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. 2012 Dec;23(3):326-32.
doi: 10.1007/s13337-012-0098-8. Epub 2012 Sep 6.

Expression of Foot-and-Mouth Disease Virus Non-Structural Protein, 3D in Insect Cells and its Application in Detection of Anti-FMDV Antibodies

Rakesh Kumar  1 , M Hosamani, B P Sreenivasa, Anil Kotyal, R Venkataramanan
Affiliations

Expression of Foot-and-Mouth Disease Virus Non-Structural Protein, 3D in Insect Cells and its Application in Detection of Anti-FMDV Antibodies

Rakesh Kumar et al. Indian J Virol. 2012 Dec.

Abstract

Non-structural proteins (NSPs) based diagnostics are useful for large-scale sero-surveillance of foot-and-mouth disease (FMD) and to monitor viral activity as a follow up to the vaccination campaign in FMD endemic countries like India which aim at disease control through vaccination. These diagnostics are also handy in the serology of import/export of cloven-footed animals. In the present study, non-structural protein RNA polymerase (3D gene) of FMD virus (FMDV) was expressed using baculovirus expression system. Protein expression was analyzed by SDS-PAGE and confirmed by its immuno-reactivity with serum from a FMDV infected bovine, in the western blot. Recombinant 3D protein was purified and evaluated in the indirect ELISA with 1072 cattle serum samples. Diagnostic sensitivity and specificity of the assay were found to be 92 and 100 %, respectively, when tested with cattle sera of known FMD status. The 3D based ELISA developed here is useful for screening the animals as an adjunct to other NSP based diagnostics available for routine serosurveillance of FMD.

Keywords: 3D; Baculovirus; ELISA; Foot-and-mouth disease virus; Non-structural protein.

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Figures

Fig. 1
Fig. 1
Expression of 3D of FMDV in insect cells. A SDS-PAGE analysis of baculovirus expressed proteins in TN-5 cells. Lane 1 Polyhydrin protein (29 kDa) expressed by AcMNPV (wild type baculovirus). Lane 2 3D protein (53 kDa) of FMDV expressed by recombinant baculovirus in insect cells. Lane M Prestained protein molecular weight marker (MBI, fermentas life sciences). Lane 3 3D protein (53 kDa) fraction purified by nickel affinity chromatography. B Western blotting for expressed 3D protein. Lane 1 3D protein 3D protein (53 kDa) of FMDV expressed by recombinant baculovirus in TN-5 insect cells. Lane M Prestained molecular weight marker (Biorad)
Fig. 2
Fig. 2
Reactivity of the control sera at various dilutions to check the sensitivity of the test system
Fig. 3
Fig. 3
Frequency distribution of the T/N values of serum samples (n = 1072), collected from naïve, infected, vaccinated and field animals collected at random
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