The use of equine influenza pseudotypes for serological screening.
Abstract: Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. In order to produce infective pseudotype particles it was necessary to additionally co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave the HA. High titre pseudotype virus (PV) was then used in PV antibody neutralization assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% correlation between the results of the two assays, with the PVNA assay appearing slightly more sensitive. Future work will extend the testing of the PVNA with a larger number of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre.
Publication Date: 2012-12-31 PubMed ID: 23515229PubMed Central: PMC3601075
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research study outlines the development and initial testing of a new method for screening horse flu antibodies using a simulated virus. This new method offers a potential solution for challenges faced with standard screenings, including complex processes and inconsistencies between laboratories.
Background and Purpose of the Study
- The researchers are trying to address the issues faced by standard serology methods used to test for influenza. These include lab-to-lab variability, complicated protocols, and the requirement for handling potentially dangerous virus strains in high biological containment facilities.
- To overcome these challenges, the scientists have successfully used avian and human influenza HA pseudotyped retroviruses in antibody neutralization tests in the past. Their current study tests the feasibility of using a similar approach for horse flu, by creating an equine influenza pseudotyped lentivirus.
Methodology
- The researchers generated the equine influenza pseudotyped lentivirus by co-transfecting HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol, and firefly luciferase reporter genes, and then harvested the virus from the supernatant.
- To produce infective pseudotype particles, they also co-transfected a plasmid encoding the TMPRSS2 endoprotease to cleave the HA.
Analysis and Results
- The high-titre pseudotype virus (PV) they produced was then used in PV antibody neutralization tests (PVNAs) on horses, both vaccinated and non-vaccinated.
- Their sera were also tested with single radial haemolysis (SRH) assay. The researchers found a 65% correlation between the results of the PVNA and SRH assays. The PVNA assay seemed slightly more sensitive.
Future Directions
- While the initial results of the new method are promising, the research team recognizes the need for further testing. They propose extending the use of the PVNA with a larger number of samples for more robust sensitivity/specificity assessments.
- The researchers also express the importance of evaluating inter-laboratory and intra-laboratory variability and defining a protective titre in future studies. Addressing these areas would provide a more comprehensive understanding of the applicability and limitations of the new serology screening method.
Cite This Article
APA
Scott S, Molesti E, Temperton N, Ferrara F, Böttcher-Friebertshäuser E, Daly J.
(2012).
The use of equine influenza pseudotypes for serological screening.
J Mol Genet Med, 6, 304-308.
Publication
Researcher Affiliations
- Viral Pseudotype Unit, School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime, ME4 4TB, UK.
Grant Funding
- G0600369 / Medical Research Council
Conflict of Interest Statement
None declared.
References
This article includes 29 references
- Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes.. J Exp Med 2003 Mar 3;197(5):633-42.
- Böttcher E, Freuer C, Steinmetzer T, Klenk HD, Garten W. MDCK cells that express proteases TMPRSS2 and HAT provide a cell system to propagate influenza viruses in the absence of trypsin and to study cleavage of HA and its inhibition.. Vaccine 2009 Oct 23;27(45):6324-9.
- Böttcher E, Matrosovich T, Beyerle M, Klenk HD, Garten W, Matrosovich M. Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium.. J Virol 2006 Oct;80(19):9896-8.
- Corti D, Voss J, Gamblin SJ, Codoni G, Macagno A, Jarrossay D, Vachieri SG, Pinna D, Minola A, Vanzetta F, Silacci C, Fernandez-Rodriguez BM, Agatic G, Bianchi S, Giacchetto-Sasselli I, Calder L, Sallusto F, Collins P, Haire LF, Temperton N, Langedijk JP, Skehel JJ, Lanzavecchia A. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins.. Science 2011 Aug 12;333(6044):850-6.
- Daly JM, Lai AC, Binns MM, Chambers TM, Barrandeguy M, Mumford JA. Antigenic and genetic evolution of equine H3N8 influenza A viruses.. J Gen Virol 1996 Apr;77 ( Pt 4):661-71.
- Daly J, Daas A, Behr-Gross ME. Collaborative study for the establishment of a candidate equine influenza subtype 2 American-like strain A/EQ/South Africa/4/03 - horse antiserum biological reference preparation.. Pharmeuropa Bio 2007 Dec;2007(1):7-14.
- Daly JM, MacRae S, Newton JR, Wattrang E, Elton DM. Equine influenza: a review of an unpredictable virus.. Vet J 2011 Jul;189(1):7-14.
- Garcia JM, Lai JC. Production of influenza pseudotyped lentiviral particles and their use in influenza research and diagnosis: an update.. Expert Rev Anti Infect Ther 2011 Apr;9(4):443-55.
- Garten W, Klenk HD. Cleavage Activation of the Influenza Virus Hemagglutinin and Its Role in Pathogenesis.. Avian Influenza 2008.
- Hsu M, Zhang J, Flint M, Logvinoff C, Cheng-Mayer C, Rice CM, McKeating JA. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.. Proc Natl Acad Sci U S A 2003 Jun 10;100(12):7271-6.
- Ito H, Watanabe S, Takada A, Kawaoka Y. Ebola virus glycoprotein: proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies.. J Virol 2001 Feb;75(3):1576-80.
- John TJ, Fulginiti VA. Parainfluenza 2 virus: increase in hemagglutinin titer on treatment with Tween-80 and ether.. Proc Soc Exp Biol Med 1966 Jan;121(1):109-11.
- Katz JM, Lim W, Bridges CB, Rowe T, Hu-Primmer J, Lu X, Abernathy RA, Clarke M, Conn L, Kwong H, Lee M, Au G, Ho YY, Mak KH, Cox NJ, Fukuda K. Antibody response in individuals infected with avian influenza A (H5N1) viruses and detection of anti-H5 antibody among household and social contacts.. J Infect Dis 1999 Dec;180(6):1763-70.
- Morley PS, Hanson LK, Bogdan JR, Townsend HG, Appleton JA, Haines DM. The relationship between single radial hemolysis, hemagglutination inhibition, and virus neutralization assays used to detect antibodies specific for equine influenza viruses.. Vet Microbiol 1995 Jun;45(1):81-92.
- Mumford JA, Wood J. Establishing an acceptability threshold for equine influenza vaccines.. Dev Biol Stand 1992;79:137-46.
- Nie Y, Wang G, Shi X, Zhang H, Qiu Y, He Z, Wang W, Lian G, Yin X, Du L, Ren L, Wang J, He X, Li T, Deng H, Ding M. Neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection.. J Infect Dis 2004 Sep 15;190(6):1119-26.
- OIE Equine influenza, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2012
- Paillot R, Hannant D, Kydd JH, Daly JM. Vaccination against equine influenza: quid novi?. Vaccine 2006 May 8;24(19):4047-61.
- Steinhauer DA. Role of hemagglutinin cleavage for the pathogenicity of influenza virus.. Virology 1999 May 25;258(1):1-20.
- Temperton NJ, Chan PK, Simmons G, Zambon MC, Tedder RS, Takeuchi Y, Weiss RA. Longitudinally profiling neutralizing antibody response to SARS coronavirus with pseudotypes.. Emerg Infect Dis 2005 Mar;11(3):411-6.
- Temperton NJ, Hoschler K, Major D, Nicolson C, Manvell R, Hien VM, Ha do Q, de Jong M, Zambon M, Takeuchi Y, Weiss RA. A sensitive retroviral pseudotype assay for influenza H5N1-neutralizing antibodies.. Influenza Other Respir Viruses 2007 May;1(3):105-12.
- Temperton NJ, Wright E. Retroviral Pseudotypes. eLS 2009.
- Webster RG. Are equine 1 influenza viruses still present in horses?. Equine Vet J 1993 Nov;25(6):537-8.
- Wool-Lewis RJ, Bates P. Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines.. J Virol 1998 Apr;72(4):3155-60.
- Wright E, Hayman DT, Vaughan A, Temperton NJ, Wood JL, Cunningham AA, Suu-Ire R, Weiss RA, Fooks AR. Virus neutralising activity of African fruit bat (Eidolon helvum) sera against emerging lyssaviruses.. Virology 2010 Dec 20;408(2):183-9.
- Wright E, McNabb S, Goddard T, Horton DL, Lembo T, Nel LH, Weiss RA, Cleaveland S, Fooks AR. A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in Africa.. Vaccine 2009 Nov 27;27(51):7178-86.
- Wright E, Temperton NJ, Marston DA, McElhinney LM, Fooks AR, Weiss RA. Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison.. J Gen Virol 2008 Sep;89(Pt 9):2204-2213.
- Yamagishi H, Nagamine T, Shimoda K, Ide S, Igarashi Y, Yoshioka I, Matumoto M. Comparative measurement of equine influenza virus antibodies in horse sera by single radial hemolysis, neutralization, and hemagglutination inhibition tests.. J Clin Microbiol 1982 Apr;15(4):660-2.
- Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.. Nat Biotechnol 1997 Sep;15(9):871-5.
Citations
This article has been cited 8 times.- Kinsley R, Pronost S, De Bock M, Temperton N, Daly JM, Paillot R, Scott S. Evaluation of a Pseudotyped Virus Neutralisation Test for the Measurement of Equine Influenza Virus-Neutralising Antibody Responses Induced by Vaccination and Infection.. Vaccines (Basel) 2020 Aug 21;8(3).
- Scott SD, Kinsley R, Temperton N, Daly JM. The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus.. Pathogens 2016 Dec 15;5(4).
- Grehan K, Ferrara F, Temperton N. An optimised method for the production of MERS-CoV spike expressing viral pseudotypes.. MethodsX 2015;2:379-84.
- Trombetta CM, Perini D, Mather S, Temperton N, Montomoli E. Overview of Serological Techniques for Influenza Vaccine Evaluation: Past, Present and Future.. Vaccines (Basel) 2014 Oct 13;2(4):707-34.
- Carnell GW, Ferrara F, Grehan K, Thompson CP, Temperton NJ. Pseudotype-based neutralization assays for influenza: a systematic analysis.. Front Immunol 2015;6:161.
- Bentley EM, Mather ST, Temperton NJ. The use of pseudotypes to study viruses, virus sero-epidemiology and vaccination.. Vaccine 2015 Jun 12;33(26):2955-62.
- Sawoo O, Dublineau A, Batéjat C, Zhou P, Manuguerra JC, Leclercq I. Cleavage of hemagglutinin-bearing lentiviral pseudotypes and their use in the study of influenza virus persistence.. PLoS One 2014;9(8):e106192.
- Wang B, Wang B, Liu P, Li T, Si W, Xiu J, Liu H. Package of NDV-pseudotyped HIV-Luc virus and its application in the neutralization assay for NDV infection.. PLoS One 2014;9(6):e99905.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
