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

Cecilia Di Genova (1) , Gabrielle Sutton (2, 3) , Romain Paillot (2, 3) , Nigel Temperton (1) , Stéphane Pronost (3, 2) , R Scott Pirie (4)

1 Viral Pseudotype Unit
2 LABÉO - LABÉO, Pôle d’analyses et de recherche de Normandie
3 BIOTARGEN - Biologie, génétique et thérapies ostéoarticulaires et respiratoires
4 Edin. - University of Edinburgh

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.

Domaines: Médecine vétérinaire et santé animale

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. 11th International Equine Infectious Diseases Conference, Sep 2021, Online, France. pp.55-56, ⟨10.1111/evj.81_13495⟩. ⟨hal-03374610⟩