Epistatic interactions between neuraminidase mutations facilitated the emergence of the oseltamivir-resistant H1N1 influenza viruses

ARTICLE

Received 19 Jan 2014 | Accepted 19 Aug 2014 | Published 9 Oct 2014

Epistatic interactions between neuraminidasemutations facilitated the emergence of theoseltamivir-resistant H1N1 influenza virusesSusu Duan1, Elena A. Govorkova1, Justin Bahl2,3, Hassan Zaraket1, Tatiana Baranovich1,

Patrick Seiler1, Kristi Prevost1, Robert G. Webster1 & Richard J. Webby1

Oseltamivir-resistant H1N1 influenza viruses carrying the H275Y neuraminidase mutation

predominated worldwide during the 2007–2009 seasons. Although several neuraminidase

substitutions were found to be necessary to counteract the adverse effects of H275Y, the

order and impact of evolutionary events involved remain elusive. Here we reconstruct H1N1

neuraminidase phylogeny during 1999–2009, estimate the timing and order of crucial amino

acid changes and evaluate their impact on the biological outcome of the H275Y mutation. Of

the 12 neuraminidase substitutions that occurred during 1999–2009, 5 (chronologically,

V234M, R222Q, K329E, D344N, H275Y and D354G) are necessary for maintaining

full neuraminidase function in the presence of the H275Y mutation by altering protein

accumulation or enzyme affinity/activity. The sequential emergence and cumulative effects of

these mutations clearly illustrate a role for epistasis in shaping the emergence and sub-

sequent evolution of a drug-resistant virus population, which can be useful in understanding

emergence of novel viral phenotypes of influenza.

DOI: 10.1038/ncomms6029

1 Department of Infectious Diseases, St Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, Tennessee 38105, USA.2 School of Public Health, The University of Texas Health Science Center at Houston, 1200 Pressler Street, Houston, Texas 77030, USA. 3 Program inEmerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, 8 College Road, Singapore 169857, Singapore.Correspondence and requests for materials should be addressed to R.J.W. (email: [email protected]).

NATURE COMMUNICATIONS | 5:5029 | DOI: 10.1038/ncomms6029 | www.nature.com/naturecommunications 1

& 2014 Macmillan Publishers Limited. All rights reserved.

mailto:[email protected]

http://www.nature.com/naturecommunications

The evolutionary course of influenza A viruses is shaped byinterplays among mutation, reassortment and naturalselection1. Influenza A viruses, similar to all RNA

viruses, have a high mutation rate2; whether a mutation canspread at a population level (epidemiologic fitness) is dependenton its impact on viral biologic fitness (replication fitness within ahost and transmission fitness between hosts)3. Understanding theimpact of mutations and interaction of mutations on viral fitnessis, therefore, critical for a mechanistic understanding of viralphenotype emergence.

The influenza virus neuraminidase (NA) inhibitors oseltamivirand zanamivir are the options currently approved in the UnitedStates for immediate control of influenza virus infection. Theirclinical use, however, provides a selection force to driveemergence of resistance within treated individuals. Before 2007,resistant viruses were detected only infrequently during NAinhibitor treatment4–7 and very rarely during surveillance7–9,suggesting that those drug-driven resistant viruses had littleepidemiologic fitness. However, during the 2007–2009 influenzaseasons, oseltamivir-resistant H1N1 viruses surged from o1% to490% prevalence worldwide10–12. Such spread of resistance atpopulation level was not attributed to oseltamivir use inindividuals, but to global transmission of the resistant virusescarrying the NA H275Y mutation13,14, suggesting these H275Y-mutant viruses had acquired advantageous epidemiologic fitness.A mechanistic understanding of such drug-independentresistance spread would give us insights to the adaptability andevolution of drug-resistant influenza viruses.

Recent studies have advanced our understanding of thebiological properties of the H275Y-mutant viruses related totheir different epidemiologic fitness outcomes. Genetically, theNA genes of most H275Y-mutant viruses were closely associatedwith the genetic 2B clade (represented by A/Brisbane/59/2007[BR07]) of H1N1 viruses but not with the other three clades(clade 1, represented by A/New Caledonia/1999 [NC99]; clade2A, represented by A/Solomon Island/23/2006 [SI06]; and clade2C)15–19. This clade-specific resistance distribution suggested alink between biologic fitness and genetic context of the H275Ymutants. Indeed, phenotypically, NC99-like H275Y mutantsmanifested greater biological cost relative to their respectivewild-type counterparts than did BR07-like mutants, as measuredby growth in cells, mice and ferrets20–23, and by their NAaffinity15,16,19 and cell surface accumulation24.

Several mutations have been identified elsewhere in the NAthat can counteract the adverse effects of the H275Y mutation. Ithas been found that the D344N16,25, R222Q and V234M24 NAsubstitutions can counteract the reduced NA affinity and surfaceaccumulation caused by the H275Y mutation; therefore, thesemutations are ‘permissive’ for the H275Y mutation. Anotherstudy confirmed that changing the permissive substitutions to thenon-permissive substitutions (Q222R, M234V) compromised thereplication fitness of a clade 2B H275Y-mutant virus in vitro andin ferrets26. Although illuminating, the identification of these NApermissive mutations has not provided a full understanding of theevolutionary path and molecular process involved in the fitnesschanges of the H275Y-mutant viruses.

Here we reconstruct the molecular evolutionary path of the NAprotein of seasonal H1N1 viruses from the NC99 to BR07 geneticlineage, during 1999–2009. We then evaluated the biologicaloutcomes of the H275Y mutation in different NA geneticcontexts at different stages of the path. We further investigatethe chronological order and nature of NA mutations and theirimpact on the phenotypic outcome of the H275Y mutationin vitro and in vivo. These biologic fitness assessments includeNA functional activity (accumulation and substrate affinity),replication in cells (plaque morphology), and viral growth and

transmissibility in ferrets. We find that multiple mutations,having either permissive or compensatory epistatic interactionswith the H275Y, sequentially alter the outcomes of the H275Ymutation in NA functionality and virus biological fitness. Ourfindings show biologic evidence for epistasis in which thephenotypic outcome of a mutation depends on the absence orpresence of others. These results support a role for epistasis inshaping the evolution of influenza viruses, and in the emergenceand spread of viral phenotype.

ResultsNA protein sequence evolution from 1999 to 2009. The NAsequences of seasonal H1N1 viruses isolated during 1999–2009showed a major genetic divergence, from the NC99 (clade 1)to BR07 (clade 2B) lineage15,16,19. We first sought to identifychanges in the NA protein sequence during this lineage transitionthat would explain the different H275Y mutation outcomes in theNC99 and BR07 lineages. Comparison of the consensus NA genesequences revealed ten amino acid substitutions (H45N, K78E,E214G, R222Q, V234M, G249K, T287I, K329E, D344N andG354D, excluding H275Y) differentiating the lineages(Supplementary Table 1) and one substitution (D354G)differentiating wild-type and H275Y-mutant NA sequenceswithin the BR07-like lineage. None of the substitutions residedat the NA active site (Supplementary Fig. 1).

We reconstructed the N1 NA protein’s evolutionary path,focusing on the period 1999–2009, and estimated the timeand order of occurrence of the amino acid substitutions byusing a tip-calibrated relaxed molecular clock phylogenetic treereconstruction method27 (Fig. 1). The NC99-like NA population(the tree trunk in Fig. 1) acquired most of the mutations insuccession during 1999–2007, rather than through a singleselective sweep, evolving into the BR07-like NA population inearly 2007; multiple mutations were seemingly acquired at a fewtime points. Several intermediate NA populations (clustered awayfrom the trunk Fig. 1) that had acquired some of the mutationsco-circulated for some time but were extinguished before 2007.The H275Y mutation occurred only sporadically in thosepopulations, in which it ended as an NA evolutionary terminus(the tip of a branch Fig. 1). In mid-2007 (2007.485, Bayesiancredible intervals, 2007.302–2007.698), soon after the BR07-likeNA population was established, the H275Y was fixed intothe population. Only one mutation (D354G) was acquiredafter the H275Y, thereby completing the NA protein evolutionsuch that oseltamivir resistance dominated the populationduring 2008–2009.

Different outcome of the H275Y mutation in NA functionality.The NA protein sequence changes seen in our phylogenetic cal-culations prompted us to evaluate the possible accompanyingphenotypic changes in NA functionality. To this end, weexpressed the NA proteins (using equal quantities of NA-con-taining plasmids to partially mimic NA expression by equalquantities of infectious virus particles) and assessed theirtotal activity and accumulation. To quantify the different NAproteins, we adapted a method for uniformly epitope-taggingrecombinant proteins24. After comparing untagged, carboxy-terminal-tagged and amino-terminal-tagged recombinantNABR/59 proteins, we chose the C-terminal epitope tag for usein all NA proteins, as it did not significantly affect NAaccumulation or activity (Supplementary Fig. 2). We comparedlineage-representative NA proteins as well as intermediateproteins that had partial complements of the ten substitutions(Supplementary Tables 2, 3).

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6029

2 NATURE COMMUNICATIONS | 5:5029 | DOI: 10.1038/ncomms6029 | www.nature.com/naturecommunications



We first evaluated NA enzyme activity (Fig. 2a). Across the twolineages, the BR07-like wild-type NABR/59 had enzyme activityB3–4 times that of two NC99-like wild-type proteins (NAGA/17

and NAMEM/13), while the intermediate NAMS/03 (it had acquiredthe R222Q and D344N substitutions but not V234M yet, andtherefore it had atypical intermediate genotype) had activitynear that of NABR/59. Within the NC99-like lineage, the threepairs of wild-type and H275Y-mutant NA proteins differed onlyat position 275; the H275Y-mutants (NAGA/20 (H275Y) andNAMEM/13-H275Y) had activity of only B50% that of theirwild-type counterparts, while the activity of the intermediatemutant NAMS/03 (H275Y) was reduced least (B20%). Within theBR07-like lineage, the activity of H275Y-mutant proteinsdid not significantly differ from that of wild-type NABR/59,although the two representative mutants (NANJ/15 (H275Y) andNANY/1692 (H275Y)) had slightly higher activity and twointermediates (NABR/59-H275Y and NANY/3467 (H275Y)) that hadnot acquired the D354G or D344N mutations showed slightlylower activity.

We next examined whether the observed difference in the NAactivities reflected differences in their protein amount. Across thetwo lineages, two BR07-like wild-type proteins (NABR/59 andNACA/27) showed higher accumulation than three NC99-like wild-type proteins (NAGA/17, NAMEM/13 and NAMS/03) (Fig. 2b andSupplementary Fig. 3a). Within the NC99-like lineage, accumula-tion of the three H275Y-mutant proteins was substantially lowerthan that of their wild-type counterparts, but accumulation of theintermediate NAMS/03-H275Y was reduced least. Within the BR07-like lineage, accumulation of the five H275Y-mutant NA proteins

was similar to that of the wild-type NABR/59. Cell surfaceaccumulation of the NA proteins showed the same trends ofchange as observed in total NA accumulation (Fig. 2c).

As the protein amount differed among the NAs, we nextexamined each NA-specific activity by standardizing each NA’sactivity relative to its protein level, which reflected each NA’sintrinsic conversion ability (Supplementary Fig. 3b). Thedifferences in the standardized NA-specific activities showedsimilar patterns to those in the total NA activity. Across the twolineages, the standardized activity of wild-type NABR/59 wasB2–3 times of that of the two representative NC99-like wild-typeNAs (NAGA/17 and NAMEM/13). Within each lineage, all theH275Y NAs showed slightly lower standardized activity thantheir wild-type counterparts, except that two representativeBR07-like H275Y NAs (NANJ/15(H275Y) and NANY/1692 (H275Y))showed higher standardized activity than the NABR/59. Thedistinct levels of NA-specific activity between the two lineagesreflected that the NAs had varying intrinsic conversion ability inaddition to their different expression level.

Enzyme Km is an intrinsic enzyme property and its value isinversely correlated with the enzyme substrate-binding affinityand enzyme conversion ability: the higher Km value indicateslower binding affinity and reaction rate, leading to lower activity.We next examined whether the different levels of standardizedNA-specific activity reflect different Km values. Across the twolineages, the Km values of two BR07-like wild-type proteins(NABR/59 and NACA/27) were only B25–30% those of two NC99-like wild-type proteins; the intermediate NAMS/03 protein had anintermediate Km (Fig. 2d). Within lineages, the H275Y mutant

R222QV234M

R222Q

V234M

H1N1 virusesNA H275(Oseltamivir susceptible)

H1N1 virusesNA H275Y(Oseltamivir resistant)

201020052000199519901985

V234M

V234M

D344N

R222Q

R222Q

R222QE214G

G354DT287I

K329EH45N

D344NG/R249K

D354GH275Y

K78E

D344N

D344N

D344N

D354D

H275Y

H275Y

K329E

K329E

Figure 1 | Temporally structured maximum clade credibility phylogenetic tree of the NA gene of seasonal H1N1 viruses, showing the timeline of

amino acid substitutions. Ancestral state reconstruction of tested amino acids is annotated on the tree backbone to indicate when specific mutations were

fixed in the population. The trunk colour change indicates a 495% posterior probability of amino acid fixation. Purple bars on tree nodes indicate

the Bayesian credible intervals of ancestral divergence time estimates (450% posterior probability). Isolates with H275Y mutation before H275Y fixation

into the population are indicated with red branches.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6029 ARTICLE




proteins had consistently higher (B2-fold) Km values than theirwild-type counterparts; only the intermediate NANY/3467 (H275Y),which had not acquired the D344N, had an unusually high Kmwithin the BR07 lineage. The lineage-associated Km levelscorroborated previous reports that BR07-like viruses had greaterNA affinity than the NC99-like viruses15,19,28.

Taken together, these results revealed two NA phenotypictransformations during the NC99-to-BR07 NA evolution. First,the accumulation and substrate affinity of the wild-type BR07-likeNA proteins increased greatly, resulting in inherently higherfunctionality. Second, the outcome of the H275Y mutation in netNA function differed across lineages: the H275Y-generated NAdefects were severe in the NC99-like NA proteins but minimal orabsent in the BR07-like NA proteins. These results indicate thatcertain mutations acquired by the BR07-like NA proteinscontributed to the observed NA phenotypic transformations.

Different outcome of NA H275Y in virus transmissibility. Theobserved NA phenotypic transformations during NC99–BR07NA evolution prompted us to assess the transmissibility ofrepresentative viruses isolated at different stages of the evolutionpath, an essential factor for virus spread. To this end, we chose aferret model to recapitulate virus human transmission. Although

so far it is recognized as the best model to recapitulate humantransmission of influenza virus, ferret model has limitationssuch as outbred model and random factors in sneezing activityand so on; hence, we chose to assess multiple wild-type andH275Y-mutant viruses of each lineages, looking for commonobservations for each lineage. We assessed the growth andtransmissibility of selected viruses via direct contact andrespiratory droplets in influenza-naive ferrets. Transmission wasindicated by nasal virus shedding and seroconversion (Fig. 3 andSupplementary Table 4).

In the NC99 lineage, both the wild-type (MEM/13/06) andH275Y-mutant (GA/20/06 and MS/03/02) viruses caused pro-ductive infection in three of three inoculated donor ferrets, butthe two H275Y mutants were shed at much lower titres on day 1post inoculation (p.i., B3 and 1.5 log10TCID50 lower, respec-tively) (Fig. 3a). At day 1 post contact (p.c.), only the wild-typevirus had been transmitted to three of three recipients by directcontact, and neither H275Y mutant had been transmitted to allrecipients and their titres were B3 log10TCID50 lower (Fig. 3b).The wild-type virus was transmitted via respiratory droplets totwo of three ferrets at days 3 and 5 p.c., while both the H275Ymutants were transmitted to only one of three ferrets at days 7and 9 p.c. (Fig. 3c). Therefore, the transmission of the twoNC99-like H275Y mutants was commonly slower and less

Rep Rep

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NA

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t)

Figure 2 | NA protein phenotypes of seasonal H1N1 influenza viruses of the NC99 and BR07 lineages. (a) Neuraminidase enzyme activity of total NA

proteins expressed by equal quantities (0.5mg) of the respective plasmids. Rep, representative NA protein of the indicated lineage; Int, intermediate NA

protein of the indicated lineage, fully or partially conforming to the ten consensus lineage residues. The residue at the NA protein 275 position (wild-type

(wt) or H275Y) is indicated in parentheses. NA-H275Y is shown when the H275Y was introduced by mutagenesis. Data were normalized to NABR07

protein. Dotted and dashed lines indicate the activity level of lineage-representative NAGA/17 and NABR/59 proteins, respectively. (b) Representative

western blotting showing total accumulation of NA protein variants expressed by equal quantities (0.5mg) of the respective plasmids. Black and grey

indicate wt and H275Y-mutant NA proteins, respectively. Panels show detection by anti-HA-tag (upper), anti-b-actin (middle) and anti-GFP (lower).

(c) Representative flow cytometric plots of cell surface accumulation of NA protein variants expressed by equal quantities (0.5mg) of respective plasmids.

The pairs differed only at position 275, with the exception of the last panel, the BR07-like NAs. (d) Km values of the NA proteins. The dotted and

dashed lines indicate the value of lineage-representative NAGA/17 and NABR/59, respectively. All graphs show mean±s.e.m. from three to five times

independent experiments. *Po0.05, two-tailed t-test, versus NABR/59; wPo0.05, two-tailed t-test, versus its counterpart wt NA at left side.





efficient than the NC99-like wild-type virus, suggesting that thetransmissibility of the NC99-like H275Y mutants was impaired,consistent with their severe NA defects observed.

In the BR07 lineage, the wild-type (BR/59/07) and threeH275Y-mutant viruses (HW/28/07, NY/3467/09 and NY/1692/09) caused productive infection in all inoculated donors (three ofthree). Virus shedding between wild-type and H275Y-mutantvirus only differed in the intermediate NY/3467/09 virus (B1.5log10TCID50 lower in the mutant virus at day 1 p.i.) (Fig. 3a).Each virus had been transmitted to three of three recipients bydirect contact at day 1 p.c. (Fig. 3b), showing similar transmissionkinetics, although the intermediate HW/28/07 virus was trans-mitted later to one recipient. Each virus was transmitted to two of

three recipients via respiratory droplets, with similar kinetics(Fig. 3c), except that the representative H275Y-mutant NY/1692/09 virus was transmitted earlier (day 3 p.c.) than the wild-typeBR/59/07 (day 7 p.c.) to one recipient. Therefore, the transmis-sion efficiency of the three BR07-like H275Y mutants wascommonly similar to the BR07-like wild-type virus, showing thatthe transmissibility of BR07-like H275Y mutant viruses isminimally impaired or not impaired, consistent with theirminimal or absent NA defects observed.

We next generated two H275Y mutant reverse-genetics (rg)viruses, rgBR/59/07-NAH275Y and rgBR/59/07-NAH275YþD354G,to represent the evolutionary acquisition of the H275Y andD354G mutations within the BR07 lineage. Both H275Y mutants

MEM/13/06 (wt)(NC99-like, representative)

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MEM

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5Y

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NC99-like BR07-like rgBR/59/07

Figure 3 | Transmissibility of seasonal H1N1 influenza viruses of the NC99 and BR07 lineages in ferrets. (a) Virus titres in nasal wash samples from

individual donor ferrets (black), direct-contact (DC) ferrets (blue) and respiratory-droplet (RD) ferrets (red). Arrows indicate the day (day 1 p.i for

donors) when recipient ferrets were housed with donor ferrets. (b) Virus titres in DC ferret nasal wash samples at day 1 post contact. Each dot represents

an individual ferret. *Po0.05, two-tailed t-test, versus MEM/13. (c) Virus titres in RD ferret nasal wash samples on the first day of detection (the

day post contact), which is indicated above each column.





productively infected three of three inoculated ferrets (Fig. 3a).They were transmitted to three of three recipients via directcontact and to two of three via respiratory droplets, but withdifferent kinetics. At day 1 p.c., only the NAH275Y virus had notbeen transmitted to all direct-contact ferrets (were transmittedonly two of three), showing slightly slower transmission than thatof the wild-type and NAH275YþD354G viruses (Fig. 3b); theNAH275YþD354G virus was transmitted to two of three recipientsvia respiratory droplets at days 3 and 5 p.c., showing slightlyfaster transmission than did the wild-type and NAH275Y viruses(transmission at days 3 and 7 p.c.) (Fig. 3c). These results wereconsistent with the observations from the three BR07-likeH275Y-mutant viruses and further confirmed that the H275Ymutation minimally reduced the transmissibility of the BR/59/07virus and suggested that addition of D354G restored thetransmissibility of the H275Y-mutant virus to the wild-type level.Another report29 found that D354G not only increased thetransmissibility of a resistant virus in guinea pigs but also to alevel higher than that of the wild-type virus, suggesting that theextent of D354G increasing resistant virus transmissibility maydepend on other viral protein context.

In summary, the growth and transmissibility of NC99-like andBR07-like wild-type viruses was indistinguishable despite theirdifferent NA phenotypes. However, the growth and transmissi-bility outcome of the H275Y-mutant variants differed greatlybetween the two lineages, as did the extent of H275Y-generatedNA defects, suggesting that the NA defects was directly correlatedwith virus transmissibility.

Mutation interactions alter phenotypic outcome of NA H275Y.We next sought to identify which of the 11 amino acid sub-stitutions that occurred during the NC99–BR07 NA evolutionaccount for the observed NA phenotypic transformations. To thisend, we examined the impact of each individual mutation on NAfunctionality with or without the H275Y mutation.

We first adopted a strategy using plaque size to evaluate NA-mediated virus release during the replication cycle. A plaque isformed from a single virus particle via multiple cycles ofreplication, and release of the progeny viruses at each cycle ismediated by NA functionality. Thus, NA mutation that couldalter NA activity sufficiently would directly affect the speed ofvirus release and consequently determine the plaque size. We firstvalidated the feasibility of this strategy by confirming that plaquesize paralleled NA enzyme activity in two pairs of rg viruses, aBR07-like pair (NABR/59 and NABR/59-H275Y) and an NC99-likepair (NAGA/17 and NAGA/20 (H275Y)) (each pair differs only atposition 275; Supplementary Fig. 4a,b). We next generated rgBR/59/07 viruses whose NA proteins carried the respective substitu-tions N45Hþ E78K, G214E, Q222R, M234V, K249G, I287T,E329K, N344D and D354G (representing a change from BR07-like to NC99-like), in the presence and absence of H275Y. Foursubstitutions (Q222R, M234V, E329K and N344D) in NABR/59

significantly reduced plaque size, with and/or without the H275Y(Fig. 4a and Supplementary Fig. 5a). These four positions and thefinal substitution site (354) were selected for further evaluation;the remaining five substitutions were unlikely to reduce virusgrowth appreciably and were not further evaluated.

BR/59-NA-wt

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Figure 4 | Plaque size, NA activity, NA accumulation and substrate affinity of the BR/59/07 NA proteins with different amino acid substitutions.

(a) Plaque size of viruses carrying ten different NA amino acid substitutions, in the absence (white) or presence (grey) of the H275Y mutation. The ten

amino acid residues were replaced with the corresponding NC99-like consensus residues. (N45Hþ E78K were substituted together, as both reside

in the stalk region.) Data represent mean±s.e.m. diameter of B10 randomly selected plaques. Dashed line indicates mean plaque diameter of wild-type

NABR/59. (b) Enzyme activity of total NABR07 and NABR07-H275Y variant proteins with five different amino acid substitutions, expressed by equal quantities

(0.5mg) of the respective plasmids. Dotted and dashed lines indicate mean activity of NABR/59and NABR/59-H275Y, respectively. (c) Representative western

blotting showing total accumulation of protein variants with substitutions; the variants were expressed by equal quantities (0.5mg) of the respective

plasmids. Black and grey indicate wt and H275Y-mutant NA, respectively. Panels show detection by anti-HA-tag (upper), anti-b-actin (centre) and anti-GFP

(lower). (d) NA Km values of rgBR/59/07 viruses carrying the respective NA amino acid substitutions in the absence and presence of the H275Y

mutation. Dotted and dashed lines indicate the values of NABR/59and NABR/59-H275Y, respectively. All graphs represent mean±s.e.m. of three to five times

independent experiments. *Po0.05, two-tailed t-test, versus NABR/59; wPo0.05, two-tailed t-test, versus NABR/59-H275Y.





The four substitutions (Q222R, M234V, E329K and N344D)variably reduced enzyme activity in NABR/59 or NABR/59-H275Y

(Fig. 4b), consistent with the reduced plaque size observed.Conversely, although not notably increasing plaque size, theD354G substitution significantly increased enzyme activity inboth NABR/59 and NABR/59-H275Y; it increased the enzymeactivity NABR/59-H275Y to a level equivalent or slightly higherthan that of NABR/59. Two substitutions (Q222R and M234V) inboth NABR/59 and NABR/59-H275Y greatly reduced the NA proteinaccumulation (Fig. 4c and Supplementary Fig. 5b) and the level ofaccumulation resulted by each mutation with H275Y was muchlower than that by each mutation alone (such as H275Y-Q222Rversus Q222R alone). Three substitutions (Q222R, E329K andN344D) in both NABR/59 and NABR/59-H275Y significantlyincreased the Km value B2-fold compared with Km ofNABR/59 and NABR/59-H275Y, respectively (Fig. 4d), and the Kmvalues resulted by each mutation with H275Y was much higherthan that by each mutation alone (such as H275Y-Q222R versusQ222R alone). The three Km-changing mutations differentlyreduced NA-specific activity with Q222R causing the biggestreduction (Supplementary Fig. 5c). The D354G substitution hadno appreciable effect on protein accumulation or Km values butsignificantly increased the NA-specific activity (SupplementaryFig. 5c). The 354 position resides at the NA surface but facestoward the virion’s membrane (Supplementary Fig. 1) andtherefore is structurally unlikely to affect substrate interactiondirectly. Thus, D354G increased enzyme conversion ability byunidentified mechanisms other than Km level. In contrast, thethree affinity-changing mutations (Q222R, E329K and N344D)are located at the NA surface and face away from the virion’smembrane and tetramer interface (Supplementary Fig. 1),structurally allowing substrate interaction.

Together, our results identified two classes of mutationinteraction in NABR/59 that sequentially altered the outcome ofthe H275Y mutation in net NA function. First, acquisition ofR222Q, V234M, K329E and D344N mutations by NABR/59 eachincreased NA activity and thus minimized subsequent H275Y-generated defects. Second, acquisition of the D354G mutation byNABR/59-H275Y increased NA activity to wild-type protein levels,resulting in unaltered net NA functionality. Thus, the higherlevels of enzyme activity (Fig. 2a and Supplementary Fig. 3b) ofNANJ/15 (H275Y) and NANY/1692 (H275Y) reflected their furtheracquisition of the D354G after the H275Y addition anddemonstrated the role of D354G in increasing H275Y NA activity.

Reconstruction of NC99–BR07 NA phenotypic evolution in vitro.We next tested whether the molecular determinants identifiedabove were sufficient to explain the NA phenotypic transforma-tions that accompanied the NC99–BR07 transition. The V234M,R222Q, K329E and D344N mutations (representing a changefrom NC99-like to BR07-like) were introduced into the NAGA/17

and NAGA/20 (H275Y) (already possessing the D354G mutation)NC99-like proteins, which differed only at position 275.All substitutions, except D344N, significantly increasedthe functional activity of NAGA/17 and, to a lesser extent, ofNAGA/20 (H275Y) (Supplementary Fig. 6a). Enzyme activity par-alleled the plaque size of the mutant viruses (SupplementaryFig. 6b). In NAGA/17, two substitutions (R222Q and V234M)greatly increased NA protein accumulation (Fig. 5a) and three(R222Q, K329E and D344N) variably reduced the Km value(Fig. 5b); all of these changes occurred to a lesser extent inNAGA/20 (H275Y). These findings confirmed that these four sub-stitutions play a role in increasing overall NA function and inreducing the H275Y-generated defects, although no single sub-stitution could alone fully transform the NA phenotype and fully

offset the H275Y-generated defects. Thus, the intermediate levelof NA accumulation observed in NAMS/03, NAMS/03 (H275Y)

(which had acquired R222Q and D344N) and unusually high Kmvalue in NANY/3467 (H275Y) (which had not acquire the D344N)(Fig. 2) can be explained by partial acquisition of these pheno-typic determinants.

We then evaluated whether sequential addition of all fourphenotypic determinants were sufficient to fully reconstruct theBR07-like NA phenotype. The four substitutions were consecu-tively added to the NC99-like NAGA/17 and NAGA/20 (H275Y)

proteins in the order indicated by the phylogenetic reconstructionof ancestral states (V234M-R222Q-K329E-D344N or V234M-R222Q-D344N-K329E) (Fig. 1). The sequential addition of thefour substitutions progressively increased the functional activityof NAGA/17 and NAGA/20 (H275Y) to a final level equivalent to thatof NABR/59 and NABR/59-H275Y, respectively (SupplementaryFig. 7a). The increased NA activity closely paralleled increasedplaque size (Supplementary Fig. 7b). Addition of V234M andR222Q, but not the two subsequent mutations, to NAGA/17 andNAGA/20 (H275Y) increased NA protein accumulation to a levelequivalent to that of NABR/59 and NABR/59-H275Y, respectively(Fig. 5c). Addition of V234M did not appreciably reduce Km, butsubsequent addition of R222Q, K329E and D344N progressivelyreduced the Km values of NAGA/17 and NAGA/20 (H275Y) (Fig. 5d)to that of NABR/59 and NABR/59-H275Y, respectively.

In summary, sequential addition of the four identifiedmolecular determinants into the two NC99-like NA proteins(already possessing the D354G mutation) fully reconstructed theBR07-like NA phenotype. The impact of the interactions of theV234M and R222Q with the H275Y on total NA accumulationcorroborated and expanded previous findings about surfaceNA accumulation24. The two subsequent substitutions,K329E and D344N, increased NA affinity and further reducedH275Y-generated NA functional losses.

The outcome of the H275Y mutation in other N1-subtype NA.As the NA H275Y mutation is associated with oseltamivir resis-tance mainly in the N1 subtype, we evaluated the functionaloutcomes of the H275Y mutation in other N1 proteins, includingthose of two human 2009 pandemic H1N1 (pdmH1N1) (NACA/

04 and NADM/524) (Fig. 6a,b) and two human H5N1 (NAVN/1203

and NAHK/213) viruses (Fig. 6c,d). The H275Y mutation causedloss of NA activity by 45–70% in these four NA proteins andgreatly diminished their surface accumulation (Fig. 6). The extentof H275Y-generated defects observed in these four N1 proteinsshowed that that the H275Y mutation remained deleterious forNA functionality in these NA genetic contexts, suggesting thatadditional mutations would be required to restore NA function-ality for these four viruses to carry H275Y mutation to spread toany degree. These results demonstrate evaluation of the biologycost by the H275Y mutation at protein level can be an infor-mative analytic tool for assessing other emerging resistant virusesin other future studies.

DiscussionThe findings presented in this study detail the molecularmechanisms and temporal process by which the biologic fitnessof oseltamtivir-resistant H1N1 viruses was altered by epistaticinteractions between successive NA mutations. We discoveredthat the NA molecular changes (in the temporal order V234M-R222Q-K329E-D344N-H275Y-D354G), which occurred duringNA genetic evolution during 1999B2009 gradually reduced andfinally mitigated H275Y-generated defects on NA functionality.The genetic context-dependent fitness outcome of the H275Y-mutant viruses is characteristic of epistasis effects. Therefore, the





sequential and cumulative epistatic interactions in NA contrib-uted substantially to the evolutionary course of the H275Ymutation-containing H1N1 viruses.

Epistatic interactions occurs when the phenotypic outcome of amutation is conditional to the presence or absence of othermutations in the genome30 and it can have either positive(alleviating) or negative (aggravating) effects30. Epistasis ininfluenza A viruses has been estimated by modelling sequenceevolution of human H3N2 and H1N1 viruses31–33, and wassuggested to be prevalent in haemagglutinin (HA) and NAproteins31. There have, however, been few biological observationsof epistatic interactions at the protein or virus phenotypic level. Inone example, two advantageous immune-escape Nucleoproteinmutations that would have otherwise been destabilizing wereacquired through interaction with other stabilizing mutations34.Our findings provide direct biological evidence of epistatic inter-actions in determining phenotypic outcome of a drug-resistantmutation during NA evolution. The interaction sites wereconcordant with a previous statistical analysis based on NAsequence alone31, supporting a role for epistasis in shapinginfluenza virus evolution.

In the context of current knowledge, our results suggest a four-stage scenario of molecular evolution of the NA protein in H1N1viruses during 1999–2009: (1) during 1999–2006, incidentalintroduction of the NA H275Y mutation into the predominant

circulating NC99-like viruses severely impaired NA functionalityand, consequently, virus fitness and ability to spread. Theseviruses became extinct. (2) Meanwhile, during 1999–2006, theNC99-like viruses gradually evolved under host pressures,including immunity. The NA protein sequentially acquired 12substitutions, of which R222Q, K329E, G249K and D344N havebeen shown to cause NA antigenic drift35, while V234M, R222Q,K329E and D344N greatly enhanced NA accumulation and/orsubstrate affinity. (3) In 2007, a new BR07 lineage, which hadundergone HA and NA antigenic drift, emerged and becameprevalent. The introduction of the NA H275Y mutation intoBR07-like viruses minimally reduced NA function due to itsincreased surface accumulation and/or substrate affinity ascompared with the NC09-like NA. (4) After the H275Ysubstitution was acquired in mid-2007, final addition of the NAD354G fully compensated for the remaining H275Y-generatedfunctional defects. Therefore, these BR07-like, oseltamivir-resistant viruses had no net loss of NA function, had biologicalfitness equal at least to that of the BR07-like wild-type viruses andspread readily during the 2008–2009 influenza season.

Our findings show the presence of two types of epistaticinteractions that collectively altered the H275Y phenotype. Fourpermissive epistatic mutations (V234M, R222Q, K329E andD344N) were selected before addition of H275Y, increasingoverall NA functionality fist and providing a permissive context

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Figure 5 | Total protein accumulation and Km values of the NC99-like NA proteins with different amino acid substitutions. (a) Representative

western blotting showing total accumulation of the NC99-like NA proteins (NAGA/17and NAGA/20 (H275Y)) with the indicated single substitutions; the

variant NAs were expressed by equal quantities (0.5 mg) of the respective plasmids. Black and grey indicate wt and H275Y-mutant NA proteins,

respectively. (b) NA Km values of rgBR/59/07 viruses carrying NAGA/17 or NAGA/20 (H275Y) with the indicated single substitutions. Dotted and dashed

lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. (c) Representative western blotting showing total accumulation of NAGA/17 and

NAGA/20 (H275Y) proteins with sequentially added substitutions (sub), the order is the same in next panel; NAs were expressed by equal quantities

(0.5mg) of respective plasmids. (d) NA Km values of rgBR/59/07 viruses carrying the NAGA/17 and NAGA/20 (H275Y) with sequentially added

substitutions. Dotted and dashed lines indicate Km values of NAGA/17 and NAGA/20 (H275Y), respectively. All graphs show mean±s.e.m. of three to five

times independent experiments. *Po0.05, two-tailed t-test, versus NAGA/17; wPo0.05, two-tailed t-test, versus NAGA/20 (H275Y).





for potential function-decreasing mutation. The D354G muta-tion, which was added to the BR07-like NA populationimmediately after H275Y acquisition, was very likely to beselected due to its ability to mitigate remaining H275Y-associatedfunctional defects in a typical epistatic compensatory manner.The combined phenotypic effects of both the permissive andcompensatory mutations were required to maintain the net NAfunction of the H275Y-mutant protein.

The emergence of the V234M and R222Q substitutions before1999 suggests that the selection forces for these substitutions hadexisted and was not drug pressure. The extensive overlap of thefour permissive NA mutations (V234M, R222Q, K329E andD344N) and the four reported antigenic-drift NA mutations(R222Q, G249K, K329E and D344N)35 suggests that host anti-NA immunity may have been the primary driver of theoverlapping mutations. All four antigenic-drift mutations arelocated at the surface of the NA globular head, opposite thetetramer interface and viral membrane (Supplementary Fig. 1),potentially allowing interaction to both antibody and substrate.Our screening did not find the G249K substitution able to affectvirus growth sufficiently to alter plaque size, but a small effect onenzyme affinity is possible. Thus, selection of antigenic-drift NAmutations probably inadvertently provided a permissive contextfor the H275Y mutation and altered its phenotypic outcome. Theantigenic drift-driven NA mutation spread can be a plausibleexplanation in addition to previously proposed genetichitchhiking process36. The non-overlapping mutation V234M,not located at the NA surface, did not affect enzyme affinity andwas not a probable antigenic-drift mutation. However, it precededa series of NA affinity-changing and antigenic-drift mutations,showing that the first altered NA phenotype was expression leveland the second was functionality, suggesting that proteinexpression level could affect introduction of function-changingmutations during protein evolution.

Our mechanistic reconstruction of the evolutionary course ofthe H1N1 viruses has direct implications for assessing thepotential fitness, evolution and spread of other H275Y-mutant

viruses of N1 subtype. In the case of 2009 pdmH1N1 viruses, thesevere defects on NA functionality generated by the H275Ymutation is consistent with the reduced transmissibility of H275Ymutants experimentally37,38 and the limited detection of suchviruses during surveillance (1%B3.5% detection rate)39–41.However, experimental transmission and community spread ofH275Y-mutant pdmH1N1 viruses has been reported40,42–44,including a very recent alarming community spread in Sapporo,Japan45, suggesting that potential permissive mutation mighthave been emerged in certain cluster of variants. Three studiesusing computational predictions or in vitro and in vivoexperiments have suggested several potential permissive NAmutations for the H275Y in pdmH1N1 viruses46–48, which areentirely different from the one identified in seasonal H1N1viruses. Given that NA genes of the seasonal H1N1 andpdmH1N1 were phylogenetically distant and had extensiveamino acid dissimilarities, it is not surprising that differentH275Y-permissive mutations would be required for pdmH1N1viruses, which is also very likely to be the case for other N1-subtype viruses. Our study suggest that the evolution course ofpdmH1N1 NA towards the potential resistance spread should bemonitored longitudinally by assessing the extent of H275Y-generated NA defects, especially in community-transmittedresistant isolates. The NA defects evaluated by a spectrum ofassays from protein expression, enzyme activity to virustransmission level would be most informative for assessingdominance potential of resistant viruses. The occurrence ofsmaller or no NA defects in all assay levels is worth a high alert.

Similarly, the deleterious outcome of the H275Y mutation onthe NA protein from H5N1 viruses is consistent with the rare caseof resistant viruses detected in the field (only three cases wheredrug treatment or prophylaxis was used)49,50. As the highlypathogenic H5N1 (hpH5N1) viruses carry unique virulencedeterminants51, H275Y-generated NA defects may not alter theiroverall pathogenicity. However, as hpH5N1 infection is asubstantial public health concern, it would be advisable tocompare the biological fitness of wild-type versus emerging

pdmH1N1CA/04/09

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Figure 6 | Defects in enzyme activity and protein accumulation caused by the H275Y mutation in other N1-subtype NA proteins. Each panel

shows a pair of NA protein variants differing only in the absence or presence of the H275Y mutation, including two pairs of pdmH1N1 NA proteins

(a,b) and two pairs of hpH5N1 NA proteins (c,d). Enzyme activity of total accumulated NA protein is shown at left in each panel. Representative flow

cytometry plots showing surface accumulation are at right. NA proteins were expressed by equal quantities (0.5mg) of respective plasmids. Data

are the mean±s.e.m. of at four determinations. *Po0.05, two-tailed t-test, versus its counterpart wild-type NA.





resistant viruses by identifying H275Y-generated NA defects. Ofspecial value in our study is the use of NA-containing plasmids toassess defects in NA protein expression and activity, as describedhere, averting the need to engineer infectious hpH5N1 virus.

Our findings of the H275Y mutation in H1N1 viruses may helpin assessing the biological outcome of other NA inhibitor–resistance markers. NA inhibitor–resistance mutations are virussubtype- and drug-dependent and may be NA catalytic residues(such as NA 118, 276, 292 and so on, and N2 numbering) orframework residues (such as NA 119, 222, 274, 294 and so on,and N2 numbering)7,52. The H275Y (274 in N2 numbering) is aframework residue mutation52. To date, surveillance and clinicalobservation have shown that R292K, E119V, N294S and I222Vmutant H3N2 viruses4,7–9, I222V and I222R mutant pandemicH1N1 viruses40,53,54, and R292K mutant H7N9 viruses 55 haveemerged only from drug-treated individuals and have not beenable to spread at the population level. Their compromisedepidemiologic fitness indicates that the biological outcome ofthese mutations on the proteins and viruses remains detrimental.Among these, resistance mutations at the catalytic residuespresumably would cause much more profound loss of NAfunctionality than framework mutations, and could consequentlycompletely eliminate the virus biological fitness. However, thesmaller loss-of-function by framework mutation might berestored more readily, such as the H275Y. Another twoframework mutations I222V and I222R in the pandemic H1N1viruses have shown less loss of NA function56,57, and its NAevolution course may therefore merit more attention.

Interaction of the H275Y mutation with permissive andcompensatory NA mutations explains the restoration of the NAfunction and the fitness of the resistant viruses to the wild-typelevel, but NA function alone does not explain why the resistantviruses outtransmitted over the wild-type viruses. Therefore, theroles of other viral proteins in conferring the greater fitness ofresistant viruses should be examined. For example, HA binding toreceptors also plays important role in influenza virus transmis-sion. The NA and HA genes of H1N1 viruses showed the samegenetic clustering and antigenic lineage patterns16–19, indicatingtheir co-evolution. The HAs of the resistant viruses exhibitedreduced reactivity to antibody inhibition58. HA mutations canpromote replication of BR07-like H275Y-mutant viruses in cells59

and drive NA mutations that alter antigenicity and NA-inhibitorsusceptibility60. All of these findings suggest the occurrence ofphenotypic interactions between the HA and NA proteins(probably to promote functional balance) during the evolutionof H275Y-mutant viruses. Comparison of the HA sequences ofthe BR07-like viruses used in the ferret experiment revealed fourHA mutations (G185V, N186D, A189T and H192R) shared bythe H275Y-mutant viruses (Supplementary Table 5). These fourpositions are located in or near to the 190 helix, which is one ofthe three structure elements enclosing the base of the HA bindingsite61; although these positions are not directly involved inreceptor binding, whether and how they can affect the receptorbinding and virus transmissibility would be of interest forfurther study. In addition, intra-subtype, inter-clade segmentreassortment events observed in the BR07-like resistantviruses18,62 suggest that a combination of internal proteininteractions plays a role in enhancing overall virus fitness.

In conclusion, we have shown how the fitness cost of a drug-resistant NA mutation in human seasonal influenza virus can bereduced or eliminated during NA evolution, allowing the resistantstrain to spread. As influenza viruses continue to evolve underimmune pressure and NA inhibitors remain the primarytherapeutic option, we suggest that NA genetics, antigenic driftand resistance mutation-generated NA functional outcomeshould be collectively and continually monitored to assess the

biologic fitness of emerging resistant viruses and their potentialevolutionary course towards resistance spread.

MethodsCells and viruses. Pandemic DM/524/09 and DM/528/09 viruses were providedby Statens Serum Institute, Copenhagen, Denmark. NY/3467/09 and NY/1692/09viruses were provided by the Wadsworth Center of the New York State Depart-ment of Health. Wild-type and NA H275Y-mutant MS/03/02 viruses were pro-vided by the Neuraminidase Inhibitor Susceptibility Network. The remainingH1N1 viruses, including CA/04/09, BR/59/07, HW/28/07, NJ/15/07, GA/17/06,GA/20/06, TX/30/07 and MEM/13/06, were provided by the US Centers for Dis-ease Control and Prevention. The NA gene segments of VN/1203/04 and HK/213/03 H5N1 viruses were generated into plasmids previously63,64, and no infectiousH5N1 viruses were used in the present study. All virus stocks used underwent aninitial limited number (1B2) of passages in MDCK cells on reception, to maintaintheir original properties. The 50% tissue culture infectious dose (TCID50) or plaqueforming unit was used to measure virus infectivity in MDCK cells as previouslydescribed37.

Sequence analysis. Viral RNA was isolated by using the RNeasy Mini kit(Qiagen). Samples were reverse-transcribed and analysed by PCR, using segment-specific primers as described previously65. Sequencing was performed by theHartwell Center for Bioinformatics and Biotechnology at St Jude Children’sResearch Hospital. DNA sequences were completed and edited by using theLasergene software package (DNASTAR).

Phylogenetic estimation of timing and sequence of mutations. All the pub-lished NA gene sequences of seasonal H1N1 viruses were downloaded from NCBI(Influenza Virus Resource). Initial visual inspection of multiple NA sequencealignments containing taxa with and without the H275Y oseltamivir resistancemutation identified 11 amino acid mutations associated with the NA lineagedivergence. To estimate the time of most recent common ancestor that possessedthe mutations of interest, we used all available full-length H1N1 NA sequences thatalso had the exact date of isolation in the accession, and generated three data setscomprising randomly selected taxa plus the past vaccine strains, referencesequences and six available pre-2007/2008 H275Y variants (ntax¼ 413 for eachdata set). The SRD06 approximate codon model of evolution with an HKY85þgamma nucleotide substitution model for codon position 1þ 2 and codon partition3 was used to estimate the phylogenetic tree. The ancestral state reconstruction ofall tested mutations was jointly estimated with the phylogeny. The NA evolutionaryhistory and the ancestral states reconstruction were estimated using a tip datecalibrated Bayesian relaxed-clock phylogenetic tree reconstruction method27. Weassigned the individual mutations as a tip observed state66. These mutations wereindicators for possible adaptive mutations. We assessed which mutations were fixedin the population, the sequence of fixation and when this fixation occurred.Phylogenetic analysis was carried out in BEAST (v1.7.3).

Plasmid construction and site-directed mutagenesis. The full-length com-plementary DNA (eight segments) of BR/59/07 virus and the NA segments of GA/17/06 and GA/20/06 viruses were cloned into the pHW2000 plasmid vector togenerate rg viruses, as described previously67. Mutations of interest wereintroduced into the corresponding NA genes by using QuickChange site-directedmutagenesis (Stratagene) and confirmed by Sanger sequencing. The full-lengthprotein-coding sequences of the NA genes of interest were cloned into thepCAGGS plasmid vector for expression of recombinant NA proteins in BHK cells,as described previously68. The HA epitope tag (YPYDVPDYA) was added tothe N or C terminus of the NA protein by incorporating the coding sequences intothe primers.

Generation of rg influenza viruses. Reverse-genetics viruses were rescued bytransfecting a 293T/MDCK cell co-culture with eight PHW2000 plasmids con-taining the eight virus segments, using TransIT LT-1 (Panvera), as describedpreviously67. Rescued viruses contained variant NA segments in the uniformbackbone from other seven segments of the BR/59/07 virus. The rescuedsupernatants from co-culture were first titrated by plaque assay and then onepassage of the supernatant in MDCK cells (multiplicity of infection¼ 0.001B0.01)was used to prepare stocks of all rg viruses.

Generation and detection of recombinant NA proteins. BHK cells were trans-fected with the respective NA gene-containing pCAGSS plasmids by using Lipo-fectamine 2000 (Invitrogen), as described previously68, and NA protein expressionwas measured by NA enzyme activity assay, western blotting or flow cytometry. Forcomparison of different proteins, BHK cells were transfected with equal quantitiesof the respective pCAGGS plasmids (0.5 mg unless indicated otherwise) in a 12-wellplate, in parallel. For western blotting, 0.05 mg of pCMV-GFP plasmid was co-transfected with the respective pCAGGS plasmid. Cells were harvested in B36 hpost transfection. For the subsequent NA enzyme activity assay, cells were lysed





with non-denaturing NA enzyme buffer containing 0.5% Triton X-100. Forwestern blotting, cells were lysed with denaturing SDS-containing RIPA buffer. Allsamples were processed in parallel. For protein expression level determination,anti-HA epitope monoclonal antibodies (Sigma, 1:20,000) were used to detect theepitope-tagged recombinant proteins in western blotting or flow cytometry. Inwestern blotting, b-actin was detected (anti-b-actin, Santa Cruz, 1:20,000) asloading control and green fluorescent protein (GFP, detected by anti-GFP, SantaCruz, 1:2,000) was used as transfection efficiency control. Fluorescent dye-labelled(IRDye680 or IRDye800) anti-mouse or -rabbit secondary antibodies (LI-COR,1:20,000) were used to detect the respective primary antibodies. The blot signalswere visualized and analysed in Odyssey imaging system (LI-COR). Full-gel imagesof the blots used in the Figures are provided in Supplementary Fig. 9.

NA enzyme activity and kinetics. The NA activity assay was based on afluorometric assay using the substrate 20-(4-methylumbelliferyl)-a-D-N-acet-ylneuraminic acid (MUNANA) (Sigma-Aldrich) in enzyme buffer containing33 mM 2- (N-morpholino) ethanesulphonic acid hydrate (Sigma-Aldrich), 4 mMCaCl2 at pH 6.5 at 37 �C for 30 min69. Hydrolysis of 1 mol MUNANA by NAliberated 1 mol fluorescent product 4-methylumbelliferone69 and the fluorescenceof liberated product was read in a Synergy 2 multi-mode microplate reader(BioTek), using excitation and emission wavelengths of 360 and 460 nm,respectively. Purified 4-methylumbelliferone (Sigma-Aldrich) was used as astandard for estimation of product generation and substrate consumption in theassay (for example, Supplementary Fig. 8a). For NA enzymatic activitymeasurement, all the harvested samples were serially diluted and were assayed inparallel with 100 mM MUNANA, and then NA quantity–activity curves weregenerated. The dilution at which all NAs were within their linear range of productgeneration and approximately consumed o20% of substrate was adopted foractivity measurement (for example, Supplementary Fig. 8b,c). Next, the activitywas normalized to a reference NA as the % relative activity (for example, Fig. 2a).The enzyme activity of all expressed NA proteins was measured by the sameprocess.

For NA kinetics assay, all viruses were standardized to a dose of 106 PFU ml� 1,a dose at which all viruses were determined to consume o10% of 100 mMMUNANA substrate in NA activity assays (for example, Supplementary Fig. 8a).We then measured NA enzyme kinetics in the enzyme buffer using serialconcentrations of MUNANA (final concentration, 800–6.25 mM, twofold serialdilution) in a total volume of 50ml. The fluorescence of released 4-methylumbelliferone was measured at 37 �C every 60 s for 40 min. Km wascalculated by fitting the data to the Michaelis–Menten equations by nonlinearregression in Prism5 software (GraphPad).

Transmission experiments in ferrets. All experiments were conducted in anABSL2þ laboratory under applicable laws and guidelines, and after approval fromthe St Jude Children’s Research Hospital Animal Care and Use Committee. Four-to 5-month-old ferrets were obtained from Triple F farm; all were tested sero-negative for contemporary influenza A H1N1 and H3N2 viruses and influenza Bviruses. Ferrets were initially housed in isolators and monitored for 3–5 days toestablish baseline body temperature and overall health. Donor and contact ferretswere housed separately. Transmission experiments (1 donorþ 1 direct-contactrecipientþ 1 respiratory-droplet recipient) were conducted in triplicate for eachvirus. Three donor ferrets were inoculated intranasally with 105 TCID50 of virus in1.0-ml sterile PBS. At day 1 p.i., each donor was co-housed with one naive direct-contact ferret. One additional naive ferret was placed in an adjacent cage separatedby double-layered (B5 cm apart) perforated dividers to assess respiratory-droplettransmission; air flow was directed from the donor cage toward the recipient cageusing a Borazine gun (Zero Toys). Nasal wash samples were collected at days 1 and2 p.i, and then every other day for 14 days by flushing both nostrils with 1.0 mlPBS. TCID50 titres of nasal washes were determined in MDCK cells. Serum sampleswere collected 3 weeks after inoculation, treated with receptor-destroying enzyme,heat-inactivated at 56 �C for 30 min and tested against the homologous virus by HIassay with 0.5% packed turkey red blood cells.

Statistical analysis. All statistical analyses were performed using Student’s t-testin Prism5 software. Data are presented as mean±s.e.m. The level of significancewas determined as Po0.05.

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AcknowledgementsThis work was supported by Contract Number HHSN266200700005C from the NationalInstitute of Allergy and Infectious Disease, US National Institutes of Health and by theAmerican Lebanese Syrian Associated Charities (ALSAC). We thank Sharon Naron forediting the manuscript, Drs Nancy Cox (U.S. Centers for Disease Control and Preven-tion) and Kristen St George (New York State Department of Health) for providing theseasonal H1N1 viruses, and Dr Charlie Russell for providing the pCAGGS plasmid andBHK cells. We thank Petr Krylov, Erik Karlsson, Heather Forrest, John Franks, LisaKercher, David S. Carey and Betsy Little for assistance in the ABSL2þ laboratory. Wethank Dr Suzanne Jackowski for helpful discussion and advice.

Author contributionsD.S. designed and conducted all experiments and wrote the manuscript. E.A.G. oversawthe experiments. J.B. performed the statistical phylogenetic analysis. Z.H. contributed tomolecular and animal experiments. B.T. and S.P. contributed to animal experiments. P.K.contributed to cellular and animal experiments. R.G.W. and R.J.W. provided conceptualdirection, designed and supervised the overall study and revised the manuscript. Allauthors commented and revised the manuscript.

Additional informationAccession codes: The full genome sequence of A/Brisbane/59/2007(H1N1) virus hasbeen deposited in Genebank database under the accession codes CY058484 to CY058491.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Competing financial interests: Although this study had no corporate funding, R.G.W.and E.A.G. are currently performing a different research study funded by F. Hoffmann-LaRoche, Ltd., Basel, Switzerland. The remaining authors declare no competing financialinterests.

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How to cite this article: Duan, S. et al. Epistatic interactions between neuraminidasemutations facilitated the emergence of the oseltamivir-resistant H1N1 influenza viruses.Nat. Commun. 5:5029 doi: 10.1038/ncomms6029 (2014).






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Epistatic interactions between neuraminidase mutations facilitated the emergence of the oseltamivir-resistant H1N1 influenza viruses

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