and clade 2.1 viruses from Indonesia using a fluorometric 4-Methylumbelliferyl N-acetyl-α-D-neuraminic acid (MUNANA) based enzyme assay. We showed that the clade 2.1 viruses circulating in Indonesia had a lower susceptibility to oseltamivir with a mean IC₅₀ of 11.5 nM (IC₅₀ = concentration of drug to inhibit enzyme activity by 50%) compared to the clade 1 isolates with a mean IC₅₀ of 0.5 nM [13]. We suggested that an H252Y difference in the NA between clade 1.1 and clade 2.1 respectively was the most likely cause of the lower oseltamivir susceptibility. This was subsequently confirmed by others using mutagenesis [20], [21]. The Y252 impairs the rotation of E276 in the NA active site, which is necessary to form the hydrophobic pocket to accommodate the pentyl ether side chain of oseltamivir. Susceptibility to zanamivir is not affected. The reduced susceptibility to oseltamivir in vitro also corresponds to reduced susceptibility in animal models [20], [22].

We also reported that some HPAI H5N1 viruses isolated from the Kandal province in Cambodia in 2005 had approximately 6–7-fold reductions in susceptibility to oseltamivir compared to the isolates from 2004, but there was no difference in zanamivir susceptibility. We did not know whether this was a regional cluster or whether this was more widespread [13]. Based on observations of Rameix-Welti et al. [15] we suggested that a S246G mutation may be associated with this reduced susceptibility, but sequence data was not available at that time. The availability of further isolates from Cambodia has now enabled us to test this hypothesis further.

We aimed to extend the scope of our previous study by evaluating the drug susceptibility of a large panel of HPAI H5N1 virus isolates from Indonesia from 2006–2008 as well as avian isolates from Cambodia and Thailand from 2004–2007. Samples were screened against oseltamivir and zanamivir using the fluorometric MUNANA-based enzyme assay. Based on box plot statistical analysis, those identified as outliers were also screened against peramivir. Overall, higher IC₅₀s were confirmed for oseltamivir in clade 2.1 HPAI H5N1 viruses circulating in Indonesia compared to clade 1viruses. But more disturbingly, eight Indonesian viruses (5%), all bearing I222 mutations were identified statistically as mild to extreme outliers for oseltamivir. None of the H5N1 isolates tested demonstrated reduced susceptibility to zanamivir (IC₅₀s all <5 nM).

We used samples selected from our previous work as control reference strains [13] for the enzyme inhibition assays. These included a clade 1.1 virus from Viet Nam (A/chicken/Vietnam/08/2004) which was sensitive to both zanamivir and oseltamivir, a clade 1 virus from Cambodia (A/goose/Kandal/2005) which had demonstrated a 6 to 7-fold reduction in oseltamivir susceptibility, and an Indonesian clade 2.1 virus A/chicken/Wates/126/2005, which had previously displayed around a 15-fold reduction in oseltamivir susceptibility compared to the clade 1.1 Viet Nam viruses we had tested [13].

The importance of surveillance of the drug susceptibilities of human influenza isolates has led to widespread testing of NAI susceptibility of circulating human influenza strains in recent years, especially since the emergence of the oseltamivir resistant H1N1 seasonal strain in 2008 [7]–[10]. There are still concerns that a variant HPAI H5N1 strain may yet cause a pandemic, hence knowledge of the spectrum of drug susceptibility of H5N1 viruses is also critical for the management of patients infected with H5N1, as well as for stockpiling strategies for a potential pandemic arising from an H5N1 variant. There has been limited availability of avian influenza isolates for testing of drug susceptibilities, although even in those few tested there have been reports of decreased susceptibility of clade 2 isolates compared to clade 1 isolates [13], and additional mutations which have affected susceptibility including I117V, V116A, S246N, S246G and I222L [11], [12], [15].

In collaboration with the Indonesian Ministry of Agriculture, CSIRO AAHL has been involved with an FAO-implemented OFFLU technical project monitoring HA genetic and antigenic changes in Indonesian clade 2.1 HPAI H5N1 isolates. In addition to the information on the effects of HA drift mutations on antigenicity, these viruses provided a valuable opportunity for screening for susceptibility to the NAIs. The HPAI H5N1 viruses were from both commercial and backyard poultry, and covered different geographic areas of Indonesia. We also performed assays on a few virus samples from Cambodia and Thailand. Testing 166 HPAI H5N1 isolates in the MUNANA based enzyme inhibition assay detected no virus with an IC₅₀>5 nM for zanamivir. In contrast we saw a higher mean and median IC₅₀ for oseltamivir for the Indonesian isolates (∼25 nM) than in our previous report (11 nM). Of more concern is that we identified a number of clade 2.1 isolates from Indonesia which were phenotypically and genotypically resistant to oseltamivir. These mutations also resulted in different binding properties in our IC₅₀ kinetics assays, with loss of slow binding to oseltamivir, a feature seen with many resistant NAs [30].

Although the H274Y mutation, which confers resistance to oseltamivir, is the most commonly detected mutation in influenza N1 viruses, there are increasing numbers of reports of mutations at residue I222 (I223 N1 numbering) including I222T/V/M/L conferring reduced NAI susceptibility in pandemic H1N1, seasonal H1N1 and H5N1 influenza strains. We identified five viruses with I222T or I222V mutations with IC₅₀s in the 40–75 nM range, and three with I222M mutations, two with IC₅₀s greater than 250 nM. Unlike the H274Y which only confers resistance in the N1 subtype, I222 mutations cause reduced susceptibility in N1 [11], [27], [33]–[40], N2 [41] and influenza B [27], [42]–[44] NAs, and have been detected after oseltamivir treatment in patients and in vitro exposure, but also spontaneously without drug exposure. Of approximately 1500 highly pathogenic H5N1 NA sequences in the NCBI Influenza Virus Resource, we found four I222V, four I222L, seven I222T, one I222Q and one I222M, an incidence of 1%. All mutations were found in clade 2 strains, with only the I222T found in clade 1 sequences. Although mutations at I222 generally only reduce susceptibility by less than 20-fold, what is of more concern is that they act synergistically to increase resistance to very high levels with H274Y [35], [37], [41] and E119V mutations. Our results suggest that the I222 mutations may also act synergistically with the H252Y mutation in the clade 2 viruses. Hurt et al. [35] generated I222V and I222M H5N1 mutants in a clade 1 background, and IC₅₀s in their MUNANA based assay were 5 and 27 nM respectively. In contrast ours were 10-fold higher than these, with an IC₅₀ greater than 250 nM for the I222M viruses, indicating that H252Y difference in the clade 2.1 background sequence also contributed to the higher IC₅₀s. Interestingly the I222M mutation only had a small impact on peramivir binding, which also requires reorientation of E276 to form the hydrophobic pocket to accommodate its side chain [32], [45]. However even the H274Y mutation has less impact on peramivir binding compared to oseltamivir [30]. Similarly the H252Y difference between clade 1 and 2 viruses had a minimal effect on peramivir binding. This lack of effect may be due to the additional strong interactions of the 4-guanidinium group on peramivir.

We identified a virus with an I117V mutation, which although statistically was not an outlier, its IC₅₀ for oseltamivir was well above the median IC_50. There are more than 40 H5N1 viruses in the public sequence data bases with this mutation, and others have reported the spontaneous emergence of this mutation in H5N1 viruses from infected untreated ferrets [34] as well as in oseltamivir treated ferrets [46]. While others have reported I117V only confers a small difference in susceptibility in H5N1 isolates [11], [12], [28], [47], it has recently been demonstrated that the I117V acts synergistically with the H274Y mutation to increase oseltamivir and peramivir resistance to levels that would be of clinical concern [48]. Our results demonstrated that this mutation also affected the kinetics of oseltamivir binding compared to the wild type, hence it also appears to be acting synergistically with the H252Y mutation to further decrease oseltamivir susceptibility. We identified three additional isolates with IC₅₀s in the 30 nM range which also demonstrated loss of slow binding. None of these had known mutations conferring altered susceptibility or mutations in conserved residues, but this demonstrates that there may be numerous changes which can also act synergistically with the H252Y to further reduce efficacy of oseltamivir binding.

We demonstrated that the decreased susceptibility to oseltamivir seen in some Cambodian isolates correlated with an S246G mutation, in agreement with Rameix-Welti et al. [15]. Residue 246 is reported to mediate hydrogen bonded interactions with the substrate and inhibitors [49]. There was a recent report that the S246G mutation had no impact on oseltamivir susceptibility in Cambodian isolates [14]. However they used the chemiluminescent assay, and relative drug susceptibility can differ between this and the MUNANA based assay we used [26]. An S246N mutation was also reported to cause a 24-fold reduction in oseltamivir susceptibility in isolates from Laos [11]. Fortunately the S246G mutation that we saw in our 2005 isolates appeared to be a limited cluster.

While the resistant viruses here were identified by statistical analysis, compared to the wild type clade 1.1 reference virus some of these viruses were between 50- and 500-fold resistant, but only 2- to 20-fold resistant to the clade 2.1 reference virus. However, there is no consensus on a definition of resistance that is known to relate to clinical failure. Many laboratories use a 10-fold change in IC₅₀ in the enzyme inhibition assay compared to the wild type IC₅₀, however since some viruses have a higher base line IC₅₀, e.g. influenza B [26] and the clade 2.1 HPAI H5N1 strains, such viruses may be clinically resistant with only a few fold increase in IC₅₀ compared to their wild type counterparts. The IC₅₀ values also vary between the chemiluminescent and fluorescent enzyme inhibition assays [19], [26]. Furthermore our IC₅₀ kinetics experiments demonstrate how the IC₅₀ can change with incubation times in the NAI assays. However, for the drugs to have some therapeutic benefit the levels in vivo would need to be significantly higher than the IC₅₀s. The levels of oseltamivir in plasma are estimated to be in the range from 400 to 1200 nmol/L [50], [51]. Levels in saliva are estimated to be less than 5% of plasma levels [52]. Thus levels in the upper respiratory tract may be significantly lower than 100 nM. With IC₅₀s for oseltamivir for many of these outliers detected here >50 nM, they could present an even greater challenge for effective treatment with oseltamivir.

There are nine conserved residues in the NA active site which contact the sialic acid substrate, and a further ten residues which provide structural stability to these residues [23], [53]. Mutations conferring altered susceptibility to the NAIs have been mostly located within these amino acids [25]. However, the more the effects of mutations on the function of the NA are analyzed, the more it is obvious that non-active site residues can have subtle, but important effects on the enzyme function and stability. This was observed recently when an oseltamivir-resistant A/Brisbane/59/2007 like H1N1 virus emerged with an H274Y mutation, which rapidly spread globally, demonstrating no loss of fitness compared to the wild type virus. It has been demonstrated that three mutations (R222Q, V234M, D344N) compensated for the impact on fitness of the H274Y mutation [54], [55]. Our demonstration of differences in the kinetics of oseltamivir binding in NAs with no previously identified mutations emphasizes the need for phenotypic surveillance to detect the subtle effects of drift mutations on NAI susceptibility.

Since the emergence of the pandemic H1N1/09 virus there appears to be more complacency about the pandemic potential of HPAI H5N1 strains. However HPAI H5N1 viruses continue to spread and evolve and surveillance is of critical importance to inform on both the antigenic variation for vaccine preparation, as well as their antiviral susceptibilities. Pending the development of a suitable vaccine, antivirals will be the first line of defence in any new pandemic. Our analysis of the NAI susceptibility of 155 virus isolates from Indonesia identified eight (5%) outliers with reduced oseltamivir susceptibility with I222 mutations and a further four viruses also demonstrated loss of slow binding of oseltamivir. In comparison, even with the use of oseltamivir, only 1% of pandemic H1N1 isolates from humans have the H274Y mutation conferring oseltamivir resistance. The higher incidence of these I222 mutations that we observed is unexpected and of concern. Widespread phenotypic analysis of susceptibility of avian influenza HPAI H5N1 viruses to the NAIs needs to be carried out where the virus is endemic in poultry, to be able to respond in a timely fashion to the emergence of any strain capable of transmitting between humans.