Research Results - (PDF, 101 mb) - USAID

Between 1980 and 1990, ten faculty
members from the College of Veterinary Medi-
cine at Colorado State University have contributed,
some in a major way, to the SR-CRSP
Animal health Project. In addition, two Peruvian
Ph.D. students, two U.S. students, and four
additional foreign Ph.D. or M.S. students have
been wholly or partially supported by the SR-
CRSP project. Research at Colorado State
University has led to research linkages within
Peru, the U.S., and to the Moredun Institute in
Scotland.
Research Results
Subproject 1. Major core protein
purification and molecular cloning of
the ovine pulmonary carcinoma
retrovirus.
Objectives
1. Purify and characterize the major core (gag)
of OPC retrovirus mganravector
protein of OP o to using an affinity
column with antibody to a heterologous type D
retrovirus,
2. Prepare polyclonal or monoclonal antibodies
to Pree p c alor olonal aD
Problem Statement and Approach
Our strategy to isolate and propagate the
ovine pulmonary carcinoma (OPC) retrovirus
depends in part on its known relationship to
already characterized type D retroviruses of
primates: Mason-Pfizer monkey virus (MPMV),
Squirrel monkey retrovirus (SMRV) and Langur
retrovirus (LRV). Based on materials available
from these viruses, we ave developed assays
(competition radioimmui,assay and
immunoblotting assay) for detection of the
putative OPC retrovirus in samples of lung
tissue and fluid from affected animals. Further
progress toward development of a serological
test for carriers of the virus and eventually a
vaccine to prevent the infection depends upon
production of large quantities of purified viral
proteins. This can be most efficiently accomplished
by in vitro propagation of the virus or
some of its genes.
Purification of homologous OPC
retrovirus major core protein from lung fluid or
tissue of OPC cases will be attempted using
affinity chromatography. Our previous work
indicates that there is sufficient homology of this
protein with p27 of SMRV in a competition RIA
so that we can use a polyclonal goat serum to
bind the protein in an affinity column. The OPC
protein can then be eluted, concentrated, and
used to prepare rabbit polyclonal antiserum or
murine monoclonal antibodies using standard
techniques. Several cloning strategies will be
employed in attempts to obtain OPC gene
sequences. A lamba gtl1 expression library
produced from OPC cDNA which had been
reversed-transcribed from virus containing
nucleic acid will be screened using heterologous
(anti-MPMV p27) or homologous (produced
above) antiserum and subcloned into a plasmid
(pSP72). If the lambda gtl1 library does
not contain OPC sequences, efforts to amplify
OPC DNA sequences using the polymerase
chain reaction will be attempted. Gag,pol, and
env gene regions highly conserved among type
retroviruses will be used as templates for
construction of oligonucleotide primers to
3. Obtain molecular clones containing OPCdectdiSohrnbtigwthMM
initiate the reaction. Amplified OPC DNA as
gene equeces.detected
in Southern blotting with MPMV
gene sequences,
probes will be cloned directly into phage vec­
tors
justification
It is widely recognized that OPC (also
known as sheep pulmonary adenomatosis or
jaagsiekte) is the most highly prevalent disease
affecting adult sheep of the Central and Southem
Sierra of Peru, and that it is also of major
importance in developing countries of Africa,
Asia, the Middle East, and the Far East. Because
of the high prevalence of OPC in many areas of
the world and the lack of diagnostic tests for
infected sheep, cull and quaranckneprocedures
have not been effective in limiting its economic
impact. Further progress in controlling this
disease is dependent upon identification and in
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