Med Microbiol Immunol (2012) 201:61–72

DOI 10.1007/s00430-011-0206-1

ORIGINAL INVESTIGATION

Neuraminidase inhibitor susceptibility of swine inXuenza A viruses isolated in Germany between 1981 and 2008

Katja Bauer · Ralf Dürrwald · Michael Schlegel · Kathrin Pfarr · Dominik Topf · Nadine Wiesener · Hans-Martin Dahse · Peter Wutzler · Michaela Schmidtke

Received: 21 March 2011 / Published online: 19 June 2011© Springer-Verlag 2011

Abstract European swine inXuenza A viruses donatedthe matrix protein 2 as well as the neuraminidase (NA)gene to pandemic inXuenza A (H1N1) viruses that emergedin 2009. As a result, the latter became amantadine resistantand neuraminidase inhibitor (NAI) susceptible. Theserecent developments reXecting the close connectionbetween inXuenza A virus infection chains in humans andpigs urge an antiviral surveillance within swine inXuenza Aviruses. Here, NAI susceptibility of 204 serologically typedswine inXuenza A viruses of subtypes H1N1, H1N2, andH3N2 circulating in Germany between 1981 and 2008 wasanalyzed in chemiluminescence-based NA inhibitionassays. Mean 50% inhibitory concentrations of oseltamivirand zanamivir indicate a good drug susceptibility of testedviruses. As found for human isolates, the oseltamivir andzanamivir susceptibility was subtype-speciWc. So, swineinXuenza A (H1N1) viruses were just as susceptible to osel-tamivir as to zanamivir. In contrast, swine H1N2 and H3N2inXuenza A viruses were more sensitive to oseltamivir thanto zanamivir. Furthermore, reduction in plaque size andvirus spread by both drugs was tested with selected H1N1

and H1N2 isolates in MDCK cells expressing similaramounts of �2.3- and �2.6-linked sialic acid receptors. Dataobtained in cell culture-based assays for H1N1 isolates cor-related with that from enzyme inhibition assays. But, H1N2isolates that are additionally glycosylated at Asn158 andAsn163 near the receptor-binding site of hemagglutinin(HA) were resistant to both NAI in MDCK cells. Possibly,these additional HA glycosylations cause a misbalancebetween HA and NA function that hampers or abolishesNAI activity in cells.

Keywords Swine inXuenza A virus · Hemagglutinin glycosylation · Neuraminidase inhibitors · Oseltamivir · Zanamivir · Resistance · In vitro

Introduction

A broad host range is characteristic for inXuenza A viruses.Besides birds and humans, pigs are infected by theseviruses. In Europe, inXuenza A viruses of subtypes H1N1,H1N2, and H3N2 are endemic in pigs and circulate all theyear round [1]. These viruses are genetically diVerent fromclassical swine inXuenza A viruses in North and SouthAmerica [2]. The latter are descendants of H1N1 inXuenzaA viruses causing the pandemic in 1918 [2], while currentlycirculating European avian-like swine H1N1 inXuenza Aviruses were introduced from birds into the pig populationin 1979 [3, 4]. European human-like swine inXuenza Aviruses of subtype H3N2 result from reassortment of ahuman H3N2 virus (HA and NA gene segments) intro-duced into the European pig population following the 1968“Hong Kong” Xu pandemic and an avian-like H1N1 swinevirus (all other gene segments) between 1983 and 1985[5, 6]. Multiple reassortment steps led to the occurrence of

K. Bauer · K. Pfarr · D. Topf · N. Wiesener · P. Wutzler · M. Schmidtke (&)School of Medicine, Department of Virology and Antiviral Therapy, Jena University Hospital, Hans-Knöll-Str. 2, 07745 Jena, Germanye-mail: Michaela.Schmidtke@med.uni-jena.de

R. Dürrwald · M. SchlegelIDT Biologika GmbH, Am Pharmapark, 06861 Dessau-Roßlau, Germany

H.-M. DahseLeibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Beutenbergstr. 11a, 07745 Jena, Germany

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62 Med Microbiol Immunol (2012) 201:61–72

human-like swine H1N2 isolates in Great Britain in 1994[7, 8]. As a result, swine H1N2 inXuenza A viruses consistof a human H1, a human N2, and six avian-like internalgene segments.

Infection of pigs by inXuenza A viruses from birds andpeople is enabled by the expression of sialic acid receptorsfor avian as well as human inXuenza A viruses in the respi-ratory tract of these animals [9]. In case of simultaneousinfection, pigs can act as a mixing vessel facilitating reas-sortment of avian, human, and/or diVerent swine inXuenzaA viruses. The genetic evolution of swine inXuenza Aviruses in Europe conWrms that [1, 5–8, 10–13].

Till 2009, only sporadic cases of transmission of swineinXuenza A viruses to human have been observed in Swit-zerland, the Netherlands, and Spain, whereby transmittedviruses did not spread among humans [14–18]. Then, newinXuenza A (H1N1) viruses emerged and caused the Wrstpandemic of the twenty-Wrst century [19, 20]. These pan-demic inXuenza A (H1N1) viruses contain a new combina-tion of gene segments from avian (PB2 and PA), human(PB1), classical swine (HA, NP, and NS), and Eurasian(M and NA) swine inXuenza A viruses [21–24]. By donat-ing the M as well as NA gene segments to pandemic inXu-enza A (H1N1) 2009 virus, Eurasian swine inXuenza Aviruses impacted remarkably on Xu treatment. The respec-tive M gene aVected adversely the therapeutic eVect of ionchannel inhibitors because it contains the genetic resistancemarker S31N in matrix protein 2 [25, 26]. Accepting this Mgene segment, pandemic inXuenza A (H1N1) virusesbecame resistant against these drugs [27]. In contrast, theNA gene of Eurasian swine inXuenza A viruses conferredneuraminidase inhibitor (NAI) susceptibility. Due to thehigh prevalence of amantadine-resistant viruses among allcirculating inXuenza A virus subtypes [27–34], NAI are theonly drugs considered for Xu therapy at the moment. Theyexhibit antiviral activity against inXuenza A as well as Bviruses [35], are well tolerated, and resistance was rare inprevious studies [35–38]. But, during the inXuenza season2007/2008, a spontaneous increase in oseltamivir resistancerates was noted among seasonal inXuenza A viruses of sub-type H1N1 worldwide [39–41]. Fortunately, these oseltam-ivir-resistant seasonal inXuenza A (H1N1) viruses werenearly completely replaced by pandemic inXuenza A(H1N1) viruses that possess the NAI susceptible N1 of Eur-asian swine inXuenza A viruses [42, 43].

These sudden changes in drug susceptibility caused byincorporation of gene segments of swine inXuenza Aviruses underline the importance of monitoring drug sus-ceptibility of swine inXuenza A viruses. With the exceptionof a pilot antiviral study including a small number of swineH3N2 isolates [44], the NAI susceptibility of Eurasianswine inXuenza A viruses is unknown until now. To Wll thisgap, a NAI susceptibility surveillance of swine inXuenza A

viruses isolated from pigs with Xu symptoms in Germanybetween 1981 and 2008 was conducted in the present study.

Materials and methods

Cells

Madin-Darby canine kidney (MDCK) cells (Friedrich-LoeZer Institute, Riems, Germany) were maintained inEagle’s minimum essential medium (EMEM) and MDCK-SIAT1 cells (Dr. Matrosovich, Marburg, Germany) inDulbeccos’s modiWed Eagle’s medium (DMEM) bothsupplemented with 10% fetal bovine serum, 100 U/ml pen-icillin, and streptomycin. The serum-free EMEM (testmedium) applied in cell culture-based assays on conXuent3-day-old cell monolayer was formulated with 2 �g/mltrypsin and 1.2 mM bicarbonate. Normal human bronchialepithelial (NHBE) cells (Lonza, Basel, CH) were subcul-tured in bronchial epithelial basal medium (BEBM) accord-ing to the manufacturer’s instructions. Cells of passage twoand three were grown on membrane supports (CostarTranswell Clear, Corning, NY) at the air–liquid interface asdescribed elsewhere [45].

Viruses

Swine inXuenza A viruses of subtypes H1N1 (n = 80),H1N2 (n = 35), and H3N2 (n = 91) were obtained duringa surveillance program based on a total of 1331 submis-sions of nasal swabs from pigs with clinical disease dur-ing 2003–2008. The surveillance comprised the entireterritory of Germany. Most isolates originated fromdensely swine-populated areas. Viruses were isolated inembryonated hen’s eggs. The H1N1 isolates A/swine/Potsdam/15/81, A/swine/Schwerin/103/89, A/swine/Bakum/5/95, and A/swine/Belzig/2/01 as well as the H1N2 iso-lates A/swine/Bakum/1832/00 and A/swine/Bakum/1833/00 were provided by Dr. Schrader (Federal Institute forRisk Assessment, Berlin, Germany [12]). Virus stockswere prepared in MDCK cells, aliquoted, and stored at¡80°C until use.

Titers of virus stocks were determined according to Reedand Muench in MDCK cells and expressed as 50% tissueculture infectious dose (TCID50) [46].

Compounds

Zanamivir (GG167) and oseltamivir carboxylate (GS4071)were kindly provided by GlaxoSmithKline (Uxbridge, UK)and F. HoVmann-La Roche AG (Basel, CH), respectively.Compound stocks were prepared in water and stored at4°C.

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Med Microbiol Immunol (2012) 201:61–72 63

Detection of �2.3- and �2.6-linked sialic acids on MDCK, MDCK-SIAT1, and NHBE cells

Cell surface expression of sialic acids was analyzed by usingdigoxigenin (DIG)-labeled lectins Sambucus nigra (SNA)and Maackia amurensis agglutinin (MAA) from the DIGglycan diVerentiation kit (Roche, Mannheim, Germany) spe-ciWc for �2.6- and �2.3-linked sialic acids, respectively.

On the one hand, FACS analysis was performed to quan-tify the level of �2.3- and �2.6-linked sialic acid expressionas described elsewhere [47]. In brief, »2 £ 106 cells/ml inTris-buVered sialine, pH 7.5, and 1 mM each of MgCl2,MnCl2, and CaCl2 were incubated for 1 h at room tempera-ture with either 1 �l SNA, 5 �l MAA, or without lectins.Lectins were detected with monoclonal mouse anti-DIGantibody (Roche, Mannheim, Germany) and either polyclonalgoat anti-mouse immunoglobulins/FITC or polyclonal rabbitanti-mouse immunoglobulins/FITC (Dako, Hamburg,Germany). Fluorescence intensity was analyzed on a BDLSR II Flow Cytometer (BD Biosciences, Heidelberg,Germany).

On the other hand, immunohistochemical staining of cellmonolayers was performed according to the instructionsfrom the DIG glycan diVerentiation kit (Roche, Mannheim,Germany) to visualize expression pattern of �2.3- and �2.6-linked sialic acids.

Chemiluminescent NA inhibition assay

Chemiluminescence-based NA inhibition assays were per-formed as previously described [42] using the NA-Star®

InXuenza Neuraminidase Inhibitor Resistance DetectionKit (Applied Biosystems, Darmstadt, Germany). BrieXy,NA activity was titered by serial twofold dilutions of thevirus to determine the virus dilution resulting in a signal-to-noise ratio between 10:1 and 40:1. All viruses were dilutedat least Wvefold to avoid signal quenching. For determina-tion of 50% inhibitory concentration (IC50), NA inhibitionwas tested with serial half-log NAI concentrations rangingfrom 10 to 0.03 nM, each concentration in triplicate. Sixuntreated virus controls were included. Chemiluminescencewas read using a MLX microtiter plate luminometer(Dynex Technologies, Chantilly, Virginia, USA).

Evaluation of NAI susceptibilities in cell culture-based assays

Plaque reduction assays were performed as described previ-ously [26]. Serial tenfold dilutions of oseltamivir andzanamivir were tested in duplicate in at least three indepen-dent assays. The 50% inhibitory concentrations (IC50) werecalculated from the dose–response curves based on plaquesize reduction.

The inXuence of NAI on virus spread was evaluated asdescribed elsewhere [44] in at least two independent assays.BrieXy, MDCK cells were infected with virus at a multi-plicity of infection (MOI) of 0.0005 TCID50 per cell thatresulted in a nearly 100% infection rate of MDCK cells inuntreated virus controls 48 h after infection. After methanolWxation, viral nucleoprotein was detected by immunostain-ing using monoclonal mouse anti-inXuenza A nucleopro-tein antibody (Acris Antibodies, Herford, Germany) andthe Dako REAL™ Detection System APAAP Mouse(Dako, Hamburg, Germany).

Sequencing

Extraction of viral RNA from MDCK cell supernatants wasperformed according to the manufacturer’s instructionswith the use of RNeasy Mini kit (Qiagen, Hilden,Germany). Viral RNA encoding the surface proteins HAand NA was ampliWed by RT–PCR and sequenced asdescribed previously [48] with fragment-speciWc primerspublished elsewhere [49].

For sequence comparisons of HA and NA genesequences of German swine inXuenza A viruses sequence,data were obtained from the PubMed GenBank (http://www.ncbi.nlm.nih.gov/genomes/FLU/Database/multiple.cgi) andaligned with the program BioEdit version 7.0.5.3 [50] andCLUASTALW.

ConWrmation of HA glycosylation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis

One- to two-day-old conXuent MDCK cell monolayersgrown in 6 cm culture dishes were infected with virusstocks of A/swine/Potsdam/15/81 (H1N1) and A/swineBakum/1832/00 (H1N2), and mock-infected for control.Furthermore, virus isolated from mouse lung tissue at day4 p.i. and passaged once in MDCK cells was included inthese studies to check the stability of glycosylation duringvirus passages. After 24 h of incubation, MDCK cellswere scraped oV from the culture dishes, suspended, andcentrifuged by 3,000 U/min for 5 min. Proteins wereextracted with NTE-BuVer (100 mM NaCl; 10 mM Tris/HCl pH 7.4) and 10% NP-40 and stored at ¡80°C untilfurther preparation. Before Western blotting, protein con-centrations were determined with the Bradford methodand Coomassie Brilliant Blue G250 (Bio-Rad, Munich,Germany).

Protein samples of 50 �g were incubated with 10£glycoprotein denaturating buVer for 10 min at 95°C.Then, denatured proteins were incubated with test buVerwithout enzyme or with either Endo H glycosidase orPNGase F (both 500 U; New England Biolabs, Frankfurt

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64 Med Microbiol Immunol (2012) 201:61–72

am Main, Germany) in test buVer for 2 h. At the end ofthe digestions, 5£ protein loading buVer (Fermentas, St.Leon-Rot, Germany) was added and all samples wereexamined by 10% SDS–PAGE analysis. After electro-blotting onto protran® nitrocellulose membrane (What-man, Dassel, Germany), non-speciWc binding sites wereblocked with skim milk dissolved in TBS-BuVer (1 MTris–HCl pH 8.0; 5 M NaCl). Tris-buVered saline plus0.5% Tween20 were used for all washing procedures.For visualization of viral proteins, membranes wereincubated successively (1) with anti-A/swine/Bakum/1832/00 (H1N2) or anti-A/swine/Potsdam/15/81 (H1N1)mouse serum (1:100, in-house), (2) rabbit anti-mouseIgG (H&L) AP-conjugated (1:2,000; Cell Signaling,USA), and (3) the NBT/BCIP detection system (Roche,Mannheim, Germany).

Hemagglutination assay

Serial twofold virus dilutions were tested in duplicate asdescribed previously [44]. HA titers were calculated as thereciprocal values of the highest virus dilution that aggluti-nated chicken erythrocytes.

Statistical analysis

The 50% enzyme inhibitory concentrations (IC50) toeither drug were analyzed using Microsoft Excel (Micro-soft Corp., Redmond, WA, USA). Then, mean and stan-dard deviation (SD) of IC50 values were determinedseparately for each virus subtype for the period 1981–2005, 2006, 2007, 2008 as well as cumulatively from1981 to 2008.

In agreement with human inXuenza virus NAI surveil-lance studies [51], the mean IC50 + 3SD criterion wasselected as a cutoV value. It was calculated for each sub-type with each NAI. Isolates with IC50 values greaterthan tenfold of the mean IC50 for each respective subtypeand drug were deWned as extreme outliers and excludedfrom statistical analysis of the overall population. Asdone for assessment of inXuenza A virus drug suscepti-bility [27, 51], box and whisker plot analyses of log-transformed IC50 values [52] were performed for eachdrug using SigmaStat 3.10 software (Systat SoftwareInc., San José, CA, USA) to determine quartiles andinterquartile ranges (IQRs) necessary for establishing astatistical cutoV for the identiWcation of outliers with sig-niWcantly increased IC50 values. The statistical cutoV wasset at 3£ the IQR to the right of the third quartile (X0.75)[51]. Descriptive statistics and one-way ANOVA of orig-inal scale IC50 values were calculated from each drugusing SigmaStat 3.10 software with statistical signiW-cance set at � = 0.05.

Results

Surveillance of NAI susceptibility of swine inXuenza A viruses

The IC50 values obtained by using chemiluminescence-based NA inhibition assays are summarized in Table 1 forthe individual study periods (1981–2005, 2006, 2007, and2008) as well as for the whole study period (1981–2008).The box and whisker plot statistical analysis summarizingthe data of the whole period is shown in Fig. 1.

The statistical cutoV was calculated based on box plotanalysis of the pooled IC50s for each subtype and for eachantiviral drug (Table 1). Neither for oseltamivir nor forzanamivir an outlier was identiWed.

There was no trend toward increased IC50s with time.From 1981 to 2005, in 2006, 2007, and 2008, the mean IC50

values for swine inXuenza A (H1N1) viruses were low toboth oseltamivir (0.13–0.24 nM) and zanamivir (0.11–0.25 nM). In contrast, the mean IC50 values for swine inXu-enza A (H1N2) as well as swine inXuenza A (H3N2)viruses were similarly low to oseltamivir (0.27–0.30 nMand 0.36–0.59 nM, respectively) but higher against zanami-vir (0.68–0.89 nM and 0.68–1.37 nM, respectively). Basedon pooled IC50s of all tested isolates, mean and SD asshown for each subtype and drug in Table 1 conWrm thesubtype-independent NA inhibitory activity of oseltamiviras well as the subtype-speciWc activity of zanamivir.

ConWrmation of receptor expression in MDCK cells

Results from FACS analysis as well as immunohistochemi-cal staining using the lectins MAA and SNA for �2.3- and�2.6-linked sialic acid detection, respectively, demonstratehigh levels of both receptors in MDCK, MDCK-SIAT1,and commercially available NHBE cells (Fig. 2). The lattertwo cell cultures were recommended for assessment of NAIsusceptibility of human inXuenza A viruses based on highlevel expression of �2.3- and �2.6-linked sialic acid recep-tors [53–55]. Because neither results from FACS analysisnor from immunohistochemical staining revealed a signiW-cant diVerence in the receptor expression between MDCK,MDCK-SIAT1, and NHBE cells, all cell culture-basedassays reported in this study were conducted in MDCKcells.

Susceptibility of selected swine inXuenza A viruses in cell culture-based assays

Previously, a good NAI susceptibility was shown forselected swine inXuenza A (H3N2) viruses in MDCK cells[44]. Here, results from plaque reduction assays demon-strate an eYcient inhibition of plaque size of all three swine

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Med Microbiol Immunol (2012) 201:61–72 65

Tab

le1

Stat

istic

al a

naly

sis

of 5

0% in

hibi

tory

con

cent

rati

on (

IC50

) va

lues

det

erm

ined

in th

e N

A in

hibi

tion

assa

y an

d gr

oupe

d ac

cord

ing

to y

ear

and

viru

s su

btyp

e

aA

ll I

C50

val

ues

are

expr

esse

d in

nM

bC

utoV

val

ue w

as d

eter

min

ed b

ased

on

the

elec

ted

crite

rion

of

IC50

>m

ean

IC50

+3S

Dc

Stat

istic

al c

utoV

val

ue w

as d

eter

min

ed b

ased

on

log 1

0 IC

50>

X0.

75+

3IQ

Rd

Pva

lue

dete

rmin

ed b

y A

NO

VA

(�

=0.

05)

Dru

gV

irus

1981

–200

520

0620

0720

0819

81–2

008

Tot

al �

204

Pva

lued

Ran

gea

Mea

n§

SDR

ange

Mea

n§

SDR

ange

Mea

n§

SDR

ange

Mea

n§

SD

Ran

geM

ean§

SD

Cut

oVa,

bS

tatis

tical

cu

toV

c

Ose

ltam

ivir

A/H

1N1

0.09

–0.3

4 (n

=17

)0.

17§

0.07

0.10

–0.6

7 (n

=27

)0.

24§

0.11

0.09

–0.4

5 (n

=20

)0.

20§

0.09

0.04

–0.3

0 (n

=18

)0.

13§

0.07

0.04

–0.6

7 (n

=82

)0.

19§

0.10

0.48

0.28

3<

0.00

1

A/H

1N2

0.10

–0.6

0 (n

=17

)0.

27§

0.16

0.10

–0.5

6 (n

=5)

0.30

§0.

170.

10–0

.65

(n=

10)

0.27

§0.

160.

08–0

.53

(n=

4)0.

27§

0.19

0.08

–0.6

5 (n

=36

)0.

27§

0.16

0.75

0.05

10.

911

A/H

3N2

0.10

–0.7

4 (n

=20

)0.

39§

0.24

0.10

–0.8

6 (n

=25

)0.

36§

0.21

0.03

–0.9

1 (n

=17

)0.

59§

0.28

0.08

–0.8

7 (n

=24

)0.

36§

0.21

0.03

–0.9

1 (n

=86

)0.

39§

0.24

1.10

0.11

90.

020

Zan

amiv

irA

/H1N

10.

09–0

.30

(n=

17)

0.17

§0.

050.

06–0

.62

(n=

27)

0.22

§0.

120.

09–0

.95

(n=

20)

0.25

§0.

200.

03–0

.21

(n=

18)

0.11

§0.

050.

03–0

.95

(n=

82)

0.19

§0.

140.

600.

990

<0.

001

A/H

1N2

0.25

–1.3

0 (n

=17

)0.

72§

0.33

0.19

–2.3

0 (n

=5)

0.89

§0.

860.

16–1

.80

(n=

10)

0.81

§0.

480.

38–1

.04

(n=

4)0.

68§

0.30

0.16

–2.3

0 (n

=36

)0.

76§

0.45

2.12

1.20

90.

863

A/H

3N2

0.15

–2.1

0 (n

=20

)1.

06§

0.68

0.18

–2.2

0 (n

=25

)0.

68§

0.50

0.03

–2.4

5 (n

=17

)1.

37§

0.60

0.23

–1.7

0 (n

=24

)0.

94§

0.35

0.03

–2.4

5 (n

=86

)1.

06§

0.68

3.10

1.31

20.

002

123

66 Med Microbiol Immunol (2012) 201:61–72

inXuenza A (H1N1) viruses by oseltamivir as well aszanamivir (Table 2). In contrast, a markedly reduced NAIsusceptibility of two H1N2 isolates was noticed using thisexperimental approach.

The results from studies on the inXuence of oseltamivir andzanamivir (both 1 �g/ml) on virus spread in MDCK cells byimmunohistochemical staining of the viral nucleoprotein undermulti-step life cycle conditions further conWrmed the diVer-ence in NAI susceptibility of the tested swine inXuenza Aviruses of subtype H1N1 and H1N2 in MDCK cells (Fig. 3).Almost all cells were virus-infected in untreated virus controls,as shown by intensive staining of cells 48 h after virus inocula-tion. Drug treatment strongly reduced cell-to-cell spread of theH1N1 isolates A/swine/Potsdam/15/81, A/swine/Schwerin/103/89, and A/swine/Bakum/5/95. In contrast to untreatedvirus controls, only small infection foci were detected. But, noinhibition of virus spread was observed in NAI-treated MDCKcells infected with the two H1N2 isolates A/swine/Bakum/1832/00 and A/swine/Bakum/1833/00.

Genetic analysis and glycosylation of A/swine/Potsdam/15/81 and A/swine/Bakum/1832/00

Genotyping is usually applied to conWrm the NAI-resistantphenotype determined in enzyme inhibition assays. Because

Fig. 2 Expression of �2.3- and �2.6-linked sialic acids on MDCK,MDCK-SIAT1, and NHBE cells. Cells were incubated with DIG-la-beled �2.3- and �2.6-linkage-speciWc lectins MAA and SNA. Afterincubation with monoclonal mouse anti-DIG antibodies and FITC-la-

beled anti-mouse immunoglobulins, cells were subjected to FACSanalysis (red control without lectins, blue NeuAc�2,3Gal, green Neu-Ac�2,6Gal) or immunohistochemical staining with NBT/BCIP,respectively (color Wgure online)

Fig. 1 Quatile box and whisker plots showing distribution of IC50 val-ues of oseltamivir (O) and zanamivir (Z) among 204 swine inXuenza Aviruses isolated in Germany between 1981 and 2008

-2,0

-1,5

-1,0

-0,5

0,0

0,5

O Z Z ZO O

IC50

, log

10nM

A/H1N1n=82

A/H1N2n=36

A/H3N2n=86

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Med Microbiol Immunol (2012) 201:61–72 67

Table 2 Susceptibility of selected swine inXuenza A H1N1 and H1N2 viruses to neuraminidase inhibitors in vitro and agglutination of chickenerythrocytes

a Inhibition of NA activity by oseltamivir carboxylate and zanamivir was tested in a chemiluminescence-based assay. Values represent the average50% inhibitory concentration (IC50) of at least two experimentsb At least three plaque assays were performed in MDCK cells. The average concentrations of oseltamivir and zanamivir reducing the plaque sizeby 50% are shown

Porcine FLUAV Subtype IC50 NA activity inhibition [nM]a IC50 plaque size [�M]b Reciprocal HA titer

Oseltamivir Zanamivir Oseltamivir Zanamivir 4°C 37°C Fold

A/swine/Potsdam/15/81 H1N1 0.16 § 0.08 0.10 § 0.02 0.02 § 0.00 0.01 § 0.00 128 64 2

A/swine/Schwerin/103/89 H1N1 0.18 § 0.01 0.20 § 0.01 0.01 § 0.00 0.13 § 0.04 16 16 1

A/swine/Bakum/5/95 H1N1 0.09 § 0.01 0.17 § 0.03 0.05 § 0.02 <0.15 64 64 1

A/swine/Bakum/1823/00 H1N2 0.19 § 0.03 0.69 § 0.07 >2.59 >150 64 0 >32

A/swine/Bakum/1833/00 H1N2 0.14 § 0.03 0.53 § 0.22 >2.59 >150 64 0 >32

Fig. 3 InXuence of treatment with oseltamivir and zanamivir onspread of swine inXuenza A viruses of subtypes H1N1 and H1N2 inMDCK cells. Virus-infected cells were detected by immunostaining of

the viral nucleoprotein 48 h after infection. They are shown as black-stained cells

swine influenza A virus untreated

virus control

oseltamivir

1 µg/ml

zanamivir

1 µg/ml

Potsdam/15/81 (H1N1)

Schwerin/103/89 (H1N1)

Bakum/5/95 (H1N1)

Bakum/1832/00 (H1N2)

Bakum/1833/00 (H1N2)

cell control

123

68 Med Microbiol Immunol (2012) 201:61–72

neither oseltamivir- nor zanamivir-resistant NA were detected,NA sequencing was performed exemplarily for swine inXu-enza viruses A/swine/Potsdam/15/81 (H1N1) and A/swine/Bakum/1832/00 (H1N2). In agreement with the NAI suscepti-ble phenotype in enzyme inhibition assays, neither NA dele-tions nor known substitutions conferring NAI resistance werefound (results not shown; JN088204 and JN088205).

Adjacent to NA, HA can have an impact on virus sus-ceptibility to NAI in cell culture-based assays [56]. Com-parison of HA sequences revealed that H1N1 (E190D) aswell as H1N2 (E190D and G225D) swine inXuenza Aviruses contain amino acid substitutions increasing bindingto �2.6-linked sialic acids (Table 3). But, the H1 of H1N1markedly diVers from the H1 in H1N2 in the number andlocation of glycosylation sites (Table 3). Two of three addi-tional potential glycosylation signals (Asn158, Asn163)detected in the swine inXuenza A viruses of subtype H1N2were located near the receptor-binding site.

To verify diVerences in HA glycosylation of H1N1 andH1N2 isolates, selected virus stocks of strains A/swine/Pots-dam/15/81 (H1N1) and A/swine/Bakum/1832/00 (H1N2)were subjected to SDS–PAGE analysis. Additionally, iso-lates passaged once in mouse lungs as well as in MDCK cellswere examined to check the stability of glycosylation duringpassages. The results of SDS–PAGE analysis shown inFig. 4 revealed the anticipated diVerence in electrophoreticmobility of HA0 molecules of A/swine/Potsdam/15/81(5 potential glycosylation sites as indicated in Table 3) andA/swine/Bakum/1832/00 (7 potential glycosylation sites asindicated in Table 3). The slowest-migrating band was pro-duced by the HA0 of virus stocks of A/swine/Bakum/1832/00 followed by the HA0 of A/swine/Potsdam/15/81(Fig. 4a). Pretreatment of the samples with PNGase F wasaccompanied by a loss of the diVerence in electrophoreticmobility of HA0 molecules of both viruses. A single intenseband of the same size was detected in all treated samples.This enzyme is an amidase that cleaves between GlcNAc andasparagine residues and removes N-glycans. No visible eVectwas observed on the electrophoretic mobility of HA0 afterhydrolysis with Endo H that cleaves between the chitobiose

core of mannose and some hybrid oligosaccharides fromN-linked glycoproteins. Glycosylation remained unchangedduring MDCK cell and mouse passages. Taken together, theobtained results conWrm the presence of additional glycans instocks of A/swine/Bakum/1832/00 (H1N2).

DiVerent binding aYnity of HA of swine inXuenza A viruses for chicken erythrocytes

Glycans near the receptor-binding site can change receptoraYnity of HA and be one explanation for the reduced NAIsusceptibility in MDCK cells [57]. Therefore, the HA recep-tor-binding properties of swine inXuenza A viruses werecomparatively studied in hemagglutination assays withchicken erythrocytes at two diVerent temperatures. Theinvestigation of receptor-binding sites revealed substantialdiVerences between the studied swine inXuenza A viruses(Table 2). H1N1 Isolates eYciently agglutinated chickenerythrocytes at both 4 and 37°C. In contrast, the reciprocalHA titer of H1N2 isolates was reduced by >32-fold.

Discussion

In the present study, baseline susceptibility of 204 swineinXuenza A viruses from diVerent geographic regions inGermany collected between 1981 and 2008 to oseltamivirand zanamivir was compared with a chemiluminescence-based NA inhibition assay. This test is highly sensitive,reproducible, and requires low virus volume for testing[58]. Obtained results demonstrate that both drugseYciently inhibit the NA of all tested isolates (Table 1).The mean IC50 values (Table 1) are in good agreement withthat of human inXuenza A viruses [27, 34, 37, 48, 51, 52] aswell as a couple of swine H3N2 isolates studied previously[44]. They conWrm the subtype-speciWc activity of oseltam-ivir and zanamivir described for human inXuenza A viruses,which are based on diVerences in 3-dimensional NA struc-tures [59]. So, swine inXuenza A viruses of subtype H1N1were just as susceptible to oseltamivir as to zanamivir,

Table 3 Amino acids in position 226, 228, 190, and 225 determining receptor-binding properties and potential glycosylation sites present in theHA1 subunit of swine inXuenza A H1N1 and H1N2 viruses selected for cell culture-based assays (H3 numbering)

* Sequence similar to that of A/Bakum/1832/00

Subtype Swine inXuenza A virus (gen bank number)

Amino acids of the receptor-binding site Potential glycosylation sites at Asn

190 225 226 228 20 21 33 94a 158 163 165 271 276 289

H1N1 Potsdam/15/81 (AAZ15840) D G Q G + + + + ¡ ¡ ¡ ¡ ¡ +

Schwerin/103/89 (AAZ15841) D G Q G + + + + ¡ ¡ ¡ ¡ ¡ +

Bakum/5/95 (AAZ17358) D G Q G + + + + ¡ ¡ + ¡ + ¡H1N2 Bakum/1823/00 (ABS53372) D D Q G + + + ¡ + + ¡ + ¡ +

Bakum/1833/00* D D Q G + + + ¡ + + ¡ + ¡ +

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Med Microbiol Immunol (2012) 201:61–72 69

whereas swine inXuenza A viruses of subtype H1N2 as wellas H3N2 were more sensitive to oseltamivir than to zanam-ivir. Two- to three-times higher concentrations of zanami-vir were necessary to inhibit activity of class 2 NAs by50%. As anticipated, conventional sequencing of NA genesegments of inXuenza viruses A/swine/Potsdam/15/81 andA/swine/Bakum/1832/00 did not reveal resistance muta-tions. In conclusion, the drug susceptibility genotype was inagreement with the phenotype determined in enzyme inhi-bition assay and conWrms the oseltamivir and zanamivirsusceptibility of the NA of tested swine inXuenza A virusesas found for human inXuenza A viruses.

The results from several studies with human inXuenza Aviruses indicate that the NAI susceptibility phenotypedetermined in NA inhibition assays and/or observed indrug-treated Xu patients not necessarily correlates with NAIactivity determined in cell culture-based assays [60–63].

Comparing the inhibitory activity of oseltamivir andzanamivir against three H1N1 and two H1N2 isolates inMDCK cells, marked diVerences were found. As inEurasian H3N2 swine inXuenza A viruses [44], the NAIsusceptibility of the studied swine H1N1 isolates in enzymeinhibition assays correlates with protection in MDCK cells(Table 2; Fig. 3). In contrast, neither plaque size nor spreadof swine H1N2 inXuenza A virus isolates were markedlyinhibited by oseltamivir and zanamivir (Table 2; Fig. 3).

The lack of inhibitory activity of oseltamivir as well aszanamivir in MDCK cells in spite of highly NAI suscepti-ble NA suggests that H1N2 progeny swine inXuenza Aviruses can escape infected cells with reduced or withoutneed of NA activity in MDCK cells. Previously, thiswas shown for human inXuenza A viruses whose HAreceptor-binding site did not match well with cellularreceptor expression pattern or whose HA was abundantly

Fig. 4 Comparative examina-tion of the eVect of glycosylation sites on HA0 electrophoretic mobility of A/swine/Bakum/1832/00 (H1N2; a and b) and A/swine/Potsdam/15/81 (H1N1; a and c). MDCK cells were infected with virus stocks of both swine inXuenza A viruses used for antiviral studies in vitro as well of virus stocks derived after one mouse lung passage and propagation in MDCK cells applied for antiviral studies in vivo. Uninfected MDCK cells were included as controls. After protein extraction and quantiW-cation, 50 �g of denatured protein were treated with buVer, PGNase F, or Endo H for 2 h and analyzed under reducing conditions in 10% SDS–PAGE followed by Western blotting. The arrowheads indicate the positions of the HA0 molecules depending on their glycosylation after visualization with virus-speciWc mouse antibodies, AP-conjugated rabbit anti-mouse IgG, and the NBT/BCIP detection system. The protein standard on the left indicates the molecular masses in kilo Daltons (kDa)

b

130 kDa

95 kDa

72 kDa

HA0

HA0 (non glycosylated)

PNGase F

Endo H

- + - - + - - + -- - + - - + - - +

A/swine/Bakum/1832/00virus stock + one passage in mouse

lung and MDCK cells

cell control

c

PNGase F

Endo H

- + - - + - - + -- - + - - + - - +

A/swine/Potsdam/15/81virus stock + one passage in mouse

lung and MDCK cells

cell control

130 kDa

95 kDa

72 kDa

HA0

HA0 (non glycosylated)

a

HA0(7 potential glycosylation sites)

HA0(5 potential glycosylation sites)

130 kDa

95 kDa

72 kDa

pm

pm

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70 Med Microbiol Immunol (2012) 201:61–72

glycosylated near the receptor-binding site [36, 57, 64–66].Due to reduced need of NA activity for virus release, NAIdid not act accordingly in MDCK cells [60–63]. Accordingto the FACS analysis as well as immunohistochemicalstaining results (Fig. 3), MDCK cells used in the presentstudy are characterized by nearly similar expression of�2,3- as well as �2,6-linked sialic acid receptors. Moreover,amino acids of HA determining receptor speciWcity of thestudied avian-like H1N1 as well as human-like H1N2swine inXuenza A viruses (Table 3) facilitate binding to�2.3- and �2.6-linked sialic acid-containing receptors [67–69]. Amino acids Q226 and G228 enable binding to 2.3-linked sialic acids and D190 and D225 increase binding to2.6-linked sialic acids expressed on cell surfaces. This isconsistent with previous reports on receptor-binding prop-erties of swine inXuenza A viruses [68, 70–72] and with thepresence of either sialic acid receptor linkage type in swinetracheal tissue [9]. Possibly, the detected N-glycosylationsignals in the amino acid sequence of HA near to the recep-tor-binding site of H1N2 isolates (Asn158, Asn163; H3numbering) that are not present in H1N1 isolates (Table 3;Fig. 4) reduced the HA receptor-binding aYnity anddecreased viral dependence on NA function for infection.Glycan attached to Asn163 (H3 numbering) was proved toaVect severely viral HA binding to cellular receptors and toreduce viral susceptibility to oseltamivir and zanamivir inMDCK cells by >10,000-fold [57]. The reduced agglutinat-ing activity toward chicken erythrocytes of swine inXuenzaA viruses with additional glycans near the receptor-bindingsite (Table 2) is consistent with the observation that glycansadjacent to the receptor-binding site can change receptor-binding properties of inXuenza A viruses [73–75].

In conclusion, the results of the present study provide aWrst insight into the NAI susceptibility of Eurasian swineinXuenza A viruses illustrated by isolates circulating inGermany between 1981 and 2008, whereas drug resistancewas not observed in enzyme inhibition assays. The datacomplement human inXuenza virus surveillance activities.Even though no NAI-resistant swine inXuenza A viruseswere detected till 2008, the monitoring should be continuedto early recognize oseltamivir-resistant swine inXuenza Aviruses that could represent a threat for human health.

Acknowledgments This study was supported by the GermanBundesministerium für Bildung und Forschung (01 KI 07143 and 01KI 07142) awarded to Ralf Dürrwald and Michaela Schmidtke. Wethank Birgit Jahn for excellent technical assistance.

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