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Department of Virology, Sri Venkateswara University, Tirupati-517 502, A. P., India(Received on December 9, 2009; Accepted on February 13, 2010)Papaya ringspot virus (PRSV) causes the most wide-spread and devastating disease in papaya. Isolates ofPRSV originating from different geographical regionsin south India were collected and maintained on naturalhost papaya. The entire coat protein (CP) gene of Papayaringspot virus-P biotype (PRSV-P) was amplified by RT-PCR. The amplicon was inserted into pGEM-T vector,sequenced and sub cloned into a bacterial expressionvector pRSET-A using a directional cloning strategy.The PRSV coat protein was over-expressed as a fusionprotein in Escherichia coli. SDS-PAGE gel revealed thatCP expressed as a ~40 kDa protein. The recombinantcoat protein (rCP) fused with 6x His-tag was purifiedfrom E.coli using Ni-NTA resin. The antigenicity of thefusion protein was determined by western blot analysisusing antibodies raised against purified PRSV. Thepurified rCP was used as an antigen to produce hightiter PRSV specific polyclonal antiserum. The resultingantiserum was used to develop an immunocapturereverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels withELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test follow-ed by dot-blot immunobinding assay (DBIA) and platetrapped ELISA.Keywords :IC-RT-PCR, Papaya ringspot virus, polyclonalantibodies, recombinant coat proteinThe papaya and watermelon biotypes of Papaya ringspotvirus (PRSV-PW Varma, 1988). The disease hasbecome a major threat to papaya cultivation throughoutIndia by rendering orchards economically unproductive.PRSV was first reported in western India in 1958 (Capoorand Vrama, 1958) and since then it has spread to differentgeographical locations irrespective of the agro-climaticconditions result in crop losses of up to 85-90% (Hussainand Varma, 1994). PRSV, a definitive member of the genusPotyvirus of the family Potyviridae (van Regenmortel,2000), is non-persistently transmitted by numerous speciesof aphids in nature and particles are flexuous filamentousrods of about 780×12 nm in size (Gonsalves and Ishii,1980). The virions contain a positive sense, single strandedRNA genome approximately 10.3 kb in length surroundedby a coat protein (CP) consisting of a single protein subunitof molecular weight 36 kDa (Yeh et al., 1992). Based on thehost range specificity, PRSV is classified into two types thatcannot be distinguished by serological methods. PRSV typeP infects papaya and has a limited experimental host rangein cucurbits. PRSV type W infects wide range of economi-cally important cucurbit crops, but, it does not infect papaya(Purcifull et al., 1984). Although both types are serologi-cally related, observations suggested that papaya is themajor source for the spread of PRSV type P (Yeh et al.,1984).The efficient production of good quality antiserum topotyviruses is an uphill task under conventional methodsdue to difficulty to purify sufficient quantities of theirfilamentous particles. The virus particles are relatively lowstable and they tend to aggregate with plant debris oramong the elongated virus particles themselves. This leadsto considerable losses of virus during the purification pro-cess, thereby requiring only gentle and highly labor-inten-sive purification methods, with special attention to removalof plant proteins. The purification of PRSV from papayaleaf was shown to be difficult as papaya latex causesaggregation of virus particles during purification steps. Athigher temperatures, especially in south Indian climaticconditions, propagation of PRSV on alternative cucurbi-taceous plants for purification is also not practical. Poly-clonal antibodies raised against purified virions contaminat-ed with host tissue components, cross react with hostantigens and often give variable background reactions, thuslimiting their use in ELISA based diagnostic methods. Thedifficulties associated with the development of antibodiesof good quality against virions purified by conventional

plant pathology journal/the plant pathology journal

Development of Recombinant Coat Protein Antibody Based IC-RT-PCR and Comparison of its Sensitivity with Other Immunoassays for the Detection of Papaya Ringspot Virus Isolates from India