Citrus tristeza virus: Molecular characterization of isolates for use in mild strain cross protection, localization of the 5'-terminus and heterologous encapsidation

The purpose of this research was to validate the use of recently developed methods which permit selective differentiation of mild and severe strains of citrus tristeza virus (CTV) to speed the selection of mild CTV strains potentially useful for mild strain cross protection (MSCP) against citrus tristeza virus. The molecular evaluation for differentiation of CTV strains can be applied for the detection of mild and severe strains from the original in planta culture collected from surviving trees in CTV infected areas as well as from single aphid transmitted sub-isolates. The application of these methods of CTV strain differentiation should enable faster and better selection of mild strains which may be useful for MSCP programs.;The localization of the capsid protein (CP) and minor CP in relation to the genomic RNA termini of purified CTV virions in planta was examined, regarding the polar architecture of CTV virions, by specifically immunocapturing one termini with polyclonal antisera prepared against the bacterial expressed CTV minor CP, p27. Immuno-capture reverse transcriptase-PCR (IC-RT-PCR) conditions were optimized for the amplification of the first 510 nt at the 5'terminus, and the last 899 nt at the 3 '-terminus of the CTV genome. The 5'-terminus of CTV was consistently amplified when virions were immunocaptured using the p27 CP specific antiserum. CTV was purified, sonicated to fragment the virions, and then fractionated on a 10--40% rate zonal sucrose gradient. Gradient fractions were tested by ELISA and used to recover the fragments which were then tested for termini amplification by IC-RT-PCR. The results demonstrate that the minor CP, p27, encapsidates the 5'terminus of the CTV genomic RNA.;Heterologous encapsidation studies were performed by inoculating two mild isolates of CTV, T30a and T11a, into transgenic citrus plants which were expressing the CP gene of a T36 decline isolate of CTV. Heterologous encapsidation of the genome of the mild isolates by the CP of the severe strain, T36, was examined by using the MCA13 antisera specific to severe Florida isolates, and by selectively immunocapturing virions by the p27 CP using the p27 specific antiserum, UF 34, then IC-RT-PCR followed by strain specific hybridization probes. Two of twenty six plants were MCA13 positive, showing heterologous encapsidation between virions and the T36 CP expressed in planta.