Development of a high-throughput cell based 384-well influenza A quantification assay for interpandemic and highly pathogenic avian strains

doi:10.4236/health.2010.21006

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Vol.2, No.1, 32-37 (2010)

doi:10.4236/health.2010.21006

SciRes

Health

Development of a high-throughput cell based 384-well

influenza A quantification assay for interpandemic and

highly pathogenic avian strains

Melicia R. Gainey, Ann M. Wasko, Jennifer N. Garver, David J. Guistino, Eric M. Vela*, John E.

Bigger

Battelle Biomedical Research Center West Jefferson, OH 43162, USA; velae@battelle.org

Received 25 November 2009; revised 1 December 2009; accepted 2 December 2009.

ABSTRACT

Influenza remains a world wide health threat,

thus the need for a high-throughput and robust

assay to quantify both seasonal and avian in-

fluenza A strains. Therefore, a 384-well plate

format was developed for the median tissue

culture infectious dose assay (TCID50) utilizing

the detection of nucleoprotein by an in situ en-

zyme linked immunosorbent assay (ELISA)

which was optimized for sensitivity in this assay.

Highly pathogenic avian influenza, A/Vietnam/

1203/04 (H5N1), and interpandemic strains, A/

New Caledonia/20/99 (H1N1) and A/Brisbane/

10/07 (H3N2), were quantified using this high-

throughput assay. Each 384-well plate can be

used to analyze ten viral samples in quadrupli-

cate, eight dilutions per sample, including all

necessary assay controls. The results obtained

from 384-well plates were comparable to tradi-

tional 96-well plates and also demonstrate re-

peatability, intermediate precision, and assay

linearity. Further, the use of 384-well plates in-

creased the throughput of sample analysis and

the precision and accuracy of the resulting titer.

Keywords: Avian Influenza; ELISA;

High-Throughput Assay; Interpandemic Influenza A;

TCID50

1. INTRODUCTION

Interpandemic, or seasonal, influenza takes a toll on

public health resources, economic productivity, and

causes up to 500,000 deaths annually world-wide [1].

Highly pathogenic avian influenza (HPAI) strains cause

heavy economic losses in countries dependent upon

poultry revenues and have caused more than 250 deaths

globally [2]. In addition, HPAI strains could potentially

give rise to a pandemic. Given the economic and health

implications of these viral strains, many new vaccines

and therapeutics are being developed to counter this

threat. In order to determine efficacy of these novel

products, it is necessary to quantify antibody responses

and influenza A viruses in various matrices, ideally in a

high-throughput format. Currently, several high throu-

ghput assays to detect influenza virus exist which utilize

reverse transcription polymerase chain reaction (RT-PCR)

alone [3], and coupled with flow cytometery [4] or utilize

a latex turbidimetric immunoassay [5]. There are also a

few high-throughput screening methods to determine

putative antiviral compounds [6] and influenza antago-

nists [7]. Although these methods are able to analyze

many samples for the presence of influenza or screen

potential therapeutics, these assays do not quantify in-

fluenza virus. For high-throughput quantification, a

reverse-phased high performance liquid chromatography

has been used to enumerate the amount of hemagglutinin

in several types of viral stocks [8], but does not provide

an infectious titer. A branched DNA technology can be

used for quantification of influenza A viruses, but this

assay was developed for use as an antiviral assay [9] and

not for viral quantitation. Median tissue culture infectious

dose assays (TCID50 assay) utilizing Alamar blue in

96-well plates have also been described [10]. Analysis by

this assay provides an infectious titer by indirect means;

determining the metabolism of uninfected healthy cells

instead of detecting the presence of viral infection. To

date, 384-well plate assays have been described for the

detection of influenza via RT-PCR [11] and for the

screening of antivirals utilizing Flash plate technology

[12] or cell based luminescence assays [13]. Thus,

384-well plates have not been harnessed to provide as-

sessment of infectious titers of influenza viruses.

As no world wide standard exists for the quantification

of influenza viruses, the World Health Organization’s

(WHO) Manual on Animal Influenza Diagnosis and

Surveillance [14] was chosen as the basis for this work.

The manual recommends that viral samples be analyzed

M. R. Gainey et al. / HEALTH 2 (2010) 32-37

in quadruplicate by the TCID50 assay in a 96-well mi-

croplate format. These assays may be interpreted by the

visual observation of cytopathic effect (CPE) or the de-

tection of influenza nucleoprotein by an in situ ELISA. To

increase the assay throughput and efficiency, a 384-well

microplate assay with an ELISA readout was developed

based on recommended WHO manual TCID50 procedures.

Titration of HPAI and interpandemic influenza A viruses

in a 384-well microplate was compared to the traditional

96-well format. Based on preliminary qualification data,

the 384-well format measures accurate and precise titers

with less variability and higher assay linearity than the

96-well plates, suggesting this method provides more

reliable titer data. Further, the use of 384-well plates

reduces the time and effort required for analysis to 24

minutes per sample, a reduction of 80%. This assay for-

mat has also been adapted for micro-neutralization assays

and is amenable for use with manual or robotic liquid

handlers.

2. MATERIALS AND METHODS

2.1. Metabolic Assay

384-well plates were seeded with MDCK cells at 2.5 x

105 or 3.0 x 105 cells/mL and incubated overnight at 37°C

and 5.0% CO2. A 5% (w/v) Tetrazolium salt, 3- [4,5-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

(MTT) solution was prepared in 1X phosphate buffered

saline (PBS), added to each well and incubated 2 hr at

37°C. The reduced MTT was released from the cells by

adding Solubilization Buffer (50% dimethyl formamide

solution containing 20% w/v sodium dodecyl sulfate) and

incubating for 2 hr at 37°C. The plates were read at 550

nm with a 690 nm reference. All subsequent experiments

utilized a seeding density of 3.0 x 105 cells/mL due to

lower variability, which was analyzed by calculating the

coefficient of variance according to the location on the

plate.

2.2. Viruses and Cells

Each viral strain was received from the Centers for Dis-

ease Control (Atlanta, GA) and propagated in 8-10 day

old specific-pathogen free chicken embryos (Charles

River Franklin, CT) at 37°C for 24 hr for A/ Vietnam/

1203/04, 37°C for 72 hr for A/New Caledonia/20/99, and

35°C for 48 hr for A/Brisbane/10/07. The harvested al-

lantoic fluid was stored at ≤-70°C. Madin-Darby canine

kidney (MDCK) cells (Salisbury lineage, Sigma St. Louis,

MO), were maintained in complete Eagle’s minimal es-

sential media (EMEM) containing 1% Penicillin- Strep-

tomycin and 10% fetal bovine serum. MDCK plates

seeded at 70% confluency were purchased from Diag-

nostic Hybrids, Inc. (Athens, OH).

2.3. Viral Quantification

Viral stocks were quantified by inoculating serial dilu-

tions in quintuplicate on 96-well plates or quadruplicate

on 384-well plates onto confluent MDCK monolayers.

TPCK-treated trypsin at 2.0 μg/mL (US Biological

Swampscott, MA) was added to the inoculation media for

the interpandemic strains. Each plate contained cell cul-

ture control (CC) wells containing inoculation media

alone. The inoculated plates were incubated at 37°C and

5.0% CO2 in a humidified incubator for 20 ± 1 hr prior to

fixation.

2.4. In Situ Influenza A Nucleoprotein ELISA

The ELISA was performed as previously described [14]

with the following noted changes. Plates were fixed by

the addition of 1 ½ volumes of 80% cold acetone in water

directly to the inoculum and incubated at room tempera-

ture (RT) for 30 min. All subsequent incubations were

increased from the recommended RT to 37°C in a hu-

midified incubator to increase the sensitivity of the assay.

For the primary incubation, an equal mixture of mouse

anti-influenza A nucleoprotein monoclonal antibody

clone MAB8257 and MAB8258 (Chemicon International;

Bellirica, MA) was used. The ABTS Microwell Peroxi-

dase Substrate System (Kirkegaard and Perry Laborato-

ries, prepared according to manufacturer’s instructions)

substrate and stop solution was used according to the

manufacturer’s instructions. The plates were read at 405

nm with a 490 nm reference. The positive threshold was

calculated as the average optical density (OD) of the CC

wells plus two standard deviations. Each sample well was

scored as positive for infection if the OD was above the

positive threshold and negative for infection if the OD

was less than or equal to the positive threshold. The me-

dian infectious dose for all samples was calculated using

the Spearman Kärber formula [15].

3. RESULTS

3.1. Optimization of the 384-Well Plate

To assess the suitability of 384-well plates for a cell-

based infectivity assay, MDCK cells were seeded at two

concentrations and the metabolic activity was determined

by reduction of MTT. The metabolism of cells seeded in

the outer wells was identical to the cells seeded in the

inner wells (see Table 1). The variability was analyzed by

calculating the coefficient of variance (CV) by location

on the plate and found to be less than 13% for inner and

outer wells as well as the entire plate. Therefore, 384-well

plates were found suitable for this assay due to the low

variability demonstrated between inner and outer wells.

Further, seeding the plate at 3 x 105 cells/mL provided

less variability (CV < 8%, see Table 1).

M. R. Gainey et al. / HEALTH 2 (2010) 32-37

Openly accessible at

Table 1. 384-well plate variability measured by metabolism. 384-well plates were seeded with two concentrations of MDCK cells and

metabolism was determined by the reduction of MTT. The delta OD (difference between OD at 550 nm and 690 nm) was averaged for

the outer and inner wells in comparison to the entire plate. The standard deviation (SD) and coefficient of variance (CV) were deter-

mined for each location.

2.5 x 105 cells/mL 3.0 x 105 cells/mL

Outer Wells Inner Wells Entire Plate Outer Wells Inner Wells Entire Plate

Average ΔOD 0.28 0.29 0.29 0.35 0.35 0.35

SD 0.04 0.03 0.03 0.03 0.03 0.03

CV 13% 11% 11% 8% 7% 8%

Table 2. In situ nucleoprotein ELISA threshold calculation. Changes made to the WHO animal influenza diagnosis and surveillance

ELISA threshold obtained titers comparable to visual determination of CPE.

CPE Readout WHO ELISA ELISA

(AVG CC + 2SD)

5.7 5.0 5.6

Different plate Same plate analyzed with different thresholds

The in situ nucleoprotein ELISA was adapted for this

plate format and optimized for sensitivity, efficiency, and

safety (see Materials and Methods 2.4). Removing the

inoculum and washing the monolayer prior to fixation

was not desirable for the 384-well plate due to the in-

volvement of multiple pipetting steps and manipulation

of potentially infectious materials. Thus, the critical step

for making this assay feasible and safe is the addition of

the fixative directly to the inoculated wells. Tests dem-

onstrated no adverse effects to the reported titer or in-

crease in the background of the assay by changing the

fixation step (data not shown). To further optimize the

ELISA, the incubation temperature was raised which

reduced the concentration of conjugate required thus

increasing the sensitivity of the assay. The use of the

ABTS microwell peroxidase substrate system improved

the environmental impact of this assay by eliminating the

waste generated from the o-phenylenediamine dihydro-

chloride (OPD) substrate, which is both toxic to humans

and dangerous to aquatic systems according to the mate-

rial safety data sheet. Use of the ABTS substrate in-

creased the positive signal without significantly increas-

ing the background of negative wells (data not shown).

Unlike OPD, the ABTS substrate does not change color

upon the addition of the stop solution thus eliminating

concerns about the timing of stopping the reaction. Fi-

nally, the positive threshold was defined as the average of

optical density (OD) of the CC wells plus two standard

deviations which provides titers consistent with side by

side CPE readout experiments (Table 2).

Samples and controls are diluted in a standard 96-tube

microtiter box (see Figure 1A). Inoculation of the

384-well plate is efficient and simple when utilizing a

24-channel pipette (see Figure 1B). The content of each

microtiter tube is inoculated into four wells on the

384-well plate. This process can be adapted easily to

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AS1-1S2-1S3-1S4-1S5-1S6-1S7-1S8-1S9-1S10-1PC-1NC-1

BS1-2S2-2S3-2S4-2S5-2S6-2S7-2S8-2S9-2S10-2PC-2NC-2

CS1-3S2-3S3-3S4-3S5-3S6-3S7-3S8-3S9-3S10-3PC-3NC-3

DS1-4S2-4S3-4S4-4S5-4S6-4S7-4S8-4S9-4S10-4PC-4NC-4

ES1-5S2-5S3-5S4-5S5-5S6-5S7-5S8-5S9-5S10-5PC-5 CC

FS1-6S2-6S3-6S4-6S5-6S6-6S7-6S8-6S9-6S10-6PC-6 CC

GS1-7S2-7S3-7S4-7S5-7S6-7S7-7S8-7S9-7S10-7PC-7 CC

HS1-8S2-8S3-8S4-8S5-8S6-8S7-8S8-8S9-8S10-8PC-8 CC

(a)

123456789101112131415161718192021222324

A1111111111 11 111 11 11 11 111

B1111111111 11 111 11 11 11 111

C2222222222 22 222 22 22 22 22 2

D2222222222 22 222 22 22 22 22 2

E3333333333 33 333 33 33 33 333

F3333333333 33 33 333 33 33 33 3

G44444444444 44 44 444 44 44 44

H4444444444 44 444 44 44 44 44 4

I5555555555 55 55 555 55 55 5CCCC

J55555555555 55 55 555 55 55CCCC

K6666666666 66 666 66 66 66 6CCCC

L6666666666 66 66 666 66 66 6CCCC

M7777777777 77 77 777 77 77 7CCCC

N7777777777 77 777 77 77 77 7CCCC

O88888888888 88 88 888 88 88CCCC

P8888888888 88 888 88 88 88 8CCCC

S9S10PC NC/CCS5S6S7S8S1 S2 S3 S4

(b)

(a) TITER-BOX. Samples and controls are diluted in a standard 96-tube

microtiter box. Each color represents a different viral sample or control,

thus ten samples (S1 through S10) with a positive control virus (PC),

sample matrix matched negative control (NC), and cell culture control

(CC) are prepared. Eight dilutions of each sample and PC are prepared

with four dilutions for the NC.

(b) 384-WELL PLATE: Each tube is inoculated into four wells in the

384-well plate. The number in each well represents the sample/control

dilution inoculated into that location

Figure 1. 384-well plate set up and layout.

M. R. Gainey et al. / HEALTH 2 (2010) 32-37

Table 3. Intra-assay and inter-assay variability data for TCID50 assays. The repeatability (intra-assay variability) and intermediate

precision (inter-assay variability) of each plate format was assessed for three strains of influenza A.

Repeatability

(Intra-Assay Variability)1

Intermediate Precision

(Inter-Assay Variability)2

384-well 96-well 384-well 96-well

Expected Titer3

A/Vietnam/1203/04 (H5N1)

GEOMEAN

7.6

0.2

7.8

0.2

7.6

0.3

7.9

0.4

7.6

A/New Caledonia/20/99 (H1N1)

GEOMEAN

4.7

0.0

4.5

0.1

4.9

0.3

5.3

0.8

15%

5.1

A/Brisbane/10/07 (H3N2)

GEOMEAN

6.3

0.1

5.7

0.2

6.3

0.2

5.5

0.3

6.1

1) Value reported is the geometric mean of the titer of three iterations by one operator on one testing day

2) Value reported is the geometric mean of the titer of three iterations by two operators on two testing days

3) The expected titer was based on prior titer certification on 96-well plates by two operators over three testing days using nine randomly selected

aliquots of the stock

robotic or manual liquid handler capabilities and has been

used in our facility.

3.2. Assay Acceptability

To compare the new 384-well plate with the traditional

96-well plate, the repeatability (intra-assay variability)

and intermediate precision (inter-assay variability) were

evaluated (see Table 3) for three influenza strains (H1N1,

H3N2, and H5N1). Use of three different influenza vi-

ruses demonstrated the robustness of the assay for quan-

tification of any influenza A strain. The expected titer of

each strain was established using 96-well plates prior to

the experiment. To establish repeatability and precision,

the mean titer obtained must be within 0.5 log of the ex-

pected titer.

The repeatability was assessed by three iterations of the

same viral lot by one operator on a single day of testing.

The geometric mean and standard deviation were calcu-

lated for each plate type and viral strain (see Table 3). For

the 384-well plates, all mean titers were within 0.4 log of

the expected titer and were deemed repeatable for all three

strains. Further, the standard deviations were less than or

equal to 0.2 log TCID50/mL which demonstrates low

variability regardless of the influenza strain quantified on

the 384-well plate. The 96-well plates produced titers

within 0.6 log of the expected titer, indicating less re-

peatability with this plate format.

The intermediate precision for each assay format and

viral strain was assessed by three iterations of the same

viral lot by two operators on two testing days. The geo-

metric mean and standard deviation of the resulting 12

data points were calculated (see Table 3). All of the mean

titers obtained with 384-well plates were within 0.2 log of

the expected titer, thus establishing the intermediate pre-

cision of the 384-well plate. The 96-well plate demon-

strated less precision with all mean titers falling within 0.6

log of the expected titer. In addition, the standard devia-

tions for the 384-well plates were calculated to be 0.3 log

TCID50/mL or less, compared with 0.8 log TCID50/mL or

less for 96-well plates, indicating lower variability be-

tween assays, operators, and testing days for the high-

throughput plate format. All strains performed as ex-

pected in comparison to titers established using 96-well

plates, thus verifying the robustness of the 384-well plate.

The linearity of an assay measures the ability to obtain

results proportional to the concentration of the sample.

For each assay format, the linearity was assessed by pre-

paring four serial dilutions of the viral stock. Each dilu-

tion was then quantified in each plate type. The predicted

titer for each dilution was calculated based on the dilution

from the certified stock titer. The observed titer was

plotted against the predicted titer and a linear regression

analysis was performed (see Figure 2). For the purposes

of this study, an r2 ≥ to 0.85 was considered linear. Al-

though the 96-well plate for both the A/ Vietnam/1203/04

and A/New Caledonia/20/99 strains demonstrated linear-

ity, r2 >0.96, the 384-well plate for both strains demon-

strated greater linearity, r2 >0.99 which suggests the use of

a 384-well plate format provides more reliable data.

However, both 96-well and 384-well plates are expected

to produce results proportional to the concentration of the

sample quantified.

M. R. Gainey et al. / HEALTH 2 (2010) 32-37

Openly accessible at

r2 = 0.9942

r2 = 0.9865

r2 = 0.9959

r2 = 0.9640

0.5

1.5

2.5

3.5

0.511.522.533.54

Predicted log Titer (TCID50/mL)

Observed log Titer (TCID

50/mL)

A/Vietnam/1203/04 384-wellA/Vietnam/1203/04 96-well

A/New Caledonia/20/99 384-wellA/New Caledonia/20/99 96-well

Four serial dilutions of each strain were prepared and then quantified in each assay format. The observed log titer was compared

with the predicted titer for each dilution. The linear regression for each format was determined. An r2 >0.85 was considered linear

for each assay format.

Figure 2. Assay linearity for influenza A (H5N1 and H1N1).

3.3. Advantage of High-Throughput 384-Well

Plate

To compare efficiency of the new 384-well plate format

with the standard 96-well plate, the average time required

to analyze one sample in a TCID50 assay from seeding the

plate to analysis of the data was determined. The average

time for the 96-well assay (plates seeded in house) was 2

hr per sample from start to finish. For the most efficient

384-well assay, commercially available seeded plates

were used, removing the need for cell culture mainte-

nance. An additional advantage of these pre-seeded plates

is that more assays can be conducted each week without

the need of multiple cell lines being maintained for har-

vest. The average time for the analysis of one sample on a

pre-seeded 384-well plate was 0.4 hr. Thus, using pur-

chased 384-well plate increases efficiency of the TCID50

assay by 80%, thereby reducing cost significantly.

4. DISCUSSION

HPAI infections in animals cause economic losses in

countries dependent on poultry revenues [16]. Further,

HPAI and interpandemic (seasonal) influenza infections

in humans burden public health resources [17]. A myriad

of vaccines and therapeutics are under development;

however, assays that test influenza virus titers and screen

the efficacy of vaccines and therapeutics cannot be used

to quantify influenza virus and those that do measure

infectious virus, do so by indirect methods [9, 10]. The

TCID50 assay directly quantifies infectious influenza A

viruses by utilizing an in situ nucleoprotein ELISA that

detects the influenza A nucleoprotein in infected mon-

olayers [14]. Therefore, a 384-well plate TCID50 assay

with an ELISA readout was developed and tested for

robustness, accuracy, precision, and reliability. The time

and effort required for sample testing was also deter-

mined. For this study, two plate formats were examined: a

traditional 96-well plate and a high throughput 384-well

plate. To demonstrate the robustness of the assays, three

influenza A strains were quantified: A/ Vietnam/1203/04

(H5N1), A/New Caledonia/20/99 (H1N1), and A/ Bri-

sbane/10/07 (H3N2). All viral strains performed as ex-

pected without modification of the procedure, demon-

strating the robustness of the new 384-well plate format

when compared with the traditional 96-well plate. Addi-

M. R. Gainey et al. / HEALTH 2 (2010) 32-37

tionally, each assay format was tested for intra- and in-

ter-assay variability to ascertain the precision of the assay

and for linearity to verify the overall performance of the

plate format. Both 96-well and 384-well plate formats

tested were found to be repeatable, precise, and linear.

The 384-well plate appears to be more accurate, precise

and linear than the traditional 96-well plate and demon-

strated less overall variability. Further, the efficiency

afforded by using purchased pre-seeded 384-well plates

was substantial. The increase in efficiency greatly lowers

the cost and time required to obtain an infectious titer for

a variety of viral samples without sacrificing precision.

The 384-well plate format with ELISA readout offers a

high-throughput, more efficient alternative for the de-

termination of infectious influenza A titers in compliance

with WHO recommendations for the assay. The 384-well

plate has also been adapted for automation via liquid

handling, considerably increasing the throughput of

sample analysis in order to determine the efficacy of

novel vaccines and therapeutics.

5. ACKNOWLEDGEMENTS

The authors would like to acknowledge Dr. Hank Lockman and Mr.

Aaron Martin for their assistance in the production of this manuscript. In

addition, the authors would like to thank Drs. Herb Bresler, Catherine

Smith, and Carol Sabourin for providing their support and technical

assistance. This work was funded through the Battelle Independent

Research and Development Program.

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