Top

Virology Journal

Published in:

Open Access 01-12-2012 | Methodology

An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting

Authors: Yvonne van Remmerden, Fang Xu, Mandy van Eldik, Jacco GM Heldens, Willem Huisman, Myra N Widjojoatmodjo

Published in: Virology Journal | Issue 1/2012

Abstract

Background

Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable.

Methods

Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described.

Results

The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed.

Conclusions

The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.

Available only for authorised users

Falsey AR: Respiratory syncytial virus infection in elderly and high-risk adults. Exp Lung Res. 2005, 31 (Suppl 1): 77-PubMed

Glezen WP, Paredes A, Allison JE, Taber LH, Frank AL: Risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level. J Pediatr. 1981, 98: 708-715. 10.1016/S0022-3476(81)80829-3.PubMedCrossRef

Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, et al: Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet. 2010, 375: 1545-1555. 10.1016/S0140-6736(10)60206-1.PubMedPubMedCentralCrossRef

Elliot AJ, Fleming DM: Influenza and respiratory syncytial virus in the elderly. Expert Rev Vaccines. 2008, 7: 249-258. 10.1586/14760584.7.2.249.PubMedCrossRef

Schickli JH, Dubovsky F, Tang RS: Challenges in developing a pediatric RSV vaccine. Hum Vaccin. 2009, 5: 582-591.PubMedCrossRef

Anderson LJ, Hierholzer JC, Bingham PG, Stone YO: Microneutralization test for respiratory syncytial virus based on an enzyme immunoassay. J Clin Microbiol. 1985, 22: 1050-1052.PubMedPubMedCentral

Siber GR, Leszcynski J, Pena-Cruz V, Ferren-Gardner C, Anderson R, Hemming VG, et al: Protective activity of a human respiratory syncytial virus immune globulin prepared from donors screened by microneutralization assay. J Infect Dis. 1992, 165: 456-463. 10.1093/infdis/165.3.456.PubMedCrossRef

Parnes C, Guillermin J, Habersang R, Nicholes P, Chawla V, Kelly T, et al: Palivizumab prophylaxis of respiratory syncytial virus disease in 2000–2001: results from the palivizumab outcomes registry. Pediatr Pulmonol. 2003, 35: 484-489.PubMedCrossRef

McKimm-Breschkin JL: A simplified plaque assay for respiratory syncytial virus–direct visualization of plaques without immunostaining. J Virol Methods. 2004, 120: 113-117. 10.1016/j.jviromet.2004.02.020.PubMedCrossRef

10.

Zielinska E, Liu D, Wu HY, Quiroz J, Rappaport R, Yang DP: Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis. Virol J. 2005, 2: 84-10.1186/1743-422X-2-84.PubMedPubMedCentralCrossRef

11.

Biacchesi S, Skiadopoulos MH, Yang L, Murphy BR, Collins PL, Buchholz UJ: Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression. J Virol Methods. 2005, 128: 192-197. 10.1016/j.jviromet.2005.05.005.PubMedCrossRef

12.

de Graaf M, Herfst S, Schrauwen EJ, van den Hoogen BG, Osterhaus AD, Fouchier RA: An improved plaque reduction virus neutralization assay for human metapneumovirus. J Virol Methods. 2007, 143: 169-174. 10.1016/j.jviromet.2007.03.005.PubMedCrossRef

13.

Cosma A, Buhler S, Nagaraj R, Staib C, Hammarin AL, Wahren B, et al: Neutralization assay using a modified vaccinia virus ankara vector expressing the green fluorescent protein is a high-throughput method to monitor the humoral immune response against vaccinia virus. Clin Diagn Lab Immunol. 2004, 11: 406-410.PubMedPubMedCentral

14.

Yoder SM, Zhu Y, Ikizler MR, Wright PF: Role of complement in neutralization of respiratory syncytial virus. J Med Virol. 2004, 72: 688-694. 10.1002/jmv.20046.PubMedCrossRef

15.

Yang DP, Zielinska E, Quiroz J, Madore D, Rappaport R: Preparation of a respiratory syncytial virus human reference serum for use in the quantitation of neutralization antibody. Biologicals. 2007, 35: 183-187. 10.1016/j.biologicals.2006.09.004.PubMedCrossRef

16.

Widjojoatmodjo MN, Boes J, van Bers M, van Remmerden Y, Roholl PJ, Luytjes W: A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats. Virol J. 2010, 7: 114-10.1186/1743-422X-7-114.PubMedPubMedCentralCrossRef

17.

Hardy RW, Harmon SB, Wertz GW: Diverse gene junctions of respiratory syncytial virus modulate the efficiency of transcription termination and respond differently to M2- mediated antitermination. J Virol. 1999, 73: 170-176.PubMedPubMedCentral

18.

Tran KC, Collins PL, Teng MN: Effects of altering the transcription termination signals of respiratory syncytial virus on viral gene expression and growth in vitro and in vivo. J Virol. 2004, 78: 692-699. 10.1128/JVI.78.2.692-699.2004.PubMedPubMedCentralCrossRef

19.

Hallak LK, Spillmann D, Collins PL, Peeples ME: Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection. J Virol. 2000, 74: 10508-10513. 10.1128/JVI.74.22.10508-10513.2000.PubMedPubMedCentralCrossRef

20.

Chen M, Chang JS, Nason M, Rangel D, Gall JG, Graham BS, et al: A flow cytometry-based assay to assess RSV-specific neutralizing antibody is reproducible, efficient and accurate. J Immunol Methods. 2010, 362: 180-184. 10.1016/j.jim.2010.08.005.PubMedPubMedCentralCrossRef

21.

Walsh EE, Hruska J: Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein. J Virol. 1983, 47: 171-177.PubMedPubMedCentral

Title: An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting
Authors: Yvonne van Remmerden
Fang Xu
Mandy van Eldik
Jacco GM Heldens
Willem Huisman
Myra N Widjojoatmodjo
Publication date: 01-12-2012
Publisher: BioMed Central
Published in: Virology Journal / Issue 1/2012
Electronic ISSN: 1743-422X
DOI: https://doi.org/10.1186/1743-422X-9-253

Live Webinar | 27-06-2024 | 18:00 (CEST)

Keynote webinar | Spotlight on medication adherence

Reserve your place

Live: Thursday 27th June 2024, 18:00-19:30 (CEST)

WHO estimates that half of all patients worldwide are non-adherent to their prescribed medication. The consequences of poor adherence can be catastrophic, on both the individual and population level.

Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.

Prof. Kevin Dolgin

Prof. Florian Limbourg

Prof. Anoop Chauhan

Developed by: Springer Medicine

Obesity Clinical Trial Summary

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.

Developed by: Springer Medicine

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 1/2012

Effect of antiviral therapy on the survival and incidence of major complications in HBV-associated cirrhotic patients after splenectomy for hypersplenism and portal hypertension

Identification of nonessential regions of the nsp2 protein of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus for replication in marc-145 cell

In vitroinfection with classical swine fever virus inhibits the transcription of immune response genes

Analysis of protein expression profiles in the thymus of chickens infected with Marek’s disease virus

Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6

Rabies molecular virology, diagnosis, prevention and treatment

Keynote webinar | Spotlight on medication adherence

Live: Thursday 27th June 2024, 18:00-19:30 (CEST)

At a glance: The STEP trials