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

Abstract

Highlights• The genotype composition of a CTV source was determined.• Four techniques used and results compared.• RT-PCR and Illumina sequencing provided relatively comparable results.• The presence of RB, VT and B165 could be confirmed within the source.• Challenges encountered discussed and solutions proposed. AbstractCitrus 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 2 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.

Viral Metatranscriptomic Analysis to Reveal the Diversity of Viruses Infecting Satsuma Mandarin (Citrus unshiu) in Korea

“…However, its effectiveness has been limited to specific regions or certain varieties and has not been universally applicable. Current research is concentrated on identifying CTV isolates suitable for cross protection, and understanding the characteristics of CTV sources within citrus production regions is particularly crucial ( Kleynhans and Pietersen, 2016 ; Lubbe, 2015 ). Hence, this study holds the potential to contribute significantly to these ongoing efforts.…”

Transmission of Diverse Variants of Strawberry Viruses Is Governed by a Vector Species

“…For mixed virus infections, both antagonistic and synergistic interactions have been described [ 5 ], but strains of the same species might be under greater pressure as they exploit the same replication pathways. Interestingly, antagonistic interactions of strains were applied for plant ‘vaccination’ purposes when plants infected by a mild viral strain were protected from infection with an aggressive one, a phenomenon called ‘cross-protection’ [ 11 , 12 , 13 ]. Based on a similar principle, genetically modified crops expressing viral capsid proteins were developed to introduce resistance to related viruses [ 14 ].…”

“…So far, our current knowledge about multistrain infections is based on studies of citrus tristeza virus (CTV; Closteroviridae ) [ 11 , 12 , 15 ] and potato virus Y (PVY; Potyviridae ) [ 16 , 17 ]. Although infections with more than one strain, or genotype, in a single host organism have been known for some time, recent studies have documented a growing number of cases [ 9 , 10 ].…”

Detection of ‘ancestral’ western lineage of Citrus tristeza virus virulent genotype in declining Arunachal Wakro orange

“…As per the available ICTV taxonomy report, three distinct strains of CTV have been identified, namely the T30 (mild isolate), T36 (intermediate severity), and VT (severe isolate) (Martelli et al 2012). However, the availability of complete genome sequences and detailed phylogenetic analyses has led to the identification of at least eight different CTV genotypes distributed throughout the globe, namely the T36, T30, T3, VT, B165, HA16-5, T68, and the resistance breaking RB (Kleynhans and Pietersen 2016). The genotype of infecting CTV is considered an important factor that determines the magnitude of damage (Moreno et al 2008;Harper 2013).…”