Development of a recombinant ELISA for ovine herpesvirus 2, suitable for use in sheep

George C Russell,Ann Percival,Dawn M Grant,Kathryn Bartley,Dylan Turnbull,Kevin Mclean,Julia Lienhard,Claudia Bachofen

doi:10.1016/j.jviromet.2021.114329

George C Russell, Ann Percival + Show 6 more

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https://doi.org/10.1016/j.jviromet.2021.114329

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Abstract

The minor capsid protein of ovine herpesvirus 2, identified as a potential antigen for serological testing, was over-expressed and purified to allow its assessment in ELISA. The corresponding gene sequence (OvHV-2 orf65, Ov65) was modified to incorporate epitope tags and internal restriction enzyme sites in an E. coli codon-optimised version of the gene. This codon-optimised gene was then subject to internal deletions to identify regions of the protein that could be removed while maintaining protein solubility and antigenicity. It was found that a derivative with deletion of the conserved 5’-end of the gene (Ov65delB) expressed a polypeptide that was soluble when over-expressed in bacteria and was detected by OvHV-2 specific sera. Proteomic analysis of the affinity purified Ov65delB showed that it contained multiple predicted Ov65 tryptic peptides but also showed contamination by co-purifying E. coli proteins.An indirect ELISA, based on this affinity-purified OV65delB, was optimised for use with sheep and cattle samples and cut-off values were established based on known negative serum samples. Analysis of groups of samples that were either presumed infected (UK sheep) or tested OvHV-2 positive or negative by PCR (cattle MCF diagnostic samples) showed that the assay had 95 % sensitivity and 96 % specificity for sheep serum; and 80 % sensitivity and 95 % specificity for cattle serum. The lower sensitivity with cattle samples appeared to be due to a lack of serological response in some MCF-affected cattle.This recombinant antigen therefore shows promise as the basis of an inexpensive, simple and reliable test that can be used to detect OvHV-2-specific antibody responses in both MCF-affected animals and in OvHV-2 reservoir hosts.

Highlights

Malignant catarrhal fever (MCF) is a fatal systemic disease of cattle and other ungulates caused by infection with a group of related viruses classified as: family Herpesviridae; sub-family Gammaherpesvirinae; genus Macavirus (Davison et al, 2009)
In this paper we describe the development of a recombinant ELISA that can detect antibody responses to ovine herpesvirus 2 (OvHV-2) infection in both reservoir species and MCF-affected animals with high sensitivity and specificity
Established serological tests for MCF in either MCF-susceptible or reservoir species, irrespective of the virus responsible, have to date used reagents derived from strains of alcelaphine herpesvirus 1

Summary

Introduction

Malignant catarrhal fever (MCF) is a fatal systemic disease of cattle and other ungulates caused by infection with a group of related viruses classified as: family Herpesviridae; sub-family Gammaherpesvirinae; genus Macavirus (Davison et al, 2009). MCF is characterised by the expansion of virus-infected lymphoid cells, both within the circulation and in most organs, leading to death following infiltration of multiple tissues with infected lymphoid cells (Dewals et al, 2008; Dewals and Vanderplasschen, 2011) This virus expansion in clinical MCF cases makes the detection of infection by PCR of DNA from blood or tissue samples a reliable diagnostic method (Baxter et al, 1997; Bremer et al, 2005; Dungu et al, 2002; Flach et al, 2002; Hussy et al, 2001), and real-time fluorogenic PCR assays for both AlHV-1 and OvHV-2 have been described (Hussy et al, 2001; Russell et al, 2012; Traul et al, 2005). All serological tests for MCF virus infection developed to date used AlHV-1 as the source of antigen, mainly because this virus is readily propagated in culture (Li et al, 2011)

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