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A GENETICALLY ENGINEERED CELL LINE EXPRESSING AN INFLUENZA A VIRUS-INDUCIBLE REPORTER GENE AS A TOOL FOR DETECTING AND QUANTIFYING INFECTIOUS VIRUS Yunsheng Li1, Andrew Pekosz3 Teresa Curtiss1, Audrey Larrimer1, Abby Jones1, Paul Olivo2, Julie Dyall2 and Dave Scholl1 1 Diagnostic Hybrids Inc., Athens, OH 2 Apath, LLC, St, Louis, MO 3 Depts. Of Molecular Microbiology and Pathology & Immunology, Washington University in St. Louis, School of Medicine, St, Louis, MO Abstract

RLU/S

1E1-1B10 1E1-1C5 1E1-1C8 1E1-1H7

1 E1

Clone Number

B 293THygromycin resistant cells

293T Cells

pIAV-GFP

Hygromycin

Viruses induced the GFP reporter gene in IAV NP GFP expressing 293T cells

Figure 1. Schematic diagram for construction of influenza A detecting cell lines VIRGS-containing plasmids with reporter genes were permanently transfected into human 293T cells. After hygromycin selection, the positive clones were identified by infection with influenza A virus to induce reporter (luciferase or GFP) gene expression.

MHIA 1

MHIA 1

MHIA 1

No GFP expressing cells

Mass

Mass

Mass

Mass

60000

RLU 17074

136

92

108 112

79

Influenza virus

50000 40000 30000 10000

52569

Protected 1:120 20358

20000

3953

11703 545

130

40

0 No virus

80 16 0 32 0 64 0 12 80 25 60

IA M H

Figure 2. Specificity of 293T/IAV-Luc cell line following infection with influenza B virus. The 293T/IAV-Luc cell line was infected with ten different strains of influenza B virus (multiplicity of infection , MOI= 0.5-1). Influenza A MHIA (MOI=0.025) was used as a positive control The cells were analyzed for luciferase activity 24 hours post-infection. The Luciferase assay results showed the cell lines had high specificity for influenza A virus. Most influenza B strains could not turn on the luciferase gene even with a high MOI.

60265 52474

60000

0

IA V

M D HI A00 2 Ta 3 iw an

30000 10000

M

2

IB V 3 IB V 4 JH 00 1 M as s

L

HK

IB V

G

V IA

40000

71522

70000

44483

Protected 1:480

20000

0

0

80000

62217 57653

52474

50000

1000

1733 8386 1846 600 220 2439 160 272 164 5954

Influenza virus

70000

2000

10

200000

3000

RLU

300000

Se ru m V onl iru y so nl y

400000

Table 1. Luciferase assay of the 293T/IAV-Luc cell line with influenza A virus infection. Thirty-four lab and clinical strains of influenza A virus were tested on the cell line. The cells were analyzed for luciferase activity 24 hours post-infection (hpi). The range of induction of luciferase by the various influenza virus strain or isolates was from 1.8 RLU/virus (CDC 006) to 4312 RLU/virus (ODH-0351).

B.

A.

G L HK IB V 2 IB V 3 IB V 4 JH 00 1 M as s M M HI D A00 2 Ta 3 iw an

Ratio of S/B

500000

HI A

MHIA 1

Figure 4. 293T/IAV-GFP cell line infected with influenza A and B viruses A. The cells were infected with influenza A (strain MHIA1) at an MOI of approximately 2.0. At the indicated times after infection fluorescent cells were detected by fluorescent microscopy. Influenza A virus could be detected as early as 6 hours after infection; detection peaked around 48 hours and declined after 72 hours. Similar results were obtained with two other IAV strains (KCWA2548 and KCWA2560). B. The cells were infected with influenza B Mass at an MOI of approximately 3.0. At the indicated times after infection fluorescent cells were detected by fluorescent microscopy. The influenza B virus induced very low GFP expression. Similar results were obtained with two other influenza B virus strains (ODH0035 and R-Chin)

4000

600000 531757

M

MHIA 1

6705 bps

5'UTR pol I T'

Fold of Serum Dilution

No s erum

20

40

80

160

320

640

Fold of S erum Dilution

Figure 5. Antibody neutralization assay using 293T/IAV-Luc cells A. Neutralization of New Caledonia (NC) virus by serum from a NC-vaccinated individual. Dilutions of serum were incubated with virus (5000 infectious units) for one hour at 370C and inoculated onto 293T/IAV-Luc cells. Luciferase activity was measured on cell lysates 20 hours after infection. The TCID50 was approximately 1:480. B. Neutralization of New Caledonia (NC) virus by serum from a non-vaccinated individual. The TCID50 was approximately 1:120.

Table 2. Specificity testing of 293T/IAV-Luc cell line.

50000 0 0

200

400

600

800

1000

1200

Virus input

Summary of Results

16

100000

1

150000

4

200000

Seventeen non-influenza respiratory viruses were used to infect the 293T/IAV-Luc cell line (MOI=0.1). Influenza A ODH-0023 (MOI=0.1) was used as a positive control. The cells were analyzed for luciferase activity 24 hours post-infection. None of the respiratory viruses induced luciferase significantly above background.

0. 25

R2 = 0.9823

1,000,000 900,000 800,000 700,000 600,000 500,000 400,000 300,000 200,000 100,000 0

0. 06

250000

Luciferase RLU

RLU

72 hours

or Amp

3'UTR

100000

48 hours

A

Viruses induced the luciferase reporter gene in IAV NP Luc expressing 293T cells

0. 01 5

Generation of cell lines Plasmids containing the virus-inducible reporter gene segment (VIRGS) with either the luciferase gene (pIAVLuc) or GFP gene pIAV-GFP) were transfected into human kidney 293T cells using Hyfect (Denville) and clones were selected with 150mg/ml hygromycin B. Clones were then screened for IAV-inducible reporter gene activity following infection with IAV strain MHIA007 (MOI 0.5). Infection was conducted in serum free medium with TPCK trypsin (0.5 mgm/ml).

0

Hygromycin

or

EGFP

19 hours

8,000,000 4,000,000

Hygromycin

Luciferase RLU

Methods:

6 hours

12,000,000

Amp

pol I P

Virus input on Rmix 19hours

16,000,000

Influenza A infection

3' UTR pIAV-Luc 7886 bps pol I promoter

Background: Influenza virus infection has been one of the major causes of morbidity and mortality worldwide. Consequently, developing influenza vaccines, therapies, and diagnostic tools are critical in the prevention, diagnosis and treatment of influenza infections and pandemics. Unfortunately, traditional methods for quantifying influenza A virus titers are time-consuming and inefficient. Reliable, sensitive, and efficient methods are needed for the quantification of influenza A virus titers, for the performance of influenza A virus antibody neutralization assays, and for antiviral drug susceptibility testing. Improvements in the efficiency, and accuracy of these assays could expedite the development of influenza vaccines. To achieve these goals, a better understanding and creative utilization of influenza genome information is important. The genome of influenza virus, a negative strand RNA virus, contains eight RNA segments. The untranslated regions (UTRs) at the 5’and 3’ terminals of the influenza virus genomic segments are highly conserved and have been shown to be necessary and sufficient cis-acting elements for driving viral RNA replication and transcription. They can also be used to express nonviral or reporter genes (Andrew Lutz, et al. Journal of Virological Methods 126 (2005), 13-20). When influenza A virus infects cells that contain a reporter gene under the control of the influenza UTR, the viral RNA polymerase recognizes its target UTR resulting in transcription and translation of the reporter gene with high specificity and efficiency.

Luciferase Assay

r pol I terminato 5' UTR

Luc

20

Traditional methods for determining influenza A virus titers are very time-consuming. In order to make these assays more efficient, a genetically engineered human cell line expressing either green fluorescent protein (GFP) or luciferase (luc) under control of the untranslated regions (UTRs) of the influenza A virus nucleoprotein (NP) RNA segment (Lutz, et al. Journal of Virological Methods 126 (1-2), 13-20) was generated. The RNA transcript is expressed via a human RNA polymerase I promoter/terminator cassette. Reporter gene activity is detected as early as 8 hours post-infection with influenza A virus and was virus dose-dependent. The cell line detected more then forty influenza A strains including both lab strains and clinical strains. In contrast, about twenty influenza B strains as well as a panel of other respiratory viruses did not induce reporter gene activity. This cell line can also help detect and quantify influenza A virus antibody neutralization titers and antiviral drug resistance by providing a simple, rapid and sensitive assay when compared to traditional methods.

Am atadine concentration (!M)

Figure 3. Dose-response of 293T/IAV-Luc cell line following infection with influenza A virus. The 293T/IAV-Luc cell line was infected with nine different concentration of influenza A virus ODH99-154. The cells were analyzed for luciferase activity 24 hours post-infection. There was a linear correlation between luciferase activity and virus inoculum.

Figure 6. Amantadine susceptibility testing using 293T/IAV-Luc cells. The 293T/IAV-Luc cells were infected with IAV (isolate ODH; 100,000 infectious units) in the presence of different concentrations of amantadine. At 17 hpi the cells were analyzed for luciferase activity. The IC50 was approximately 0.3mM.

VIRGS-containing cell lines 293T/IAV-Luc and 293T/IAV-GFP detect influenza A virus with very high sensitivity and specificity. The reporter gene activity is detectable as early as 6-8 hours post-infection. Luciferase activity after IAV infection of 293T/IAV-Luc cells is virus dosedependent. There was in no loss of virus-inducible reporter activity after 28 passages.

Conclusion: This system for influenza virus detection is simple, fast, and amenable to automation. It can also be used to titer neutralizing antibody as well as for antiviral drug susceptibility testing. This method could be extremely useful for vaccine researchers and manufacturers, and potentially including H5N1 vaccines.


A Genetically Engineered Cell Line Expressing an Flu A Virus-Inducible Reporter Gene