The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus.
Abstract: Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology. The aim of this study was to optimise production of equine influenza PVs. Co-transfection of the HAT protease to activate the haemagglutinin (HA) yielded a higher titre PV than TMPRSS2 with the HA from A/equine/Richmond/1/2007 (H3N8), whereas for A/equine/Newmarket/79 (H3N8), both proteases resulted in equivalent titres. TMPRSS4 was ineffective with the HA of either strain. There was also an inverse relationship between the amount of protease-expression plasmids and the PV titre obtained. Interestingly, the PV titre obtained by co-transfection of a plasmid encoding the cognate N8 NA was not as high as that generated by the addition of exogenous neuraminidase (NA) from to allow the release of nascent PV particles. Finally, initial characterisation of the reliability of PV neutralisation tests (PVNTs) demonstrated good intra-laboratory repeatability. In conclusion, we have demonstrated that equine influenza PV production can be readily optimised to provide a flexible tool for studying EIV.
Publication Date: 2016-12-15 PubMed ID: 27983716PubMed Central: PMC5198168DOI: 10.3390/pathogens5040068Google Scholar: Lookup
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Summary
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The research article discusses the optimization of pseudotyped viruses (PVs) to enhance the understanding of equine influenza virus (EIV) biology. Researchers have explored the potential of PVs as powerful tools and emphasized on the co-transfection process to achieve the best results.
Objectives and Motivation of the Study
- The primary aim of this study was to optimize the production of equine influenza pseudotyped viruses (PVs). PVs are synthetic viruses that are created by replacing the envelope proteins of a virus with those from another virus. They are powerful tools in studying various virus biology aspects, as they allow the mimicry of the viral entry process in a safer and more controlled environment.
- The research particularly focused on equine influenza virus (EIV), a significant pathogen in the horse population worldwide, causing respiratory disease, poor performance, and economic loss in the equine industry.
Methods and Techniques
- The study began with the co-transfection of cells with plasmids that express lentiviral core proteins and viral envelope proteins. This method is one of the ways to produce pseudotyped viruses.
- The paper reports using different proteases, including TMPRSS2, TMPRSS4, and HAT. Different ratios of protease-expression plasmids were used to identify the ideal quantity for maximizing the PV titre.
- The research also involved the co-transfection of a plasmid encoding the cognate N8 NA and the addition of exogenous neuraminidase (NA) to boost the release nascent PV particles.
Key Findings
- The researchers found that co-transfecting the HAT protease for the activation of haemagglutinin (HA) yielded a higher PV titre compared to TMPRSS2 with the HA from specific EIV strains.
- It was also observed that TMPRSS4 was ineffective with the HA of either strain. Thus, illustrating that optimized PV production requires careful protease selection.
- The research indicated that there was an inverse relationship between the volume of protease-expression plasmids and the resultant PV titre, implying that more isn’t necessarily better.
- Interestingly, the PV titre sourced by co-transfection of a plasmid encoding the cognate N8 NA wasn’t as high as that produced by the addition of exogenous neuraminidase (NA). So, additional enzymes may be required to optimize PV production.
Conclusions and Implications
- This study successfully demonstrated that equine influenza PV production could be optimized to study EIV biology in more detail, thus providing a flexible research tool.
- The PV production method’s versatility also translates into intra-laboratory repeatability, which was demonstrated by the reliability of the PV neutralisation tests (PVNTs).
- By understanding and enhancing PV production, researchers can conduct more detailed and efficient studies on EIV. This can ultimately help develop new therapeutic and vaccination strategies against equine influenza, contributing to the health of the global equine population and the equine industry.
Cite This Article
APA
Scott SD, Kinsley R, Temperton N, Daly JM.
(2016).
The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus.
Pathogens, 5(4), 68.
https://doi.org/10.3390/pathogens5040068 Publication
Researcher Affiliations
- Viral Pseudotype Unit, School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime ME4 4TB, UK. S.D.Scott@kent.ac.uk.
- Viral Pseudotype Unit, School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime ME4 4TB, UK. rk320@kent.ac.uk.
- Viral Pseudotype Unit, School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime ME4 4TB, UK. n.temperton@kent.ac.uk.
- School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington LE12 5RD, UK. janet.daly@nottingham.ac.uk.
Conflict of Interest Statement
The authors declare no conflict of interest.
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Citations
This article has been cited 4 times.- Zeng L, Liu Y, Nguyenla XH, Abbott TR, Han M, Zhu Y, Chemparathy A, Lin X, Chen X, Wang H, Rane DA, Spatz JM, Jain S, Rustagi A, Pinsky B, Zepeda AE, Kadina AP, Walker JA 3rd, Holden K, Temperton N, Cochran JR, Barron AE, Connolly MD, Blish CA, Lewis DB, Stanley SA, La Russa MF, Qi LS. Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.. Nat Commun 2022 May 19;13(1):2766.
- Oladunni FS, Oseni SO, Martinez-Sobrido L, Chambers TM. Equine Influenza Virus and Vaccines.. Viruses 2021 Aug 20;13(8).
- 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).
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