﻿Antiviral resistance among highly pathogenic influenza A (H5N1)
viruses isolated worldwide in 2002­2012 shows need for
continued monitoring
Elena A. Govorkovaa, Tatiana Baranovicha, Patrick Seilera, Jianling Armstronga, Andrew
Burnhama, Yi Guanb, Malik Peirisb, Richard J. Webbya, and Robert G. Webstera,*
aDepartment of Infectious Diseases, St. Jude Children's Research Hospital, Memphis,
Tennessee, 38105-3678, USA
bJoint Influenza Research Center (Shantou University Medical College & Hong Kong University),
Shantou University Medical College, Shantou, Guangdong 515031, P.R. China
Abstract
Highly pathogenic (HP) H5N1 influenza viruses are evolving pathogens with the potential to
cause sustained human-to-human transmission and pandemic virus spread. Specific antiviral drugs
can play an important role in the early stages of a pandemic, but the emergence of drug-resistant
variants can limit control options. The available data on the susceptibility of HP H5N1 influenza
viruses to neuraminidase (NA) inhibitors and adamantanes is scarce, and there is no extensive
analysis. Here, we systematically examined the prevalence of NA inhibitor and adamantane
resistance among HP H5N1 influenza viruses that circulated worldwide during 2002­2012. The
phenotypic fluorescence-based assay showed that both human and avian HP H5N1 viruses are
susceptible to NA inhibitors oseltamivir and zanamivir with little variability over time and ~5.5-
fold less susceptibility to oseltamivir of viruses of hemagglutinin (HA) clade 2 than of clade 1.
Analysis of available sequence data revealed a low incidence of NA inhibitor­resistant variants.
The established markers of NA inhibitor resistance (E119A, H274Y, and N294S, N2 numbering)
were found in 2.4% of human and 0.8% of avian isolates, and the markers of reduced
susceptibility (I117V, K150N, I222V/T/K, and S246N) were found in 0.8% of human and 2.9 %
of avian isolates. The frequency of amantadine-resistant variants was higher among human
(62.2%) than avian (31.6%) viruses with disproportionate distribution among different HA clades.
As in human isolates, avian H5N1 viruses carry double L26I and S31N M2 mutations more often
than a single S31N mutation. Overall, both human and avian HP H5N1 influenza viruses are
susceptible to NA inhibitors; some proportion is still susceptible to amantadine in contrast to
~100% amantadine resistance among currently circulating seasonal human H1N1 and H3N2
viruses. Continued antiviral susceptibility monitoring of H5N1 viruses is needed to maintain
therapeutic approaches for control of disease.
© 2013 Elsevier B.V. All rights reserved.
Reprints or correspondence: Robert G. Webster, Department of Infectious Diseases, St. Jude Children's Research Hospital, 262
Danny Thomas Place, Memphis, TN 38105-3678; robert.webster@stjude.org; Telephone: 901-595-3400; Fax: 901-595-8559.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
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Conflict of interest: The authors have no personal or financial affiliation with a commercial entity that might pose a conflict of
interest.
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Antiviral Res. 2013 May ; 98(2): 297­304. doi:10.1016/j.antiviral.2013.02.013.
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Keywords
H5N1 influenza virus; neuraminidase inhibitors; resistance; oseltamivir; zanamivir; zanamivir
Introduction
Highly pathogenic (HP) influenza A (H5N1) viruses predominantly affect avian species but
occasionally cross the species barrier and infect humans. Since 2003, outbreaks of H5N1
influenza viruses have been reported in domestic poultry and wild birds in 63 countries/
territories (WHO/OIE/FAO, 2012). Human H5N1 virus infections were first reported in
1997, and since the virus reappeared in humans in 2003, it continues to cause sporadic
infections of humans in Southeast Asia, the Middle East, Europe, and Africa with a total of
>600 human cases and >60% mortality rates (www.who.int).
To date, multiple clades/subclades of H5N1 influenza viruses have been distinguished by
phylogenetic analysis of the hemagglutinin (HA) gene, some of which have a distinct
geographical distribution. Clade 1.1 viruses, which evolved from clade 1, continue to
circulate in Vietnam and Cambodia. Clade 2 viruses are the most diverse group of H5N1
viruses and can be divided into 5 circulating subclades: 2.1.3 variants circulate in Indonesia,
2.2 viruses circulate in India and Bangladesh, 2.2.1 viruses are found in Egypt, 2.3.2
encompasses viruses found from Southeast and Central Asia to Eastern Europe, and 2.3.4
viruses circulate in Vietnam, Laos, Thailand, and China (Marinova-Petkova et al., 2012).
Virus clade 2.3.2 in its various forms is now considered the dominant type in China,
although clade 2.3.4 has not disappeared (WHO/OIE/FAO, 2012). Clade 7 viruses are also
found in circulation in Vietnam and China. Since 1997, human infections have been caused
by H5N1 viruses of clades 0, 1, 2.1, 2.2, 2.3, and 7; however, infections in 2010­2012 have
been predominantly caused by viruses of clades 2.3.2 and 2.3.4.
The current pandemic in poultry and wild birds is a potential threat to human health due to
continuous evolution and genetic diversity of circulating avian influenza H5N1 viruses and
the contribution of genetic material from avian influenza viruses to the emergence of human
pandemic virus. In response to this threat, major efforts have been made to develop
immunogenic cross-clade­protective H5N1 vaccines (Subbarao and Luke, 2007; Prieto-Lara
and Llanos-Méndez, 2010). In the absence of an effective vaccine, antiviral prophylaxis and
treatment can play an important role. The neuraminidase (NA) inhibitors oseltamivir and
zanamivir are the recommended antiviral drugs against influenza. Patients infected with
H5N1 viruses have been predominantly treated with oral oseltamivir (Writing Committee,
2008; Adisasmito, 2010). Analysis of Avian Influenza Registry data from 10 countries
showed that the strongest impact on survival among H5N1-infected patients was observed
when treatment was initiated 2 days after symptom onset (Chan et al., 2012), and there was
an increased likelihood of survival when treatment was initiated as late as 3­5 days after
symptom onset but before respiratory failure occurred (Adisasmito et al., 2010).
The effectiveness of antiviral drugs will depend on the susceptibility of the pandemic strain,
should it emerge. Oseltamivir carboxylate (the active ingredient of oseltamivir) was shown
to be active in vitro against human and avian H5N1 influenza viruses (McKimm-Breschkin
et al., 2007; Hurt et al., 2007). NA enzyme inhibition assays revealed that clade 2.1 viruses
from Indonesia have a naturally occurring 15- to 30-fold lower sensitivity to oseltamivir
carboxylate in vitro than clade 1 viruses from Vietnam (McKimm-Breschkin et al., 2007),
which was attributed to NA's H252Y amino acid difference between the clades (Ramiex-
Welti et al., 2006). In contrast, Hurt and colleagues (2007) found little variation in the
sensitivity of the NAs of 51 avian H5N1 isolates collected over a similar time in similar
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regions. Overall, available data on in vitro sensitivity to oseltamivir carboxylate generally
show sensitivities to drug concentrations well below the minimum in vivo concentrations
achieved during therapy (Le et al., 2008). Susceptibility of H5N1 viruses to zanamivir was
not affected (McKimm-Breschkin et al., 2007).
Resistance to oseltamivir in clinically derived seasonal influenza A viruses was associated
with H274Y or N294S amino acid substitutions in the N1 NA subtype (N2 numbering used
here and throughout the text) and E119V, R292K or N294S substitutions in the N2 NA
subtype (McKimm-Breschkin, 2012). Oseltamivir-resistant H5N1 influenza viruses with an
H274Y NA mutation were reported in 3 patients during oseltamivir treatment or prophylaxis
(de Jong et al., 2005; Le et al., 2005). One of these patients had a mixed population of wild-
type NA and both H274Y and N294S mutations (Le et al., 2005). Our previous studies
showed that some Egyptian H5N1 isolates from humans have had an N294S NA mutation,
which confers a 12- to 15-fold increase in the IC50 value in an NA inhibition assay (Earhart
et al, 2009; Kayali et al., 2011). Screening of 29 HP H5N1 viruses of clade 2.3.2 from the
Republic of Laos in 2006­2008 identified three outliers with reduced NA inhibitor
susceptibility with different mutations (V116A, I222L, and S246N) (Boltz et al., 2010). A
minor subpopulation of drug-resistant clones with I117V and E119A NA mutations (the
latter being associated with zanamivir resistance in the N2 NA subtype) were detected in
human A/Turkey/65-1242/2006 (H5N1) virus (Govorkova et al., 2009).
The worldwide emergence of drug-resistant seasonal H3N2 and H1N1pdm09 influenza
variants (Bright et al., 2006; Deyde et al., 2007; Gubareva et al., 2010) restricted the use of
another class of specific anti-influenza drugs, M2 ion channel inhibitors (amantadine and
rimantadine). Although the antiviral activity of amantadine against HP H5N1 was seen in
vitro and in vivo (Ilyushina et al., 2007), the data on its therapeutic effectiveness is lacking.
The WHO guidelines from 2007 do not recommend treatment of H5N1 virus­infected
patients with amantadine unless the infecting virus is known to be susceptible or other drugs
are unavailable (Schunemann et al., 2007). Molecular markers of resistance to adamantanes
are amino acid substitutions at residues L26, V27, A30, S31, and G34 within the
transmembrane domain of the M2 protein (Hay et al., 1986; Pinto et al., 1992; Li et al.,
2004). Previous publications reported emergence and geographic diversity in the distribution
of amantadine-resistant H5N1 influenza viruses (Cheung et al., 2006; Monne et al., 2008;
Tosh et al., 2011). Amantadine-resistant H5N1 viruses were reported in Saudi Arabia,
Thailand, Vietnam, Cambodia, Malaysia, Indonesia, China, and India, although the
prevalence of resistant viruses varies in different geographical areas (Cheung et al., 2006;
Hurt et al., 2007; Monne et al., 2008; Tosh et al., 2011).
There are ongoing concerns that H5N1 viruses may yet cause a pandemic; hence, antiviral
surveillance of HP H5N1 is essential for the determination of our options for the control of a
pandemic. Here we provide a comprehensive analysis of the antiviral susceptibility of the
HP human and avian H5N1 influenza viruses isolated during the past decade (2002­2012)
based on phenotypic analysis and determination of amino acid changes occurring at the
conserved or semi-conserved NA residues (GenBank data) that may confer either a resistant
or reduced susceptibility genotype. The incidence of amantadine resistance among multiple
and evolving HA clades of H5N1 viruses was analyzed based on the sequence data
generated in the current study and that available in GenBank.
2. Materials and methods
2.1. Viruses
Ten human and 85 avian (originating from wild birds, ducks, geese and chickens) HP H5N1
influenza viruses representatives of HA clades 1 and 2 and isolated in 2002­2011 were
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obtained through the WHO network. The viruses were propagated in the allantoic cavities of
10-day-old embryonated chicken eggs at 35°C for 40 hours. All experiments were conducted
in biosafety level 3+ conditions in compliance with applicable laws and guidance.
2.2. Compounds
The NA inhibitors oseltamivir carboxylate (oseltamivir, [3R,4R,5S]-4-acetamido-5-
amino-3-[1-ethylpropoxy]-1-cyclohexene-1-carboxylic acid) and zanamivir (4-guanidino-
Neu5Ac2en) were provided by Hoffmann-La Roche, Ltd. (Basel, Switzerland). Stocks of
oseltamivir and zanamivir were prepared in distillated water, filter-sterilized, and stored in
aliquots at -20°C.
2.3. NA inhibition assay
NA activity of the H5N1 influenza viruses was measured in a fluorescence-based assay
using the fluorogenic substrate 2'-(4-methylumbelliferyl)- -D-N-acetylneuraminic acid
(MUNANA) (Sigma-Aldrich, St. Louis, MO) (Potier et al., 1979). Fluorometric
determinations were quantified with a Synergy 2 multi-mode microplate reader (BioTek
Instruments, Winooski, VT) based on the release of the fluorescent product 4-methyl-
umbelliferone using excitation and emission wavelengths of 360 and 460 nm, respectively.
Viruses were standardized to equivalent NA enzyme activity in the linear range of the curve
and were mixed with various concentrations of inhibitor in 96-well flat-bottom black opaque
plates (Corning Costar, NY). The final reaction mixture concentrations of the NA inhibitors
ranged from 0.05 nM to 5,000 nM. The virus-inhibitor mixture was incubated at 37°C for 30
min prior to the addition of MUNANA substrate and then incubated at 37°C for 30 min. The
reaction was terminated by the addition of the stop solution (0.1 M glycine in 25% ethanol,
pH 10.7). The concentration of NA inhibitor that reduced NA activity by 50% relative to a
control mixture with no NA inhibitor (IC50) was determined by plotting the percent
inhibition of NA activity as a function of the compound concentrations calculated using
GraphPad Prism 5 software (GraphPad Software, La Jolla, CA). IC50 values were recorded
as the means of 2­3 independent determinations. Reference oseltamivir-susceptible and -
resistant A/Mississippi/3/2001 (H1N1) influenza strains (mean IC50, 0.53 and 299.58 nM,
respectively) were obtained from the Antiviral Group, International Society for Influenza
and Other Respiratory Virus Diseases, and were included in each assay and showed an IC50
variability of 8% over 6 separate assays.
2.4. Data analysis
The obtained IC50 values were analyzed for oseltamivir and zanamivir to determine
statistical cutoffs for identification of potentially resistant viruses (outliers) using GraphPad
Prism 5 software (GraphPad Software, La Jolla, CA). Quantile box-and-whisker plots were
used to display the distribution of log-transformed IC50 values. The bottom and the top of
the box marked the 10th and 90th percentiles, and the line across the box marked the 50th
percentile or median. Whiskers were added to identify potential outliers and extended above
and below the box by 1.5 times the interquartile range. Mild outliers had IC50 values above
the statistical cutoff, which was >3-fold but <10-fold greater than the mean IC50 for the
drug. The isolates with 10-fold of the mean IC50 value were considered extreme outliers
and were excluded from statistical analysis of the overall population.
2.5. Susceptibility to the adamantanes
Genetic analysis of the transmembrane region of the M2 protein was conducted, and
substitutions of five residues (L26, V27, A30, S31, and G34) were used to screen for
molecular markers of adamantine resistance. RNA extraction was performed using the
RNeasy kit (QIAGEN, Valencia, CA), and RT-PCR was performed using the SuperScript III
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One-Step RTPCR System with Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA)
according to the manufacturer's protocol. Sequencing was performed by the Hartwell Center
for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital. DNA
sequences were completed and edited using the Lasergene sequence analysis software
package (DNASTAR, Madison, WI).
In addition, the alignments using ClustalW were performed in BioEdit 7 (Ibis Biosciences,
Carlsbad, CA), which was also used to scan all of the above-mentioned mutations. The 28
M2 gene sequences from this study as well as 208 human and 1433 avian sequences of HP
H5N1 viruses obtained from the GenBank database (www.ncbi.nlm.nih.gov as of October
2012) were included in the analyses.
2.6. NA sequence analysis
All viruses with elevated IC50 values (in this study, mild outliers with IC50 >3-fold but <10-
fold greater than the mean IC50 for oseltamivir) were subject to sequence analysis and
characterization of individual virus clones (28­30 clones) by using a TOPO TA Cloning Kit
for Sequencing (Invitrogen, Carlsbad, CA). The position of the identified NA mutations in
relations to the proximity to the residues located within catalytic site and within 3 Å of the
drug bound sites was analyzed as described elsewhere (Maurer-Stroh et al., 2009). In
addition, the NA gene sequences of 287 human and 1716 avian HP H5N1 viruses (those
with full-length NA sequences and those covering amino acids from 116 to 345 of the NA
obtained from the GenBank database were analyzed for the presence of NA mutations
associated with either NA inhibitor­resistant genotype in different NA subtypes (E119V/I/
A/G, H274Y, R292K, and N294S) or reduced susceptibility genotype to NA inhibitors
(V116A, I117V, K150N, D198N/G/E/Y, I222V/T/R/K/M, S246N, and E276D). The HAs of
all H5N1 influenza viruses included in the analysis had a multibasic amino acid motif (R-X-
R/K-R) in the HA connecting peptide.
2.7. Nucleotide sequence accession numbers
The NA (2 viruses, accession numbers KC436119 and KC436121) and M gene (28 viruses;
accession numbers KC436109 - KC436118, KC436120, KC436123 - KC436119) sequences
determined in this study were deposited into the GenBank database.
HA clade determination--The corresponding HA sequences of H5N1 influenza viruses
with M2 protein resistance markers were obtained from GenBank, and H5N1 viruses
representative of the HA clades were obtained from the WHO website (WHO/OIE/FAO,
2012). The human, avian, and reference HA sequences were edited using BioEdit 7 and
aligned using MUSCLE (Edgar 2004). Neighbor-joining phylogenetic trees were generated
using MEGA software (MEGA v5.05) (Tamura et al., 2011), and the HA clades were
determined for human and avian amantadine-resistant viruses.
3. Results
3.1. Susceptibility of H5N1 viruses to NA inhibitors
This study was undertaken to evaluate the antiviral susceptibility of the HP H5N1 influenza
viruses isolated during past decade (2002­2012) to NA inhibitors, and to determine the
incidence of drug-resistance associated mutations among these rapidly evolving viruses. A
total of 95 H5N1 viruses (10 human and 85 avian) representing HA clades 1 and 2 were
screened in a phenotypic fluorescence-based assay and were found to be fully susceptible to
oseltamivir and zanamivir, although the IC50 values for oseltamivir were ~2-fold higher than
those for zanamivir (mean IC50, 1.48 and 0.65 nM, respectively). The human and avian
isolates were equally susceptible to oseltamivir (mean IC50, 1.53 and 1.43 nM, respectively),
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although the number of human isolates tested was limited to 10 strains. No differences were
observed in the susceptibility to zanamivir. Analysis was also undertaken by year of H5N1
virus isolation to determine whether the distribution of IC50 values varied in different years
and whether a significant increase in mean IC50 values could be observed (Fig. 1A, B). The
H5N1 viruses isolated in 2005­2007 had the highest IC50 values to both NA inhibitors, but
we did not see a significant linear tread to sensitivity over time.
Although the mean IC50 values varied little, there were variant outliers with sensitivities
both slightly higher and lower than the means. Only a few viruses had IC50 values above the
upper quantile limit for oseltamivir (4 of 95) and zanamivir (2 of 95). Our phenotypic assay
identified 3 mild outliers for oseltamivir among avian H5N1 viruses and none for zanamivir
(Table 1). The subsequent clonal analysis of NA genes found that 4­5 individual clones
among 28­30 sequenced had different single NA amino acid substitutions. These NA
substitutions were random and did not occur in the positions reported previously to be
associated with either NA inhibitor resistance or reduced susceptibility phenotype. The
location of E228D NA mutation was close to the NA enzyme active site and may be
important for future analysis.
Analysis of the susceptibility distribution among HP H5N1 viruses representative of
different HA clades showed that viruses of clade 1 were ~5.5-fold more sensitive to
oseltamivir and ~1.7-fold more sensitive to zanamivir (mean IC50, 0.31 ± 0.26 nM and 0.40
± 0.04 nM, respectively; 30 viruses tested) than viruses of HA clade 2 (mean IC50, 1.72 ±
0.32 nM and 0.69 ± 0.07 nM, respectively; 65 viruses tested) (Fig. 1C, D). The viruses of
clades 2.3.2 and 2.3.4, which are predominant H5N1 variants circulating in poultry in
Southeast Asia (WHO/OIE/FAO, 2012), were susceptible to both NA inhibitors. Overall,
our data revealed that most H5N1 viruses retained susceptibility to oseltamivir and
zanamivir with little variability over time, although with some variability among divergent
HA genetic groups.
3.2. NA molecular markers of resistant or reduced susceptibility phenotype
Analysis of N1 NA sequences of 287 human HP H5N1 viruses isolated in 2002­2012
identified 7 viruses (2.4%) with NA inhibitor­resistant NA mutations (Table 2), although A/
Hanoi/30408/2005 (H5N1) virus had clones carrying both the H274Y and N294S NA
mutations (Le et al., 2005) and was counted as a single NA inhibitor­resistant variant. In
addition, 7 human H5N1 viruses (2.4%) had I117V, K150N, or I222V/T/K NA mutations.
Analysis of N1 NA sequences of 1716 avian HP H5N1 viruses identified 13 strains (0.8%)
with either oseltamivir-resistant (H274Y and N294S) or zanamivir-resistant (E119A) NA
mutations (Table 2). Two H5N1 viruses isolated in 2008 had both I117V and E119A NA
mutations. The NA mutations reported to reduce the susceptibility of influenza viruses to
NA inhibitors (V116A, I117V, K150N, D198N, I222V/T/M, and S246N) were identified in
50 avian viruses (2.9%) (Table 2). Double mutant viruses with I117V and D198N, I222T
and S246N, and K150N and S246N NA mutations were isolated in 2005, 2006, and 2007,
respectively. No NA inhibitor resistance­associated mutations were detected among human
and avian H5N1 viruses isolated in 2010­2012, although the number of isolates was low.
Thus, available N1 NA sequence data suggest a low incidence of mutations that affect
susceptibility to NA inhibitors among both human and avian H5N1 viruses.
3.3. Susceptibility to M2 inhibitors
A total of 1669 M2 gene sequences (208 human and 1433 avian sequences obtained from
GenBank database and 28 generated in this study) were analyzed for the presence of
molecular markers of amantadine resistance. The incidence of amantadine resistance among
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human H5N1 influenza viruses varied in different time periods (Fig. 2A). The majority
(36/37, 97.3%) of human H5N1 viruses isolated in 2002­2004 were amantadine-resistant;
however, the number of amantadine-resistant isolates decreased to 58.2% (82/141) and
38.7% (12/31) among viruses isolated in 2005­2007 and 2008­2012, respectively. The
amantadine-resistant phenotype of human H5N1 influenza viruses was predominantly
caused by either a single V27A or S31N amino acid substitution or double L26I + S31N or
V27A + S31N M2 mutations. We did not identify human H5N1 viruses with amino acid
substitutions at positions 26, 30, and 34 previously reported to be associated with resistance.
Although most drug-resistant human seasonal A (H1N1) and A (H3N2) viruses contain an
S31N substitution, only 10.1% of amantadine-resistant human H5N1 viruses contained this
substitution (Fig. 2A). The strong association of L26I and S31N M2 mutations reported
previously in H5N1 viruses continues to be present in viruses isolated in 2008­2012 and
was detected at a similar frequency as a single S31N substitution. Overall, double L26I +
S31N M2 mutations were more predominant among human HP H5N1 influenza viruses
isolated during 2002­2012 than a single S31N substitution.
Among avian H5N1 influenza viruses, the incidence of amantadine resistance markers was
lower than that in human viruses. The frequency of isolation of amantadine-resistant variants
was the highest (193/388, 49.7%) among viruses circulated in 2002­2004 and decreased in
the following years (197/825, 23.9% and 71/247, 28.7% in 2005­2007 and 2008­2012,
respectively, Fig. 2B). As in human isolates, avian H5N1 viruses isolated during 2002­2012
had double L26I + S31N M2 mutations at a higher frequency than a single S31N mutation,
although viruses with single S31N and V27A mutations were identified (Fig. 2).
Interestingly, the distribution of amantadine-resistant variants among human H5N1 viruses
was restricted to HA clade 1 during 2002­2004 (36/36, 100%), a diverse distribution pattern
in 2005­2007, and back to predominant isolation from clade 1.1 (10/12, 83.3%) in recent
years (Table 3). The proportion of amantadine-resistant avian influenza H5N1 viruses from
clade 1 has reduced over time from 83.9% in 2002­2004 to 43.2% in 2008­2012, with clade
2 representing 51.4% of isolates in 2008­2012. Taken together, the amantadine-resistant
human variants isolated from 2002­2012 belong equally to HA clades 1 and 2 (46.2% and
53.8%), but avian variants were most frequently isolated from clade 1 (~70%). Continued
diversification of avian HP H5N1 virus is also evidenced by the increasing number of clades
from which amantadine-resistant avian variants were isolated, from 4 clades in 2002­2004
to 7 clades in 2008­2012.
4. Discussion
The acquisition of NA inhibitor resistance by H5N1 influenza viruses is a serious public
health concern and monitoring the susceptibility of these viruses to available drugs is an
important part of surveillance studies and an informative aspect of risk assessment for a
pandemic. In this study, we focused on the set of questions regarding antiviral susceptibility
and resistance in the highly pathogenic H5N1 influenza viruses. We addressed what is the
level of susceptibility to NA inhibitors and adamantanes over time and among different HA
clades, what is the level of antiviral resistance among human and avian H5N1 viruses, and
whether there is evidence for an increased selection of antiviral resistance in recent years.
Our phenotypic analysis showed that human and avian H5N1 viruses are highly susceptible
to NA inhibitors oseltamivir and zanamivir. Comparison of the HA clade distribution of
susceptibility revealed ~5.5-fold higher susceptibility of viruses of clade 1 than those of
clade 2, and this difference was more pronounced (~10-fold) when we compared clade 1 and
clade 2.1 viruses. Although the number of clade 2.1 viruses was limited, we confirmed the
data reported previously (McKimm-Breshkin et al., 2007) about the lower susceptibility of
viruses of clade 2 to NA inhibitors in a phenotypic assay. However, this difference in
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susceptibility is attributed to a specific subclade of clade 2, and currently circulating 2.3.2
and 2.3.4 viruses are more susceptible to NA inhibitors than viruses of clade 2.1. The
observed differences in the susceptibility in different years also most likely is correlated
with the predominant HA clade circulating in this particular season, and the higher level of
susceptibility in 2002­2004 is correlated with predominant circulation of viruses of clade 1
(WHO/OIE/FAO, 2012).
Even though some NA mutations, such as H252Y, can increase the affinity of NA for
oseltamivir and thus lead to increased antiviral susceptibility (Ramiex-Welti et al., 2006;
McKimmm-Breshkin et al., 2007), the emergence of other mutations can potentially
decrease susceptibility. We therefore analyzed the available NA sequence data for the
presence of NA mutations that were previously reported to decrease susceptibility to NA
inhibitors. The screening revealed similarity between human and avian H5N1 influenza
viruses and a low level of NA mutations associated with NA inhibitor resistance. The
H274Y NA mutation is the most frequent mutation in human oseltamivir-resistant isolates of
the N1 NA subtype (Dharan et al., 2009; Weinstock et al., 2009). This mutation was
detected in 4 human and 4 avian H5N1 viruses isolated in 2002­2012. Only 4 human and 5
avian isolates contained the N294S NA mutation. The zanamivir-related mutation E119A
was detected in 4 avian isolates from 2008 and confirmed the finding by Govorkova et al.
(2009) that this mutation can be stably maintained in the N1 NA background. The amino
acid substitutions at some of the targeted residues in NA (e.g., V116, I117, K150, D198,
I222, and S246) have previously been linked to reduced drug susceptibility in avian and
human N1 viruses (Hurt et al., 2009; Ilyushina et al., 2010). However, most of the studies
were conducted with recombinant viruses, and their relevance to clinical resistance still
needs to be proven. Interestingly, one of the most predominant substitutions in both human
and avian H5N1 influenza viruses was at residue 222. This mutation alone did not confer a
resistant phenotype in seasonal H1N1 viruses (Ison, 2011). However, in combination with
mutation H274Y, the IC50 increased almost 2000 times for one H5N1 strain (Matrosovich
and Klenk, 2003). The NA I222R mutation was identified in the clinical isolates of patients
receiving oseltamivir treatment, and this mutation alone can to a small degree reduce the
H1N1pdm09 virus susceptibility to all three NA inhibitors (oseltamivir carboxylate,
zanamivir and peramivir) (Nguyen et al., 2010). The I222R + H274Y dual mutations further
enhanced the resistance level to oseltamivir and caused moderate resistance to zanamivir
(van der Vries et al., 2010; Pizzorno et al., 2011). Amino acid mutations at framework
residues such as I222 may interfere with the correct binding of NA inhibitors, thus
disrupting the natural susceptibility of influenza viruses to these agents. The wide spread of
drug-resistant variants is connected to the fitness of these viruses. The selected drug-
resistant mutations that can provide viral fitness benefits can be maintained without direct
selection for the mutation. The available reports on the fitness of HP oseltamivir-resistant
H5N1 viruses are focused on viruses of the two HA clades that are causing infection in
humans: clade 1 and clade 2.2 (Yen et al, 2007; Govorkova 2010; Ilyushina 2010; Kiso et al
2011). Experimental evidence suggests that a particular NA inhibitor resistance­associated
marker can cause different effects on fitness in different H5N1 virus genetic and virulence
backgrounds. Deficiency in NA function caused by an NA inhibitor resistance mutation may
not be deleterious for HP H5N1 viruses because of the extremely efficient replication of
these viruses. Overall, it appears that in the H5N1 population, oseltamivir resistance is not
yet undergoing positive selection. This might be because of the limited number of H5N1-
infected patients treated with oseltamivir (Adisasmito et al., 2010) and the fact that the NA
gene in H5N1 influenza viruses is evolutionarily constrained at a majority (84%) of
positions (Hill et al., 2009). This highlights that H5N1 is still primarily a disease of birds.
Monitoring of the natural selection of additional NA mutations at the conserved and semi-
conserved NA residues in addition to established molecular markers of resistance is
warranted.
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Our data revealed the disproportional levels of amantadine resistance observed in H5N1
viruses and human seasonal H3N2 and H1N1pdm09 viruses in recent years (Deybe et al.,
2007; Zaraket et al. 2010; Zhou et al., 2011). Importantly, the susceptibility of H5N1
influenza viruses to amantadine has increased in recent years (2010­2012). More likely, the
emergence and spread of amantadine-resistant H5N1 viruses occurs through positive drug
selection pressure in avian species. This conclusion correlates with the data reported
previously that there has been prophylactic use of amantadine in poultry production in some
parts of China (He et al., 2008). The application of phylogenetic methods based on analysis
of multiple gene segments, molecular evolutionary analyses, and geographic visualization
also concluded that amantadine use is driving selection for antiviral resistance in the global
H5N1 population (Wallace and Fitch, 2008; Hill et al., 2009). Our findings suggest that
amantadine-resistant lineages from other subtypes have limited introduction into the H5N1
subtype, although more sophisticated analysis is required to understand the frequency of
possible reassortment events with other influenza subtypes. Our data suggest that some
residues (L26I + S31N) in the M2 protein of H5N1 viruses are under stronger drug selection
pressure. The predominance of double M2 mutations among human and avian H5N1 viruses
also suggests a different pattern of selection than with seasonal H3N2 and H1N1pdm09
viruses, which are characterized by single S31N M2 mutations (Bright et al. 2006; Barr et
al., 2008). Interestingly, we observed disproportional distribution of amantadine-resistant
variants among avian and human viruses isolated in different years, most likely associated
with a particular HA clade circulating in a given geographic area (Bar et al., 2008; Le et al.,
2008).
In conclusion, given the expanding diversity of H5N1 viruses both geographically and
phylogenetically, continued surveillance of HP H5N1 viruses is needed to determine the
incidence of drug-resistant strains in both humans and avian species and to identify
molecular markers underlying these changes. These measures would allow maintenance of
therapeutic and possibly prophylactic regimens for antiviral control of disease.
Acknowledgments
We gratefully acknowledge the editorial assistance of David Galloway, the excellent technical support of the
Hartwell Center for Bioinformatics and Biotechnology, and Betsy Williford for help with illustrations.
Financial support: This work was supported by Contract HHSN266200700005C from the National Institute of
Allergy and Infectious Diseases and Cancer Center Support (CORE) grant P30 CA 21765 from the National Cancer
Institute, National Institutes of Health, and by the American Lebanese Syrian Associated Charities (ALSAC).
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Highlights
· Highly pathogenic H5N1 influenza viruses are susceptible to neuraminidase
inhibitors in vitro.
· A low incidence of oseltamivir-resistant markers in H5N1 influenza viruses
have been detected.
· An increase in susceptibility of H5N1 influenza viruses to adamantanes in
recent years was identified.
· Antiviral susceptibility surveillance of H5N1 influenza viruses is important.
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Fig. 1.
Plots showing the IC50 (nM) ranges of oseltamivir and zanamivir for human and avian
influenza H5N1 viruses tested by a phenotypic fluorescence-based NA inhibition assay.
Panels A and B show quantile box plots illustrating the mean IC50 values for oseltamivir (A)
and zanamivir (B) for H5N1 viruses isolated in different years. Panels C and D show
quantile box plots illustrating the mean IC50 values for oseltamivir (C) and zanamivir (D)
for H5N1 viruses representative of different HA clades.
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Fig. 2.
Prevalence of amantadine-resistant variants among highly pathogenic human and avian
influenza H5N1 viruses isolated in 2002­2012. The distribution of amantadine-resistant
variants with either single or double M2 mutations among human (A) and avian (B) HP
H5N1 influenza viruses. The numbers correspond to the number of amantadine-resistant
variants per total number of viruses isolated during different time periods.
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Govorkova et al. Page 16
Table
1
Avian
influenza
A
(H5N1)
viruses
with
elevated
IC
50
values
detected
in
this
study
Avian
H5N1
virus
HA
clade
NA
enzyme
inhibition
assay
(mean
IC
50
±
SD,
nM)
a
No.
mutant
clones/No.
total
(NA
mutation)
c
Oseltamivir
Fold
change
b
Zanamivir
Fold
change
b
A/chicken/Jogjakarta/BBVET/IX/2004
2.1.3
4.87
±
1.30
2.95
0.69
±
0.04
1.21
5/30
(G31A,
E228D,
T383A,
P420S,
F422L)
A/muscovy
duck/Vietnam/56/2007
2.3.4
9.25
±
0.33
5.61
0.56
±
0.05
0.98
4/28
(P73S,
S90F,
V267A,
G414D)
A/goose/Guandong/1051/2008
2.3.4
5.34
±
0.14
3.24
0.57
±
0.07
1.0
5/30
(I122T,
S125P,
P162S,
G210S,
I374N)
Susceptible
viruses
(n=92)
All
clades
1.48
±
0.19
N/A
0.65
±
0.07
N/A
N/A
a
The
concentration
of
NA
inhibitor
that
reduced
NA
activity
by
50%
relative
to
a
reaction
mixture
containing
virus
but
no
inhibitor.
Values
are
the
mean
±
SD
from
three
independent
experiments.
b
Compared
with
H5N1
susceptible
viruses.
c
TOPO
TA
cloning
was
performed
using
PCR
products
obtained
by
amplification
of
the
virus
inoculum.
Individual
virus
clones
were
analyzed
by
sequencing.
Each
clone
possesses
a
single
NA
mutation.
N/A
­
not
applicable:
Amino
acid
numbering
is
based
on
N2
NA.
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Govorkova et al. Page 17
Table
2
Incidence
of
NA
inhibitor­resistant
mutants
among
human
and
avian
influenza
A
(H5N1)
viruses
isolated
in
2002­2012
Origin/
Year
of
isolation
Number
of
H5N1
viruses
with
molecular
markers
of
NA
inhibitor
resistance
associated
with
a
Resistant
genotype
Reduced
susceptibility
genotype
No.
of
mutant/
No.
of
isolates
(%)
E119A
H274Y
N294S
No.
of
mutant/
No.
of
isolates
(%)
I117V
K150N
I222V/T/K
S246N
Human
isolates
2002­2004
0/44
(0)
--
b
--
--
2/44
(4.5)
--
1
1
--
2005­2007
6/173
(3.5)
--
4
c
3
c
3/173
(1.7)
--
--
3
--
2008­2012
1/70
(1.4)
--
--
1
2/70
(2.9)
1
--
1
--
2002­2012
7/287
(2.4)
--
4
c
4
c
7/287
(2.4)
1
1
5
--
Avian
isolates
2002­2004
2/387
(0.5)
--
1
1
7/387
(1.8)
4
--
3
--
2005­2007
4/953
(0.4)
--
3
1
35/953
(3.7)
d
9
11
8
11
2008­2012
7/376
(1.9)
4
e
--
3
8/376
(2.1)
4
e
3
1
2
2002­2012
13/1716
(0.8)
4
e
4
5
50/1716
(2.9)
d
17
e
14
12
13
a
Analysis
is
based
on
GenBank
data.
Amino
acid
numbering
is
based
on
N2
NA.
Additionally
one
avian
H5N1
virus
isolated
in
2007
had
V116A
and
two
viruses
isolated
in
2003
and
2005
had
D198N
NA
mutations
(results
not
shown).
Percentage
was
determined
by
the
number
of
resistant
isolates
per
the
total
number
of
viruses
isolated
during
the
particular
time
period.
b
--,
no
NA
mutations
were
detected.
c
A/Hanoi/30408/2005
(H5N1)
virus
(Le
et
al,
2005)
or
the
same
strain
designated
as
A/Vietnam/HN30408/2005
(Sleeman
et
al.,
2010)
had
clones
with
either
H274Y
or
N294S
NA
mutations.
They
were
counted
once
in
resistant
phenotype
as
H274Y.
d
Indicates
existence
of
viruses
carrying
double
NA
amino
acid
mutations.
One
H5N1
virus
isolated
in
2006
had
both
I222T
and
S246N
NA
mutations,
Three
H5N1
viruses
isolated
in
2007
had
both
K150N
and
S246N
NA
mutations.
They
were
counted
in
reduced
susceptibility
phenotype
only.
e
Indicates
existence
of
viruses
with
double
NA
amino
acid
mutations.
Two
H5N1
influenza
viruses
isolated
in
2008
had
NA
mutations
at
positions
E119A
and
I117V.
They
were
counted
in
resistant
phenotype
only.
Antiviral Res. Author manuscript; available in PMC 2014 May 01.
NIH-PA
Author
Manuscript
NIH-PA
Author
Manuscript
NIH-PA
Author
Manuscript
Govorkova et al. Page 18
Table
3
Incidence
of
amantadine-resistant
mutants
among
human
and
avian
influenza
A(H5N1)
viruses
isolates
in
2002­2012
based
on
HA
clade
Origin/
Year
of
isolation
No.
of
isolates
No.
of
amantadine-resistant
H5N1
variants
(%)
representative
of
HA
Clade
a
1
1.1
2.1.1
2.1.2
2.1.3
2.2
2.3.2
2.3.4
Others
b
Human
isolates
2002­2004
36
36
(100.0)
--
c
--
--
--
--
--
--
--
2005­2007
82
13
(15.9)
1
(1.2)
--
15
(18.3)
52
(63.4)
--
--
1
(1.2)
--
2008­2012
12
--
10
(83.3)
--
--
--
--
--
2
(16.7)
--
2002­2012
130
49
(37.7)
11
(8.5)
--
15
(11.5)
52
(40.0)
--
--
3
(2.3)
--
Avian
isolates
2002­2004
193
162
(83.9)
--
5
(2.6)
--
--
--
3
(1.6)
--
23
(11.9)
2005­2007
200
84
(42.0)
44
(22.0)
--
5
(2.5)
13
(6.5)
8
(4.0)
5
(2.5)
24
(12.0)
17
(8.5)
2008­2012
74
14
(18.9)
18
(24.3)
--
--
2
(2.7)
18
(24.3)
7
(9.5)
11
(14.9)
4
(5.4)
2002­2012
467
260
(55.7)
62
(13.3)
5
(1.1)
5
(1.1)
15
(3.2)
26
(5.6)
15
(3.2)
35
(7.5)
44
(9.4)
a
Clade
determination
based
on
phylogenetic
analysis
of
HA
gene
from
amantadine-resistant
mutants
with
WHO
reference
clade
(WHO
2012).
Percentage
was
determined
by
the
number
of
resistant
isolates
per
the
total
number
of
viruses
isolated
during
the
particular
time
period.
b
Other
include
HA
clades
0,
2.5,
3­9.
c
No
amantadine-resistant
viruses
detected.
Antiviral Res. Author manuscript; available in PMC 2014 May 01.
