Expression in Escherichia coli and sequencing of the coding region for the capsid protein of Dutch maedi-visna virus strain ZZV 1050: application of recombinant protein in enzyme-linked immunosorbent assay for the detection of caprine and ovine lentiviruses.
Maedi-visna in sheep and caprine arthritis-encephalitis in goats are caused by two closely related and widespread lentiviruses. The infections are characterized by life-long virus persistence and slow induction of antiviral antibodies. The diagnosis is based on the detection of antiviral antibodies. We have used the polymerase chain reaction (PCR) to amplify a part of the gag gene coding for the entire capsid protein and for parts of the matrix and nucleocapsid proteins. Sequencing of the PCR fragment of the Dutch maedi-visna virus strain ZZV 1050 revealed 85 and 92% homology to the DNA and deduced amino acid sequences, respectively, of the distantly related Icelandic visna virus strain 1514. The respective homologies with caprine arthritis-encephalitis virus strain CO were 76 and 80%. The PCR fragment was cloned into pGEX-2T and expressed as a glutathione S-transferase fusion protein. The recombinant protein could be detected on immunoblots by using a monoclonal antibody and polyclonal antisera and was further purified by glutathione-based affinity chromatography. Enzyme-linked immunosorbent assay with purified recombinant fusion protein is shown to be a sensitive and specific diagnostic tool for the detection of lentiviral infection in goats and sheep.
Zanoni
R G
RG
Institute of Veterinary Virology, Bern, Switzerland.
Nauta
I M
IM
Pauli
U
U
Peterhans
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006041:Goats; D016180:Lentivirus Infections / Q000175:diagnosis; D008969:Molecular Sequence Data; D011021:Pneumonia, Progressive Interstitial, of Sheep / Q000175:diagnosis; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012756:Sheep; D014790:Visna-maedi virus / Q000235:genetics* / Q000302:isolation & purification
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins