Deciphering mixed infections by plant RNA virus and reconstructing complete genomes simultaneously present...

Area of Science:

• Plant Pathology
• Virology
• Genomics

Background:

• Multiple phylogenetic lineages of rapidly evolving pathogens can co-circulate globally.
• Mixed infections in a single host plant can significantly impact disease outcomes.
• Rice yellow mottle virus (RYMV) is endemic to Africa and a major threat to rice production.

Purpose of the Study:

• To identify distinct RYMV isolates simultaneously infecting a single rice plant.
• To sequence the genomes of these co-infecting RYMV isolates.
• To develop and validate a cost-effective and portable method for analyzing mixed viral infections.

Main Methods:

• Combined direct cDNA Oxford Nanopore Technology (ONT) sequencing with the RVhaplo bioinformatics tool.
• Validated the method using artificial mixes of RYMV genomes with high similarity and varying ratios.
• Applied the method to field samples from Burkina Faso to analyze natural mixed infections.

Main Results:

• Successfully reconstructed two viral genomes differing by <2-3% in length from artificial mixes.
• Identified up to three distinct RYMV isolates within single rice plant samples from Burkina Faso.
• Obtained complete genome sequences for most identified RYMV isolates, enabling diversity and recombination analysis.

Conclusions:

• The developed method efficiently identifies and sequences multiple RYMV haplotypes within a single rice plant.
• This approach provides a rough estimate of relative frequencies and is cost-effective and portable.
• The method holds potential for broader application in endemic regions and for studying other RNA viruses affecting crops worldwide.