High-Throughput Sequencing Reveals the Genomic Characterization of Hibiscus Chlorotic Ringspot Virus and Enables RT-PCR Detection

Hibiscus chlorotic ringspot virus (HCRSV) is a major pathogen of Hibiscus rosa-sinensis, a globally valued ornamental plant. Here, we report the first complete genomic characterization of HCRSV isolates from India, achieved through high-throughput sequencing and further validated by RT-PCR. Two isolates, HRS1 (3.888 kb) and HRS2 (3.913 kb), shared 93.3 and 92.63% nucleotide identity, respectively, with the reference genome. Comparative mutational profiling revealed 264 mutations in HRS1 (185 transitions, 76 transversions, 1 insertion, and 2 deletions) and 233 in HRS2 (150 transitions, 77 transversions, 4 insertions, and 2 deletions). Phylogenetic analyses placed the Indian isolates in a distinct clade closely related to isolates from South Africa and China, while recombination analysis identified four recombinant events involving sequences from Singapore, Colombia, and Brazil. Further, to facilitate rapid diagnosis, we established sensitive one-step and two-step RT-PCR assays, enabling efficient detection of HCRSV in field samples. Screening of 19 symptomatic leaves confirmed viral presence and correlated infection with typical viral symptoms. To our knowledge, this is the first report of the complete genome sequences of HCRSV from India and introduces a novel one-step RT-PCR assay for its global detection, together expanding our understanding of HCRSV evolution and epidemiological surveillance.