Citrus yellow vein clearing virus (CYVCV) is a novel virus that poses a significant threat to the citrus industry. Currently, there is no effective therapeutic agent. The primary strategies for prevention and management involve utilizing virus-free seedlings and implementing stringent control measures against insect vectors. The objective of this study is to investigate the technology of virus-induced gene silencing (VIGS) as a means of developing antiviral “vaccines” for CYVCV, and to offer innovative approaches for the prevention and management of citrus viral diseases.

Based on the previously constructed VIGS vector pCLBV201 in the laboratory, a series of recombinant vectors were designed and developed to target the conserved regions of the CYVCV genome, specifically open reading frame 1 (ORF1), open reading frame 6 (ORF6), coat protein (CP), and triple gene block (TGB). Agrobacterium-mediated vacuum infiltration was inoculated on Eureka lemon, followed by RT-PCR detection. After acquiring several positive plants including pCLBV201-ORF1, pCLBV201-ORF6, pCLBV201-CP, and pCLBV201-TGB, CYVCV infectious clones were inoculated via Agrobacterium-mediated injection, with plants infiltrated with the pCLBV201 empty vector serving as controls. Subsequent RT-qPCR, Western blot analysis, symptom observation, and disease index statistics were conducted to elucidate the preventive and control effects of the various VIGS recombinant vectors on CYVCV.

A series of recombinant vectors of pCLBV201 were constructed, and the results of RT-PCR following inoculation indicated that multiple positive plants for pCLBV201-ORF1, pCLBV201-ORF6, pCLBV201-CP, and pCLBV201-TGB were obtained, respectively. CYVCV infectious clones were inoculated by injection. The results of RT-qPCR at 7, 14, 28, 70, and 150 dpi (days post infection) showed that the relative expression of CYVCV in the pCLBV201-CP and pCLBV201-TGB treatment groups was significantly lower than that in the control group. Western blot analyses conducted at 70 and 150 dpi showed a significant decrease in the expression of CP protein in the pCLBV201-CP and pCLBV201-TGB treatment groups. Observational symptoms indicated that the control group exhibited typical manifestations of CYVCV infection, such as severe vein clearing and leaf distortion, whereas the pCLBV201-CP treatment group displayed mild symptoms, and the pCLBV201-TGB treatment group showed no apparent symptoms. The disease index statistics at 70 and 150 dpi demonstrated that the disease indices of pCLBV201-CP and pCLBV201-TGB were the lowest, recorded at 17.6, 41.2 and 15.6, 29.1, respectively, while the control group had indices of 52.1 and 80.0, showing obvious differences.

The VIGS-based technology for the prevention and control of CYVCV was developed, which clarified that pCLBV201-CP and pCLBV201-TGB could significantly reduce the virus titers and alleviate the symptoms caused by the virus. They have the potential to be used as “vaccines” for CYVCV prevention and control.