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Clinical and Vaccine Immunology, December 2006, p. 1367-1372, Vol. 13, No. 12
1071-412X/06/$08.00+0     doi:10.1128/CVI.00273-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Development and Characterization of a Stable Vero Cell Line Constitutively Expressing Peste des Petits Ruminants Virus (PPRV) Hemagglutinin Protein and Its Potential Use as Antigen in Enzyme-Linked Immunosorbent Assay for Serosurveillance of PPRV

National Morbillivirus Referral Laboratory, Division of Virology, Indian Veterinary Research Institute, Mukteswar-263 138, Nainital District, Uttaranchal, India

Received 13 June 2006/ Returned for modification 15 September 2006/ Accepted 27 September 2006

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.

Published ahead of print on 18 October 2006.