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Generation of EHV‐1 pseudotype virus for cell tropism studies and virus‐neutralising assays

Abstract : Background: Equid herpesvirus-1 (EHV-1) can cause respiratory disease, abortion, neonatal death and myeloencephalopathy. Thus, EHV-1 represents a threat to the equine industry. EHV-1 exhibits 12 glycoproteins on its surface envelope, but those important for cell entry/host immune responses remains partially unclear. To investigate the contribution of these glycoproteins, pseudotype viruses (PVs) may provide a useful study tool. Objectives: Generate high titre EHV-1 PV particles for cell tropism studies and develop tests for virus-neutralising (VN) antibody detection in naturally/experimentally infected horses. Study design: Assay development. Methods: 5 EHV-1 glycoprotein gene sequences were obtained from an aborted fetus strain isolated during a large EHV-1 outbreak in France in 2010. Sequences were synthesised and subcloned into expression vectors and employed in lentivirus PV generation. PVs were utilised in a Pseudotype Virus Neutralisation Test (PVNT), a sensitive technique to measure levels of specific VN antibodies. Serum samples (n=48) tested were taken longitudinally (Days 0 to 18 pi) from ponies experimentally infected with EHV-1, compared with uninfected controls (n=4). Plasmids expressing PV components’ genes were co-transfected into HEK293T/17 using polyethylenimine (PEI). PV production and quantification were assessed by fluorescence and luminescence, respectively. For PVNT, two-fold serial dilution of equine sera were incubated with PV and target cells. As for traditional VN tests, the antibody titre was expressed as the highest serum dilution causing 50% inhibition (IC50). Results: Titres of EHV-1 PV were optimised and PVNT successfully performed and compared with a conventional EHV-1 VN assay (r=0.82). Main limitations: Cross-reactivity studies with other EHVs need further investigation. Conclusions: Functional EHV-1 PVs can be generated using a minimum of four glycoproteins gB, gD, gH and gL. The addition of gC neither enhances PV production nor is essential for cell entry. EHV-1 neutralising antibodies can be quantified in experimentally infected horse sera.
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Submitted on : Wednesday, October 13, 2021 - 9:18:00 AM
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Cecilia Di Genova, Gabrielle Sutton, Romain Paillot, Nigel Temperton, Stéphane Pronost, et al.. Generation of EHV‐1 pseudotype virus for cell tropism studies and virus‐neutralising assays. Special Issue: Abstracts of the 11th International Equine Infectious Diseases Conference, Sep 2021, Online, France. pp.55-56, ⟨10.1111/evj.81_13495⟩. ⟨hal-03374610⟩

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