Seasonal dynamics of citrus yellow vein clearing virus using HTS and RT-qPCR reveal correlation between viral copy number and symptom expression

Citrus yellow vein clearing virus (CYVCV) is a positive-sense, single-stranded RNA virus belonging to the family Alphaflexiviridae that poses a major threat to citrus production worldwide and is widespread in Kinnow mandarin orchards. Despite its prevalence, seasonal fluctuations in CYVCV infection remain poorly understood. This study examined the year-long (2024) temporal-dynamics of CYVCV in Kinnow plants confirmed to be singly-infected and free from other viruses, using symptom monitoring, high-throughput sequencing (HTS), and RT-qPCR. Leaves were collected quarterly (February, May, August, and November) from CYVCV-infected plants maintained at IARI, New Delhi. HTS confirmed CYVCV in symptomatic samples revealed clear seasonal trends and viral abundance, with sequencing depth and FPKM values peaking in February and November, while the virus was nearly undetectable in May and August months. Single nucleotide variant (SNV) analysis further highlighted genetic variability in viral populations across seasons. RT-qPCR analysis corroborated HTS results and revealed clear seasonal variation in viral load, with higher accumulation during cooler months and minimal detection during warmer periods. A strong association between viral load and symptom expression was observed. Additionally, a sensitive and quantitative RT-qPCR assay was developed and validated for reliable detection of CYVCV. This first detailed report of CYVCV seasonal dynamics provides critical insights for optimizing disease management and improving certification programs in Kinnow mandarin orchards. • Seasonal dynamics of citrus yellow vein clearing virus (CYVCV) were quantified across four climatic phases using integrated high-throughput sequencing (HTS) and RT-qPCR. • CYVCV RNA titres showed pronounced seasonal fluctuation, with strong suppression during summer months (May-August) coinciding with absence of visible symptoms. • Seasonal variation in CYVCV accumulation was observed and showed association with symptom expression under field conditions. • Combined molecular quantification and symptom assessment demonstrate a clear association between viral load, host phenology, and seasonal environmental conditions, informing optimal diagnostic and certification strategies.