Simultaneous visualization of two Citrus tristeza virus genotypes provides new insights into the structure of multi-component virus populations in a host

• A novel ISH technique has been optimized for the detection of a plant virus RNA.
• The first simultaneous in situ visualization of two distinct strains of a plant virus.
• The study provided new insights into the intra-host structure of CTV populations.

Complex Citrus tristeza virus (CTV) populations composed of mixtures of different strains of the virus are commonly found in citrus trees in the field. At present, little is known about how these populations are formed, maintained, and how they are structured within a host. Here we used a novel in situ hybridization approach allowing simultaneous visualization of two different RNA targets with high sensitivity and specificity to examine the distribution of two isolates, T36 and T68-1, representing phylogenetically distinct strains of CTV, in a citrus host in single and mixed infections. Remarkably, in doubly inoculated plants the two virus variants appeared to be well mixed within the infected tissue and showed no spatial segregation. In addition, both CTV variants were often found occupying the same cells. Possible mechanisms involved in shaping CTV populations and the biological significance of the observed lack of structural separation of the individual components are discussed.