Expression of Peste des petits ruminants virus immunogenic protein in Baculovirus system and evaluation of its diagnostic Potential

Abstract

The present study was envisaged for the expression of the Peste des petits ruminants virus newline(PPRV) immunogenic protein(s) in the baculovirus system and its use as a diagnostic antigen in newlineELISA as an alternative antigen to either prokaryotic E. coli expressed recombinant PPRV newline newlineprotein or the whole virus live antigen to avoid the use PPRV antigen in routine ELISA for large- newlinescale field application of these assays for surveillance and monitoring of PPR. newline newlineAs a part of the collection of the review of the literature, apart from the proposed research work, newlinea meta-analysis on the prevalence of PPR using the global data of 20 years on the role of PPR in newlinewildlife and atypical/unnatural hosts (bovine/camel) and its possible interference in the PPR newlineGlobal Eradication Program (PPR-GEP) was undertaken, which revealed a widening host range newline newlineof PPRV in recent years and its capacity to adapt to the multi-host system; nevertheless, sero- newlinesurveillance at the PPR eradication stages demands an assay free from PPRV antigen in the newline newlineemployed diagnostic screening assays. newlineThe hemagglutinin (H) and nucleocapsid (N) partial gene sequences of the PPR virus were newlinecloned into the pFastBac HT baculo vector, and the recombinant clones were characterized by newlinecolony PCR, restriction digestion, and sequencing. The recombinant pFastBac HT vector newlinecontaining the partial H and N genes of PPRV as recombinant clones were further transferred to newlineDH10Bac E. coli host separately for respective bacmid formation. The recombinant bacmid newlineclones were confirmed for the presence of the target gene by gene-specific and vector-specific newlinePCRs. The confirmed and selected bacmid clones DNA was transfected into insect cell lines newline(Sf- 21) separately, for the generation of recombinant baculovirus expressing the target cloned newlinegene product. Further, the Sf-2l cells infected with recombinant baculovirus at passage level-3 newline(P3) were harvested after 72 hours of infection, and plaque purified. Further, Tn5 insect cells newlinewere infected with P3 recombinant baculovirus at ten multiplicity of infection (m.o.i) to produce newlinerecombinant protein. The crude recombinant protein as cell lysate/Ni-NTA affinity-purified newlineprotein was characterized by SDS-PAGE and immunoblot using standard PPRV-specific newlinestandard sera.The rPPRV-NP/HP with a molecular weight of ~ 30 kDa/ ~43 kDa was expressed newlinein an insect cell system using generated recombinant baculovirus. The produced recombinant newline newlineprotein(s) appear to be specific in their native form as they respond with monoclonal, polyclonal, newlineand antiserum antibodies produced against PPRV/PPRV-specific serum. newlineFurther, the study describes the expressed rPPRV-NP use in ELISA as a diagnostic antigen.The newlinecrude protein as harvested cell lysate rPPRV-NP as a diagnostic antigen was evaluated as a newlinecoating antigen in the PPR Ab Chek kit and as a standard positive control antigen in the PPR Ag newlineChek kit/PPR s-ELISA kit as a substitute/replacement for PPRV antigen. Further, sera (n=220) newline newlinefrom small ruminants were employed to assess the diagnostic performance of PPR rBNP-c- newlineELISA baculovirus-expressed (PPRV-NP) in the insect cells used as a coating antigen. The assay newline newlineresults showed a relative diagnostic sensitivity of 87.41 % (95 % CI: 80.61 92.49 %) and 90.08 % newline(95 % CI: 83.63 94.61 %) and diagnostic specificity of 96.47 % (95 % CI: 90.03 99.27 %) and newline92.13 % (95 % CI: 84.46 96.78 %) against indigenous PPR competitive ELISA kit and PPR Ab newlineChek kit, respectively. newlineAdditionally, the present survey also provides information on the status of PPRV antibodies in newlinesheep and goats and associated possible host factors with the variation in the immunity levels newlineobserved in the epi-unit (village) level in the studied region of Odisha with the observed 64 % of newlinepopulation immunity or post-vaccination antibodies response. newlineThe use of this recombinant protein in diagnostics assays can provide a reliable source of secure newlinediagnostic reagents in the future. The study showed that the expressed rPPRV-NP in the newlinebaculovirus system could be an alternative diagnostic antigen to E. coli-expressed rPPRV-NP. newlineThe utility of this rPPRV-NP avoids using live PPRV antigen in routine ELISA diagnostics. In newlinethe future, it will be helpful for the large-scale field application of these recombinant assays for newlinesurveillance and monitoring of PPR during the eradication and post-eradication phases in newlineendemic or non-endemic countries. newline