Heterologous expression of alcelaphine herpesvirus 1 structural proteins and their use in the development of an ELISA

Abstract

Malignant catarrhal fever (MCF), a disease that is usually fatal in cattle, is caused by two distinct but related bovine herpesviruses which are members of the genus Macavirus. The wildebeest-associated alcelaphine herpesvirus-1 (AlHV-1) occurs mainly in East and southern Africa, whereas the sheep-associated ovine herpesvirus-1 (OvHV-2) has an almost worldwide distribution. The natural hosts or carriers of these two viruses are subclinically infected. The 130 kilobase pair (kbp) AlHV-1 double stranded DNA genome consists of 18 open reading frames (ORFs) coding for structural proteins and approximately 50 ORFs coding for non-structural proteins. The 18 structural ORFs encode for 4 capsid proteins, 5 tegument proteins, 8 glycoproteins and a minor capsid scaffold protein. ORF8 encoding for glycoprotein B, is the most conserved of the proteins amongst gammaherpesviruses, whereas the minor capsid protein encoded by ORF65, is amongst the most variable. Thus, the minor capsid protein is one of the antigens of choice for the development of an ELISA for detection of AlHV-1 reactive antibodies and glycoprotein B could be of importance in developing a cross-protective vaccine for gammaherpesviruses. The naming and annotation of most of the AlHV-1 ORFs is based on comparison with related gammaherpesviruses and bioinformatics. Most of these ORFs are putative as there is no direct experimental evidence confirming that they code for any particular protein. In order to investigate whether the ORFs code for any proteins, two ORFs were targeted for in vitro heterologous expression. AlHV-1, isolate C500, was grown in fetal bovine turbinate (BT) cell culture and viral genomic DNA extracted. ORF8, the putative glycoprotein B, was amplified with a PCR assay and inserted into a mammalian expression vector, pCI. VERO cells were transfected with the recombinant vector. Expression of ORF8 was confirmed by an indirect immunofluorescence assay (IFA) with AlHV-1 polyclonal sera and rabbit anti-bovine IgG (whole molecule) FITC conjugate. Truncated forms of ORF8 were further expressed as baculovirus recombinants using the Bac-to-Bac baculovirus expression system. Expression of the truncated ORF8 was confirmed by SDS-PAGE and Western blot. AlHV-1 ORF65, the minor capsid protein gene, was amplified with a PCR assay from the viral genomic DNA and cloned in frame with a histidine tag in a bacterial expression vector, pCOLD I. Expression of the minor capsid protein was confirmed by SDS-PAGE and Western blot with the histidine tag monoclonal as well as AlHV-1 polyclonal sera. Orf65 was expressed in large quantities and column purified using the histidine tag. Orf65 was also expressed as a baculovirus recombinant using the Bac-to-Bac baculovirus expression system. Expression of the protein was confirmed by SDS-PAGE and Western blot with the histidine tag and AlHV-1 polyclonal sera. ORF65 expression in the baculovirus Bac-to-Bac expression system was up-scaled and the expressed protein column purified. Antibodies raised in chicken against the purified antigen were used successfully in an indirect immunoassay to detect AlHV-1 infected cells. An indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against AlHV-1 was developed. It is based on the use of the AlHV-1 minor capsid protein as the capture antigen for antibodies. The primary antibodies are detected by the addition of enzymelabelled (horseradish peroxidase) protein G which detects bovid, ovid and wildebeest antibodies. Addition of a substrate of the enzyme, in this case, 3,3’,5,5’- tetramethylbenzidine (TMB), results in a colour reaction which is measured using spectrophotometric procedures. At a selected cut-off point of 18, the ELISA test has a sensitivity of 100% and a specificity of 100% and has been shown to detect AlHV-1 antibodies in cattle and wildebeest. The ELISA showed no cross-reactivity with sera raised in cattle against related viruses such as ovine herpesvirus 2, bovine herpesvirus 1, 2 and 4. The two expressed proteins used in this study were found to be amongst the antigens expressed in cattle suffering from malignant catarrhal fever. The experimental AlHV-1 indirect ELISA needs further validation and this research may be extended to determine the performance of these antigens as candidate subunit vaccines.