Affiliation:
1. National Morbillivirus Referral Laboratory, Division of Virology, Indian Veterinary Research Institute, Mukteswar-263 138, Nainital District, Uttaranchal, India
Abstract
ABSTRACT
We developed and characterized a stable Vero cell line constitutively expressing
Peste des petits ruminants virus
(PPRV) hemagglutinin (H) protein and assessed its potential use as diagnostic antigen in enzyme-linked immunosorbent assay (ELISA). PPRV H gene of the vaccine strain (Sungri-96) was amplified by reverse transcription (RT)-PCR, cloned into a eukaryotic expression vector (pTarget), and subsequently transfected and expressed in Vero cells. A stable Vero cell line was developed after 20 repeated passages by using G418 antibiotic selection pressure (400 to 600 μg/ml). The integration of PPRV H gene in the Vero cell genome and its genomic transcription were confirmed by PCR and RT-PCR assays, respectively, and the 70-kDa PPRV H protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The recombinant protein reacted specifically with PPRV anti-H neutralizing monoclonal and polyclonal antibody in competitive, sandwich, and indirect ELISA, respectively, indicating that the native form of the protein was expressed. Evaluation of the protein in competitive ELISA and indirect ELISA vis a vis whole virus was done using 306 and 146 goat field serum samples, respectively; comparable results were obtained with high degrees of relative diagnostic specificity (93.53% and 100%, respectively) and sensitivity (99.04% and 79.16%, respectively). This study shows that the PPRV H protein could be a sustainable source of safe antigen in countries of nonendemicity without the need to handle infectious virus for serodiagnosis.
Publisher
American Society for Microbiology
Subject
Microbiology (medical),Clinical Biochemistry,Immunology,Immunology and Allergy
Reference33 articles.
1. Abu Elzein, E. M., M. M. Hassanien, A. I. Al-Afaleq, M. A. Abd Elhadi, and F. M. Housawi. 1990. Isolation of peste des petits ruminants from goats in Saudi Arabia. Vet. Rec.127:309-310.
2. Anderson, J., and J. A. McKay. 1994. The detection of antibodies against peste des petits ruminants virus in cattle, sheep and goats and the possible implications to rinderpest control programmes. Epidemiol. Infect.112:225-231.
3. Anderson, J., J. A. McKay, and R. N. Butcher. 1991. The use of monoclonal antibodies in competition ELISA for detection of antibodies to rinderpest and peste des petits ruminants viruses, p. 43-53. In The sero-monitoring of rinderpest throughout Africa: phase I. International Atomic Energy Agency, Vienna, Austria.
4. Vet. Res. Commun.
5. Burnette, W. N. 1981. ‘Western blotting’ electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem.112:195-203.
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