Differentiation and Validation of Mild and Severe Strains of Citrus tristeza Virus through Codon Usage Bias, Host Adaptation, and Biochemical Profiling

Halima Khatoon¹Dharmappa D Chavan¹Vijay Kamal Meena²Abhijeet Shankar Kashyap³Kumar Sathiyaseelan¹Marimuthu Elangovan¹Lalit Patil¹Bharat Raj Meena¹Aarti Gauns⁴Utpal Kumar Bhattacharyya⁵Kajal Kumar Biswas^1*

• ¹Indian Agricultural Research Institute (ICAR), New Delhi, India
• ²Agriculture University Jodhpur, Jodhpur, India
• ³ICAR - National Bureau of Agriculturally Important Microorganisms, Mau, India
• ⁴Goa College of Agriculture, Ella Farm Old Goa, goa, India
• ⁵ICAR Research Complex for NEH Region, Umiam, India

Citrus tristeza virus (CTV) is one of the most economically significant citrus pathogens, causing epidemics worldwide. It comprises strains ranging from asymptomatic mild variants to highly virulent severe forms. However, the molecular basis distinguishing mild from severe strains remains poorly characterised. To understand these mechanisms, a total of 58 citrus samples were screened using RT-PCR targeting the coat protein gene, with 47 testing positive for CTV. Analysis of codon usage bias (CUB) revealed that both mutation pressure and natural selection influence codon preferences, with natural selection playing the dominant role. Hierarchical clustering dendrogram based on Effective Number of Codons (ENc) values reveals 20 mild-like isolates and 27 severe strains. Among the mild-like group, 9 mild strains exhibited higher Codon Adaptation Index with uracil at the third codon position and Relative codon usage patterns closely aligned with the Host, suggesting greater translational efficiency and reduced virulence. Quantitative RT-PCR confirmed lower viral accumulation in plants infected with mild strains, with a ninefold increase observed in those infected by severe strains. Validation through biological indexing confirmed the mild nature of these isolates and their ability to confer cross-protection upon challenge with severe strains. Phylogenetic relationships and sequence identity metrics indicated a close genetic association of mild strains with the VT strain, highlighting their genetic relatedness. Additionally, biochemical profiling revealed distinct patterns in sugar, phenol, antioxidant, and chlorophyll levels across mild, severe, and healthy plants. These findings highlight the promise of mild, well-adapted CTV strains as effective agents for cross-protection in citrus orchards.