Page 17 - VHSA - Onderstepoort 100 Years - Part 3

and led to the introduction of recombinant DNA technology, aimed at the cloning of the gene(s) coding for the immuno- genic antigen(s) in order to develop a recombinant vaccine. Once the technology was established it led
to the cloning of the other genome seg- ments of various orbiviruses and the use of these clones as genetic probes for diagnos- tic and comparative purposes. It was also the first step towards the determination of nucleotide sequences in later studies.
When Huismans was recruited by the University of Pretoria in 1987 as Professor and head of its Genetics Department he decided to continue his work on orbi- viruses, concentrating on AHSV, in close collaboration with the Onderstepoort group. He continued for the rest of his career to produce outstanding research results as well as training many students, some of whom were later employed by the Institute.
His successor as head of the Bioche-
mistry Section was Albie van Dijk, a biochemistry graduate from Potchefstroom University. She continued the molecular studies on orbiviruses, at first concentrating on the in vitro transcription and translation of BTV messenger RNA. Her group was joined by G.J. (Gerrit) Viljoen and D.H. (Dion) du Plessis who continued with the programme of cloning the genome segments of the 21 serotypes of BTV as dsDNA in an E. coli vector. Cross-hybridization of these DNA clones identified segment five as the most suitable group-
specific probe, since it was highly conserved
in all serotypes. Serotype-specific probes,
based on segment two, were developed
for several orbiviruses and applied in a
dot-spot test which revolutionized the
characterization of viral isolates.
Van Dijk was specifically interested
in the two non-structural viral proteins pre-
sent in infected cells, which were respec-
tively identified as a phosphoprotein with autophosphorylation activity and a glyco-
sylated membrane protein which is impli-
cated in cell exit and possibly in pathogene-
sis and/or virulence. Progress was also made
in the development of recombinant subunit
vaccines and in 1989 the first baculovirus/
BTV recombinants, which were produced by
the Laboratory of Molecular Biophysics, University of Oxford, UK, were tested at Onderstepoort and found to be effective. DNA clones expressing proteins can also be used to produce antigens suitable for use in diagnostic tests for the presence of antibodies. Such ELISA tests were developed and used in 1990 for the demonstration of antibodies against AHSV in zebras. In 1991 a series of sequencing studies carried out on serotypes
ONDERSTEPOORT 100
of BTV originating from various countries led to the discovery of ‘topotypes’, or geographical variants within serotypes, which can be used to determine the source of an outbreak of
unknown origin.
Aetiology of jaagsiekte
In preparation for his future research on jaagsiekte (ovine pulmonary adenoma- tosis), Verwoerd spent 1973 at the National Cancer Institute in Bethesda, USA. On his return a new team was assembled in the Molecular Biology Section consisting of E-M (Ethel-Michele) de Villiers, A-L (Anna-Lise) Williamson and S. (Susan) Coetzee on the professional side, and P. A. L. (Flip) Eksteen and E. (Liz) Meyer-Scharrer as technicians. Initially the hypothesis that a herpes virus might cause jaagsiekte was explored, based on the demonstration by other workers using electron microscopy of such a virus in affected lungs. It was soon found, however, that herpes viruses commonly occur in
normal sheep lungs without causing any disease.
An important breakthrough was the discovery that jaag- siekte could be transmitted experimentally by intra-tracheal injection of newborn lambs with lung-lavage material from an established case of the disease, one of its characteristics being the accumulation of fluid in the lungs. Density fractionation of the lung fluid resulted in an infective fraction with the typical density of retroviruses. Reverse transcriptase, an enzyme characteristic of these viruses, could also be demonstrated in the same fraction.
“A baffling characteristic of jaagsiekte was the fact that antibodies to the virus could never be detected, either in natural or experimental cases of the disease. The prospect of developing a vaccine or even a serological test for jaagsiekte therefore seemed very slim.”
Electron microscopy of the semi-purified 107 fraction confirmed the presence of viral par-
ticles with the typical morphology of type D retroviruses.
All attempts to cultivate the virus in cell cultures failed, however, although cell cultures could be established from the lung tumour which could transmit the disease. Final proof that the virus, now called the jaagsiekte retrovirus (JSRV), is the causative agent of the lung tumour was obtained by demonstrating an inverse relationship between the concentration of the virus in the inoculum, as measured by the reverse transcriptase activity, and the incubation
period of the disease. A baffling characteristic of jaagsiekte was the fact that antibodies to the virus could never be detected, either in natural or experimental cases of the disease. The prospect of developing a vaccine or even a serological test for jaagsiekte therefore seemed very slim.
A by-product of the jaagsiekte work was the discovery of a second retrovirus in many, but not all, cases of the disease. It had a different density, could be separated from JSRV and
Dr. P. (Polly) Roy at
Biochemistry, Molecular Biology, Biotechnoly and Immunochemistry
1908-2008
Years