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Running title: HA versus NP readout in influenza virus titration 1

2

3

Detection of non-hemagglutinating influenza A(H3) viruses by 4

ELISA in quantitative influenza virus culture 5

Van Baalen, C.A.1#, C. Els1, L. Sprong1, R. van Beek2, E. van der Vries2, A.D.M.E. Osterhaus1,2 6

and G.F. Rimmelzwaan1,2 7

1 Viroclinics Biosciences, Rotterdam, The Netherlands; 2 Erasmus MC Department of 8

Viroscience, Rotterdam, The Netherlands. 9

10

# Address correspondence to: baalen@viroclinics.com 11

12

Institutional addresses: 13

Viroclinics Biosciences; Marconistraat 16; 3029 AK Rotterdam; The Netherlands. 14

Erasmus MC Department of Viroscience; Dr Molewaterplein 50; 3015 GE Rotterdam; The 15

Netherlands.16

JCM Accepts, published online ahead of print on 12 March 2014J. Clin. Microbiol. doi:10.1128/JCM.03575-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract 17

To assess the efficacy of novel antiviral drugs against influenza in clinical trials it is necessary to 18

quantify infectious virus titers in patients’ respiratory tract samples. Typically, this is achieved 19

by inoculating virus-susceptible cells with serial dilutions of clinical specimens, and detecting 20

the production of progeny virus by hemagglutination since influenza viruses generally have the 21

capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin 22

(HA). This readout method is no longer adequate, since an increasing number of currently 23

circulating A(H3) influenza viruses display reduced capacity to agglutinate erythrocytes. 24

Here, we report the magnitude of this problem by analyzing the frequency of HA deficient 25

A(H3) viruses detected in the Netherlands from 1999-2012. Furthermore, we report development 26

and validation of an alternative method to monitor the production of progeny influenza virus in 27

quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This 28

method is based on detection of viral nucleoprotein (NP) in virus culture plates by ELISA, and it 29

produced similar results compared to the hemagglutination assay using strains with good HA 30

activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2) and other seasonal 31

A(H1N1), A(H1N1)pdm09 and the majority of A(H3) virus strains isolated in 2009. In contrast, 32

many A(H3) viruses that circulate since 2010 failed to display HA activity and infectious virus 33

titers could only be determined by detecting NP. The virus culture ELISA described here will 34

enable efficacy testing of new antiviral compounds in clinical trials during seasons in which non-35

hemagglutinating influenza A viruses circulate.36

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Introduction 37

Efficacy testing of novel antiviral drugs generally involves measuring infectious virus titers in 38

clinical samples at treatment baseline and during follow-up. For influenza, assessment of virus 39

titers by end-point dilution assays commonly relies on the measurement of viral 40

hemagglutination activity in culture supernatants to detect production of progeny virus. 41

However, the continuous accumulation of amino acid substitutions in the viral glycoprotein 42

hemagglutinin (HA) has complicated the detection and characterization of antigenic properties of 43

influenza A(H3N2) viruses by hemagglutination and hemagglutination inhibition (HI) assays, 44

respectively. Poor replicative capacity in Madin-Darby canine kidney (MDCK) cells has been 45

associated with low binding affinities of the viral HA molecules to their receptors (1,13). 46

Reduced receptor binding is also evidenced by failure to agglutinate erythrocytes of various 47

species efficiently, if at all (5,11,13,14,16). Amino acid substitutions in or around the receptor 48

binding site of the HA molecule of A(H3N2) influenza viruses affected the capacity to 49

agglutinate red blood cells (RBC) (13,14,16) and the receptor binding preference has evolved 50

from short, branched sialylated glycans to sialic acids on long polylactosamine chains (5). 51

The replicative capacity of some virus isolates improved upon serial passaging in MDCK 52

cells (1) or MDCK cells that over-express α2,6 SA-Gal receptor (MDCK-siat) (17). This may aid 53

performing HI assays with these viruses to assess their antigenic properties, provided that these 54

remain unaffected during passaging. However, despite additional passages, the frequency of 55

A(H3) virus isolates with impaired HA activity has increased in the Netherlands since 2010, as 56

reported herein. Moreover, serial passaging is no option in clinical trials that require data on 57

infectious virus titers in original clinical specimens. The number of PCR-confirmed samples 58

containing influenza A(H3N2) virus that tested false-negative in quantitative culture assays with 59

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HA activity as readout, has also increased since 2010, whereas such discordant results were not 60

found for samples containing influenza A(H1N1) or B viruses (unpublished observations). 61

Thus, alternative methods are required to monitor replication of currently circulating A(H3) 62

viruses in quantitative virus cultures. The viral cytopathic effect (CPE) could be scored, but this 63

procedure lacks specificity, is labor intensive, subjective and not suitable for high throughput 64

applications. Alternatively, viral antigen could be detected in cell culture by ELISA, which is 65

compatible with standardized automated procedures. Although an ELISA has been developed for 66

the measurement of residual virus replication in virus neutralization assays (21,25), it has never 67

been applied for the assessment of virus titers in clinical samples. Here we report the 68

development and validation of an ELISA for the detection of the viral nucleoprotein (NP) in 69

standard 6-day quantitative influenza A virus cultures. We investigated the kinetics of virus 70

replication using the NP-ELISA method, and compared virus titers with those obtained with the 71

conventional HA assay, using laboratory strains and low passage virus isolates with or without 72

good hemagglutination activity. Finally, the performance of both assays for the assessment of 73

infectious virus titers in clinical samples was compared, which indicated that for recent A(H3) 74

influenza viruses the NP-ELISA readout is superior. 75

76

Material and methods 77

Virus strains, virus isolates and clinical specimens. 78

Laboratory strains A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2) were obtained 79

from the NIBSC (South Mimms, Potters Bar, UK). Epidemic viruses were isolated and 80

propagated in MDCK cells at the National Influenza Center, Erasmus MC, Rotterdam, The 81

Netherlands, as described previously (19), and included the following (sub)types: A(H1N1), 82

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A(H1N1)pdm09, A(H3N2) and B, as determined by PCR. One set of respiratory clinical 83

specimens, nasal wash and sputum, was submitted for routine testing for the presence of 84

respiratory viruses at diagnostic unit of the department of Viroscience of the Erasmus MC, 85

Rotterdam, the Netherlands. Approval by the local medical ethical board was obtained before 86

experiments commenced. Informed consent was waived because patient inclusion was performed 87

retrospectively and data were anonymously stored as agreed by the Erasmus MC institutional 88

review board (MEC-2012-599). A second set of clinical specimens, nasopharyngeal swabs, was 89

submitted for the purpose of assessing virus titers as part of clinical trial WP22849 90

(www.clinicaltrials.gov), and appropriate informed consent and ethics approval was obtained. 91

Cells. Madin-Darby Canine Kidney (MDCK) cells were cultured in Eagle minimal 92

essential medium (EMEM) (Lonza, Verviers, Belgium) containing 20 mM Hepes buffer (Lonza), 93

0.075% Na-Bicarbonate solution (Lonza), 2mM L-Glutamine (Lonza), 100 IU/mL Penicillin / 94

100 µg/mL Streptomycin (Lonza), referred to as complete medium (CM), supplemented with 95

10% FBS (Bodinco BV, Alkmaar, The Netherlands). Cells were passaged to new culture flasks 96

twice weekly. One or two days before inoculation cells were seeded in flat-bottom 96-well tissue 97

culture treated microplates (Greiner Bio-one, Alphen a/d Rijn, The Netherlands). 98

HA titer determination. Duplicate serial 2-fold dilutions of 50µL MDCK culture 99

supernatant in PBS were prepared before adding 50µL of 0.5% turkey erythrocyte solution. 100

Following 1 hr incubation at 4°C, the hemagglutination patterns were scored. The reciprocal of 101

the highest dilution showing full agglutination was taken as the HA titer. 102

PCR. Nucleic acids were extracted from 190µL MDCK culture supernatant and eluted to a 103

volume of 110µL by using a MagNApure LC (Roche Diagnostics, Almere, The Netherlands) or 104

QiaSymphony (Qiagen, Venlo, The Netherlands). Before nucleic acid isolation, 10µL phocine 105

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distemper virus (PDV) was added as internal control. Cycle time (Ct) values and H1 and H3 106

subtypes were determined by real time RT-PCR on a 7500 Real-Time PCR system (Applied 107

Biosystems, Foster City, CA, USA) or a LightCycler 480 system (Roche Diagnostics, Almere, 108

The Netherlands) as described previously (23). 109

Virus titration. Confluent monolayers of MDCK cells were inoculated with replicate 110

(n=4) serial 10-fold dilutions of virus stocks or clinical samples in 96-well microtiter format in 111

infection medium (IM), which was prepared by supplementing CM with 0.3% Bovine Serum 112

Albumin fraction V (Sigma Aldrich), 2.5µg/mL Amphotericin B (Bristol-Myers Squibb, 113

Woerden, The Netherlands) and Trypsin (Lonza; optimal concentration determined for each lot). 114

After 90 minutes at 37°C in a humidified 5% CO2 incubator, inocula were removed, cells were 115

washed with IM and cultured for 6 days in a humidified 37°C 5% CO2 incubator. Medium 116

control wells (n=12) were included on each plate, and at least two laboratory virus strains with 117

predefined titers were included in each experiment for trending. Results were accepted when 118

titers of trend control viruses were within predefined limits. 119

HA readout. Replication of influenza viruses in cell culture was monitored by measuring 120

hemagglutination activity in the culture supernatants as follows: 25µL undiluted supernatant was 121

mixed with 50µL PBS and 25µL of a 1% turkey erythrocyte solution. After an incubation for one 122

hour at 4°C, hemagglutination patterns were scored, and frequencies of positive and negative 123

wells among the four replicates per dilution were used to calculate virus titers (Log TCID50/mL) 124

according to Spearman-Karber method (6). 125

NP readout. Replication of influenza viruses was also assessed by measuring the 126

production of NP by ELISA as follows: At various time points post inoculation of MDCK cells 127

the cell culture plates were washed once with PBS (Oxoid BR0014G, Fisher Scientific, 128

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Landsmeer, The Netherlands), fixed by adding 200µL acetone (80% in water) to each well, and 129

stored at -20°C. After removal of acetone, wells were washed trice with PBS/0.05% Tween20 130

(Merck Millipore, Amsterdam, The Netherlands) before adding the broadly reactive (4,15) 131

influenza A virus NP-specific mouse monoclonal antibody HB65 (EVL Woerden, The 132

Netherlands). Approximately 100 ng of antibody HB65 was added per well in a volume of 200 133

µl PBS containing 2% Skimmed Milk Powder (Sigma 70166) (PBS/SMP). After incubation for 134

one hour at room temperature, the plates were washed three times with PBS/0.05% Tween20 and 135

3 ng of a horseradish peroxidase-labelled Goat-anti-Mouse IgG antibody preparation (Invitrogen, 136

626520) was added in 100uL PBS/SMP and incubated for one hour at room temperature. 137

Following three wash steps with PBS/0.05% Tween20, ready-to-use 3,3’,5,5’-138

tetramethylbenzidine (TMB, Sigma T0440; 100µL/well) was added and plates were incubated at 139

room temperature for 30 minutes before adding 100uL stop solution (1N H2SO4; Sigma 320501). 140

The OD(450-620nm) was measured by using a TECAN F200 plate-reader and signals ≥0.5 were 141

considered positive, and indicative for widespread virus replication. For lower levels of virus 142

replication, e.g. in wells around the endpoint dilution, signals <0.5 but ≥ 0.2 were considered 143

positive when the OD value exceeded three-fold the mean + 3SD of uninfected cell control wells. 144

Because signals above 0.5 also exceeded this latter threshold, scoring of positive and negative 145

wells for TCID50 calculation was effectively based on the latter. Frequencies of positive and 146

negative wells among the four replicates per dilution were used to calculate virus titers 147

(LogTCID50/mL) according to the Spearman-Karber method (6). 148

149

150

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Results 152

Frequency of virus isolates with poor HA activity in the Netherlands. The annual 153

frequency of influenza A(H3) virus isolates received at the NIC of the Netherlands between 1999 154

and 2012 that agglutinate turkey erythrocytes poorly (HA titers ≤ 2) is shown in table 1. 155

Frequencies increased from ~0%-5% between 1999 and 2003 to ~12-26% between 2004 and 156

2008. The following two seasons were dominated by A(H1N1)pdm09 viruses and only few 157

A(H3N2) isolates were obtained, which all showed low HA activity. During the 2011/2012 158

season, the majority of isolates were of the A(H3) subtype, as determined by RT-PCR, and 159

50.9% (n=162) had HA titers of <2. Isolates with higher HA titers showed no such temporal 160

increase (data not shown). 161

To assess whether low HA activity in the 2011/2012 isolates was due to low virus levels or to 162

poor capacity to agglutinate RBC, we performed PCR, targeting the matrix gene segment, on the 163

samples without HA activity (n=141; one isolate was lost to follow-up) and on those with a HA 164

titer of 2 (n=20). As controls we processed ten isolates obtained during the same season with a 165

HA titer of 4 and ten isolates with a HA titer of 32, in the same experiment. PCR signals 166

indicated the presence of virus (Ct < 40) in two thirds (n=94; 67%) of the 141 isolates without 167

HA activity and in eleven (55%) of the 20 isolates with a HA titer of 2. The remaining cultures, 168

with Ct>40, are likely to have been inoculated with specimens containing non-infectious virus, 169

and were excluded from further analyses with respect to HA activity. 170

PCR-positive isolates with high Ct values are likely to contain insufficient virus for a positive 171

HA result. These samples may represent viruses that have poor intrinsic in vitro replication 172

capacity or that may not be viable for other reasons. The positive control isolates with HA titers 173

of 4 or 32 had median Ct values of 15.5 (range 12.8 – 25.5) and 14.4 (12.8 – 19.2), respectively. 174

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As expected, Ct values were generally higher in isolates with HA titers of 2 (16.0 (14.2 – 30.6) 175

or 0 (31.1 (16.0 – 39.0) (Fig. S1). However, 9 (82%) of the 11 PCR positive HA=2 isolates, and 176

21 (22%) of the 94 PCR-positive HA=0 isolates had Ct values that fell within the range of the 177

positive controls. These data indicate that a substantial number of A(H3) virus isolates replicated 178

well in vitro and were HA negative or displayed low HA titers because they failed to agglutinate 179

erythrocytes efficiently. 180

Detection of influenza A virus by NP-ELISA in tissue culture. In order to compare virus 181

replicative capacity with hemagglutination activity more directly, we developed an ELISA for 182

the detection of viral NP in 96-well tissue culture plates. Figure 1 shows NP signals (OD values) 183

of MDCK cultures that had been inoculated with limiting infectious doses of virus A/Vic/361/11 184

per well: With 1:108 and 1:109 diluted virus stock six and one out of eight wells were NP positive 185

respectively, six days post inoculation (dpi 6). The mean OD450-620nm of positive wells increased 186

from ~1.0 at dpi 1 to >2.5 at dpi 6. With a 10-fold higher dose for inoculation (1:107 dilution) all 187

eight wells became positive with mean signals approaching the maximum OD450-620nm level of 188

3.0 already at dpi 1. These signals did not decline up to dpi 6, indicating that the viral NP 189

remained attached to the wells for days, even after viral CPE resulted in complete elimination of 190

the cells. Signals in wells with uninfected control cells were 0.251 ± 0.002 and 0.270 ± 0.003 at 191

dpi 1 and 2, respectively, which declined to 0.007 ± 0.004 at dpi 6. These results are 192

representative for all influenza A viruses that were tested, although kinetics and levels varied 193

among different strains (data not shown). Due to the stability of the NP signal and the favorable 194

signal to noise ratio, detection of NP could provide a reliable alternative to detection of HA 195

activity in culture supernatants for scoring virus-positive and virus-negative cultures in standard 196

virus titration assays. 197

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Comparing HA and NP readout for assessing infectious virus titers. To validate the NP-198

ELISA as an alternative readout in virus titrations, we performed three independent experiments 199

with a panel of virus isolates (n=20) with low MDCK passage histories, and the laboratory virus 200

strains A/Bris/059/07 (H1N1) and A/Vic/210/09 (H3N2) as controls (Table 2). Virus titers based 201

on detection of HA activity in the day-6 culture supernatant or NP in the cognate culture plates 202

were similar (i.e. within 1 order of magnitude) for the two laboratory strains, as well as for the 203

five 2008/2009 influenza A(H3) isolates with > 104 TCID50/mL and HA titer of 128 after one in 204

vitro passage. One of the 2008/2009 isolates (#5) had a lower infectious virus titer based on both 205

HA and NP readout: ~0.75 and 2.33 Log TCID50/mL, respectively. The virus titers obtained 206

with the HA and NP-ELISA readouts were similar for all four A(H1N1) isolates tested, including 207

A(H1N1)pdm09, and ranged between 105.17 and 107.08 TCID50/mL. 208

In contrast, A(H3N2) viruses isolated (n=7) between Jul/2009 and Jan/2011 had lower HA 209

titers, and virus titers based on the HA readout were generally lower than those determined with 210

the NP-ELISA. Strikingly, two virus isolates (#7 and #8) with mean virus titers of 10 6.3 211

TCID50/mL based on NP-ELISA readout, scored repeatedly negative when HA activity was 212

used as readout. We confirmed by PCR, that virus was actually present in the supernatants of the 213

NP-positive cultures (data not shown). Collectively, these results indicate that the poor capacity 214

of these virus isolates to agglutinate RBC precluded reliable virus detection and assessment of 215

infectious virus titers. As expected, titers of the three influenza B virus isolates (#18-#20) could 216

be determined by HA assay, but not by NP-ELISA because the detecting antibody used is 217

specific for influenza A virus NP. 218

Performance of the NP-ELISA as readout for the assessment of infectious virus titers in 219

clinical samples. Next, we used the NP-ELISA as readout for determining virus titers in original 220

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clinical specimens obtained in the 2011/12 influenza season and compared the results with those 221

obtained with HA activity of the culture supernatants. The samples were submitted for routine 222

diagnostic testing for the presence of respiratory viruses (n=14; Table S1) or collected during a 223

clinical trial designed to evaluate antiviral treatment regimens (n=11; Table S2) between January 224

and May 2012. We tested influenza A(H3) virus PCR-positive samples only, since A(H1) virus 225

titers could be readily determined using the HA assay, which gave identical results as the NP-226

ELISA as readout for all A(H1) titrations performed so far (Table 2 and data not shown). 227

Thirteen out of the 14 original clinical specimens and eight out the 11 clinical trial samples 228

tested positive in the infectious virus titration with NP readout with mean ± SD titers of 3.23 ± 229

0.93 and 3.38 ± 1.08 Log TCID50/mL, respectively. In contrast, no virus was detected by the HA 230

assay in the supernatants of the majority of NP-positive wells, which therefore produced false-231

negative results for twenty of the samples (<0.75 Log TCID50/mL) or lower titers (1.00 Log 232

TCID50/mL; n=1). One out of 14 clinical samples and three out of 11 trial samples tested also 233

negative in the NP-ELISA, indicating that infectious virus titers were below the limit of 234

detection of the virus titration assay. This correlated with the relatively high corresponding Ct 235

values of these specimens. 236

237

Discussion 238

Virological surveillance of influenza in the Netherlands showed that frequencies of A(H3) 239

virus isolates displaying low HA activity increased between 1999 and 2012. Since their 240

introduction in the human population in 1968, A(H3) influenza viruses have evolved 241

continuously. Amino acid substitutions in the HA molecules affected their antigenic properties, 242

in vitro replication and capacity to agglutinate RBC (2,5,7,9,11-14,16,22). A subset of the 243

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2011/2012 A(H3) isolates that lacked HA activity, most likely replicated poorly in vitro, as 244

evidenced by high PCR Ct values. However, low virus titers could not explain the absence of HA 245

activity of many other A(H3) virus isolates. Up to 2008, turkey RBC were successfully used as 246

readout in virus titration assays using hemagglutination as readout. During influenza seasons 247

2009/2010 and 2010/2011 A(H1N1)pdm09 viruses were dominant. A(H3) viruses were isolated 248

sporadically, and these isolates agglutinated turkey RBC poorly. Influenza A(H3) viruses with 249

poor HA activity continued to circulate and became the dominant subtype again in the 2011/2012 250

season. The use of RBC from other species, including guinea pig and human, did not consistently 251

improve HA activity for monitoring antigenic drift by HI assays (data not shown). 252

The inefficient hemagglutination activity of influenza virus strains also forms a major 253

problem for the HA readout of quantitative virus cultures of primary specimens in 254

clinical/research settings, as well as in clinical trials. Hemagglutination has long been the 255

preferred readout method because it is generally a simple, fast and reliable virus detection 256

method. It is instrumental as high throughput readout to provide crucial information on the 257

duration of infectious virus shedding and the ability to reduce transmission in efficacy studies of 258

novel antiviral compounds. However the deficiency of virus in primary clinical specimens to 259

agglutinate erythrocytes after the first passage in MDCK cells, precluded the assessment of 260

infectious influenza virus titers by HA readout in the majority of cases during the 2011/2012 and 261

2012/2013 influenza seasons, when A(H3) virus strains were dominant. 262

In order to equip the diagnostic laboratories with a robust HA-independent technique for the 263

detection of progeny virus production in primary quantitative virus cultures, we employed an 264

ELISA that measures NP in multi-well virus culture plates. This NP-ELISA is based on the 265

method described for the WHO-recommended microneutralization assay (21,25). One important 266

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difference with virus titration assays is that in neutralization assays cell cultures are inoculated 267

with 100 TCID50/well of known virus strains – after pre-incubation with serial serum dilutions, 268

and virus production is monitored one day after inoculation. To propagate and quantify viruses 269

present in original clinical specimens, other plate types are used and an incubation period of six 270

days is recommended to allow for detection of slowly replicating viruses in wells around the 271

end-point dilution. Therefore, we evaluated the stability of the NP signal up to six days after 272

inoculation, and showed that it increased or remained stable, but never declined. In wells around 273

the end-point dilution, signals generally reached levels that were comparable to those achieved 274

with a 10- to 100-fold higher multiplicity of infection. 275

With HA-competent viruses, the HA and the NP readout methods detected virus replication in 276

largely the same wells and resulted in similar titers. By contrast, infectious virus titers of virus 277

isolates and clinical samples containing HA-deficient viruses could only be determined by using 278

the NP-ELISA. The HA assay may still be preferred as first readout method, since it requires less 279

time and reagents, and it detects A(H1N1), A(H1N1)pdm09 as well as influenza B viruses in the 280

culture supernatants. The tissue culture plates used for determining infectious virus titers can be 281

stored at -20°C until the subtype of the influenza A viruses is identified by PCR, allowing for 282

detection of virus replication by NP-ELISA as second readout in the same titration experiment. 283

Detection of NP relies on the binding of the NP-specific monoclonal antibody HB65 that reacts 284

with a broad range of influenza A viruses (4,15,26), including the A(H1N1) viruses from 1934 285

(A/PR/8/34) and A(H3N2) and A(H1N1)pdm09 viruses isolated in 2012, as reported here. Other 286

recent viruses that were recognized include A/Vic/361/11 (H3N2) and A/Cal/07/09 (H1N1), but 287

also avian and swine influenza viruses A(H5N1), A(H7N7)(15), and H7N9(10). The potential 288

emergence of variants that are not recognized by monoclonal antibody HB65 should be carefully 289

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monitored, but based on the documented conservedness of the epitope that is recognized, this is 290

not expected to occur any time soon. Alternatively, a cocktail of monoclonal antibodies could be 291

used to minimize this risk even further. 292

We have tested a number of influenza B virus NP-specific antibody preparations to evaluate 293

ELISA-based detection of influenza B virus-infected cultures at day 6 after inoculation. 294

Although several preparations recognized all Victoria- and Yamagata-lineage strains tested, in 295

many cases signals were too low to distinguish positive and negative wells in a robust way (data 296

not shown). Because current influenza B viruses do agglutinate erythrocytes efficiently, there is 297

no urgent need for alternative readout methods. 298

Antigenic characterization of virus isolates by HI assays form the basis for the annual 299

selection of vaccine strains (2,7). Due to the reduced capacity of A(H3N2) viruses to agglutinate 300

chicken RBCs and, more recently, turkey RBCs, HI data are currently also generated by using 301

mammalian RBC from guinea pig or human. However agglutination patterns are generally more 302

difficult to read, and turkey RBCs are still frequently used for antigenic characterization, 303

possibly leading to underrepresentation of circulating A(H3N2) virus variants for antigenic 304

characterization. Because of the factors that complicate the HI assay, including the need to 305

consider RBC from different species for different virus subtypes, VN assays are now also used 306

for antigenic characterization (18,20,24), and for evaluation of vaccines with relatively low 307

immunogenicity in naive human populations, e.g. H5 and H7 (3,8,21). The ELISA method used 308

in such VN assays detect virus irrespective of their capacity to agglutinate RBCs, a feature that 309

was successfully adapted to improve the monitoring of infectious virus titers in specimens of 310

clinical trials for antivirals. 311

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In conclusion, we provided epidemiological data on the emergence of A(H3) influenza viruses 312

that fail to agglutinate erythrocytes efficiently. The HA-deficiency of these viruses complicates 313

monitoring of virus replication in quantitative virus culture and assessment of infectious virus 314

titers in clinical specimens. In order to provide for an HA-independent readout for quantitative 315

virus cultures of primary patient samples in clinical/research and clinical trial settings, we have 316

validated the use of NP detection by ELISA on day six post inoculation of MDCK cells. For 317

currently circulating A(H3) viruses, the HA readout yielded mainly false-negative results, while 318

the NP readout proved to be a robust method that facilitates the measurement of virus titers in 319

clinical specimens, which is critical for the evaluation of new antiviral compounds and therapies 320

in clinical trials. 321

322

Acknowledgments 323

We thank Roche for sharing data on virus titrations of clinical trial specimens. The authors 324

CvB, CE and LS are employed by Erasmus MC spin-out company Viroclinics Biosciences B.V. 325

of which ADMO and GR are consultants. 326

327

Reference List 328

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Figure legends 423

424

FIG 1. NP signals increase up to day 6 post inoculation. MDCK cells were inoculated with serial 425

dilutions of A/Vic/361/11 in 96-well plates (eight replicates per dilution). On days 1, 2 and 6, NP 426

signals were determined by ELISA. Bars are OD450-620nm (mean ± SEM) of positive wells (here 427

defined as > 2-fold above the mean of mock-inoculated cell control (CC) wells) for the -7, -8 and 428

-9 Log diluted virus stock. Numbers of positive wells are above each bar. The CC bars show the 429

OD450-620nm (mean ± SEM) of the eight cell control wells. 430

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TABLE 1. Annual frequency of influenza A(H3) clinical isolates with low HA titers

A(H3) isolates (n)

HA = 0 HA = 2 HA ≤ 2

Season n % N % n %

99/00 334 1 0.3 16 4.8 17 5.1

00/01 7 - 0.0 - 0.0 - 0.0

01/02 265 - 0.0 - 0.0 - 0.0

02/03 150 1 0.7 - 0.0 1 0.7

03/04 400 - 0.0 99 24.8 99 24.8

04/05 338 1 0.3 40 11.8 41 12.1

05/06 116 - 0.0 16 13.8 16 13.8

06/07 259 1 0.4 52 20.1 53 20.5

07/08 21 1 4.8 2 9.5 3 14.3

08/09 415 51 12.3 56 13.5 107 25.8

09/10 3 1 33.3 2 66.7 3 100.0

10/11 13 6 46.2 - 0.0 6 46.2

11/12 318 142 44.7 20 6.3 162 50.9

Total 2,639 205 7.8 303 11.5 508 19.2

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TABLE 2. Virus titers of clinical isolates and laboratory virus strains determined by HA and NP readout

Sample information Log TCID50/ml Means

MDCK passage HA assay NP-ELISA < 1 Log

ID Subtype Datea number HAb Exp.1 Exp.2 Exp.3c mean SD Exp.1 Exp.2 Exp.3 mean SD difference

1 A(H3) Feb/09 1 128 3.75 4.25 nt 4.00 0.35 3.75 4.25 nt 4.00 0.35 y 2 A(H3) Jan/09 1 128 3.25 3.50 nt 3.38 0.18 3.75 4.25 nt 4.00 0.35 y 3 A(H3) Jan/09 1 128 5.00 5.00 nt 5.00 0.00 5.50 6.00 nt 5.75 0.35 y 4 A(H3) Jan/09 1 128 5.50 5.50 5.50 5.50 0.00 6.00 5.75 6.00 5.92 0.14 y 5 A(H3) Jan/09 1 128 <0.75 <0.75 0.75 0.75 - 3.00 1.25 2.75 2.33 0.95 n 6 A(H3) Jan/09 1 128 6.50 6.50 7.00 6.67 0.29 6.50 6.50 7.25 6.75 0.43 y 7 A(H3) Jul/09 1 2 <0.75 <0.75 <0.75 <0.75 - 6.25 6.00 6.75 6.33 0.38 n 8 A(H3) Apr/10 1 0 <0.75 <0.75 <0.75 <0.75 - 6.50 5.75 6.75 6.33 0.52 n 9 A(H3) Dec/10 2 32 3.50 2.25 2.00 2.58 0.80 3.50 4.00 4.00 3.83 0.29 n

10 A(H3) Jan/11 4 4 1.75 1.50 <0.75 1.63 0.18 2.50 3.00 2.75 2.75 0.25 n 11 A(H3) Jan/11 2 8 5.25 4.50 4.75 4.83 0.38 5.75 5.50 4.75 5.33 0.52 y 12 A(H3) Jan/11 4 8 0.75 <0.75 0.75 0.75 0.00 1.50 <0.75 1.25 1.38 0.18 y 13 A(H3) Jan/11 2 8 4.00 3.50 3.75 3.75 0.25 4.25 3.50 4.25 4.00 0.43 y

14 A(H1N1) Jan/09 2 32 5.25 5.00 5.50 5.25 0.25 5.00 5.00 5.50 5.17 0.29 y 15 A(H1N1) Aug/09 2 32 5.25 5.25 5.25 5.25 0.00 5.25 5.25 5.25 5.25 0.00 y 16 A(H1N1)pdm09 Jan/11 2 >32 6.75 7.25 7.25 7.08 0.29 6.75 7.25 7.25 7.08 0.29 y 17 A(H1N1)pdm09 Jan/11 2 >32 7.50 6.75 6.75 7.00 0.43 7.50 6.75 6.75 7.00 0.43 y

18 B Feb/09 1 128 3.75 3.50 3.50 3.58 0.14 <0.75 <0.75 <0.75 <0.75 - n 19 B Mar/11 1 64 6.00 5.75 5.75 5.83 0.14 <0.75 <0.75 <0.75 <0.75 - n 20 B Jan/11 1 >32 5.75 5.50 5.50 5.58 0.14 <0.75 <0.75 <0.75 <0.75 - n

A/Bris/059/07 A(H1N1) NA NA NA 5.75 5.75 5.75 5.75 0.00 5.75 5.75 5.75 5.75 0.00 y A/Vic/210/09 A(H3N2) NA NA NA 5.75 5.25 4.75 5.25 0.50 5.75 5.25 4.75 5.25 0.50 y a Not applicable b HA titers and number of MDCK passages of the virus stocks used in the virus titration experiments. c not tested

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