Comparison of multiple viral population characterization methods on a candidate cross-protection Citrus tristeza virus (CTV) source

Citrus tristeza virus (CTV) is the most economically important virus found on citrus and influences production worldwide. The 3' half of the RNA genome is generally conserved amongst sources, whereas the 5' portion is more divergent, allowing for the classification of the virus into a number of genotypes based on sequence diversity. The acknowledged genotypes of CTV are continually being expanded, and thus far include T36, T30, T3, VT, B165, HA16-5, T68 and RB. The genotype composition of the CTV populations of a potential cross protection source in Mexican lime was studied whilst comparing different techniques of viral population characterization. Cloning and sequencing of an ORF 1a fragment, genotype specific RT-PCRs and Illumina sequencing of the p33 gene as well as RNA template enrichment through immuno-capture was done. Primers used in the cloning and sequencing proved to be biased towards detection of the VT genotype. RT-PCR and Illumina sequencing using the two different templates provided relatively comparable results, even though the immuno-captured enriched template provided less than expected CTV specific data, while the RT-PCRs and p33 sequencing cannot be used to make inferences about the rest of the genome; which may vary due to recombination. The source was found to contain multiple genotypes, including RB and VT. When choosing a characterization method, the features of the virus under study should be considered. It was found that Illumina sequencing offers an opportunity to gain a large amount of information regarding the entire viral genome, but challenges encountered are discussed.