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. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/40709 |
| Date | January 2013 |
| Creators | Rachidi, Makgangtsake Dominic |
| Contributors | Van Vuuren, Moritz, rachidid@arc.agric.za, Wallace, David Brian |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2013 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
Page generated in 0.003 seconds