Shanshan Liu

Plant viruses are major plant pathogens and account for nearly half of emerging plant diseases worldwide. To date, few effective management measures are available for the efficient control of plant viral diseases. Cross-protection based on attenuated vaccine is an effective strategy to prevent plant viral diseases. The primary requirement for the development of attenuated vaccines is a vector with excellent characteristics, such as low pathogenicity, genetic stability, and the capacity for stable insertion of exogenous fragments. In this study, the cucumber mosaic virus (Fny strain) was genetically modified to serve as an attenuated vaccine vector. Based on pre-termination of the 2b protein-coding region and deletion of the 3′ UTR in CMV RNA2, six RNA2 mutants, designated R2-2bPTI, R2-2bPTII, R2-2bPTIII, R2-2bPTIV, R2-2bPTV, and R2-2bPTVI, were constructed. Experiments with different lengths tobacco phytoene desaturase (PDS) fragment insertion evaluated the capacity of each mutant to accommodate exogenous fragment. Based on the R2-2bPTIV, a viral fragment insertion mutant R2-2bPTIV-TMPYTVPX targeting cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), tobacco vein banding mosaic virus (TVBMV), and potato virus X (PVX) was constructed. This mutant provided effective cross-protection against these targeted virulent viruses. This study developed a series of CMV-based vaccine vector, providing materials and data for the development of plant viral diseases attenuated vaccines.

Plant viruses can infect various types of plants, including food and oil crops, and ornamental flowers, threatening agricultural production and food supply. Cross-protection is an efficient strategy against severe viral strains. Due to distinct infection mechanisms, cross-protection cases involving RNA viruses and DNA viruses often rely on the utilization of corresponding attenuated strains for control purposes. In this study, we utilized cucumber mosaic virus (CMV), a member of the RNA virus group, as the foundational framework for developing attenuated vaccines. We developed four vaccines by inserting relevant sequences from tomato yellow leaf curl virus (TYLCV), a DNA virus. All vaccines demonstrated effective prevention against TYLCV infection, with relative control efficacies exceeding 80%. Subsequently, we evaluated the preventive effects of these vaccines on mixed infections of CMV and TYLCV. Our findings demonstrated that CMV (R2-2bPTI-TYC1C4), CMV (R2-2bPTII-TYC1C4), and CMV (R2-2bPTIII-TYRep) displayed significant efficacy in preventing mixed infections. Following pre-inoculation with these vaccines, the disease index of tomato plants decreased from 100 to 56. This work provides theoretical foundations and tangible resources for controlling TYLCV through cross-protection while suggesting a feasible strategy for utilizing weak RNA virus vaccines to control DNA viruses.

Some debilitating mutations in RNA viruses are repairable; however, the triggering factors of mutation repair remain largely unknown. In this study, multiple triggering factors of mutation repair are identified based on genetic damage to the TLS in CMV. TLS mutations in different RNAs distinctively impact viral pathogenicity and present different types of mutation repair. RNA2 relative reduction level or RNA3 sequence change resulting from TLS mutation is correlated with a high rate of mutation repair, and the TLS mutation of RNA1 fails to be repaired at the high inoculum dose. However, the TLS mutation of RNA1 can be repaired at a low dose of inoculation, particularly around the dilution end-point or in the mixed inoculation with RNA2 having a pre-termination mutation of the 2b gene, an RNAi suppressor. Taken together, TLS mutations resulting in quality or quantity defects of the viral genome or TLS mutations at low doses around the dilution end-point are likely to be repaired. Different levels of TLS mutation repair necessarily require cell-to-cell movement, therefore implying its obligated effect on the evolution of low-fitness viruses and providing a new insight into Muller’s ratchet. This study provides important information on virus evolution and the application of mild viral vaccines.

Cucumber mosaic virus (CMV), the type species of the genus Cucumovirus in the family Bromoviridae, possesses a tripartite, positive-sense RNA genome. CMV is mainly transmitted by more than 80 species of aphids (Gallitelli, 2000) and infects more than 1000 species including a lot of vegetables such as cucumber, tomato and pepper (Roossinck, 2001). Different satellite RNAs (satRNA) associated with CMV have been identified and most of them attenuate symptoms induced by CMV (Simon et al., 2004). Although CMV was reported to infect cabbage in Bejing city of China, the sequences and function of its associated satRNA was undefined. In summer of 2016, celery cabbage samples with severe mosaic symptoms were collected from Tai'an city in Shandong province of China to detect potential CMV and its associated satRNA. CMV was detected from these samples using RT-PCR and western blot. To identify the potential existence of CMV in these samples, total RNAs were extracted from cabbage leaves using Trizol reagent, and then RT-PCR was performed using primers CM-R3-119-F (5-AGGCATGGCTTTCCAAGGTAC-3) and CM-R3-768-R (5-CTGACGGTTTCGTTTGCTCAGC-3), which define a fragment between positions 119-768 of CMV RNA3 (AY429432) . A PCR product of 650 bp was amplified and inserted into pMD18-T (Takara) followed by sequencing two individual clones using universal primers M13F . Through the BLAST analysis on NCBI website, sequences of the 650 bp PCR product shared highest similarity with CMV RNA3. This partial CMV RNA3 sequence from cabbage in Tai'an city was submitted to GenBank obtaining accession number KY646100 as TA-ca isolate. It shared 94.15% identity with RNA3 (D10538) of CMV Fny isolate and 96.92 % identity with RNA3 (AY429432) of CMV Ca isolate . In addition, western blot was performed to detect the coat protein (CP) of CMV. CMV CP with molecular weight of 24 kD can be detected from corresponding diseased cabbage leaves using monoclonal antibody of CMV CP (Du et al., 2013) as specific antibody at 1:8000 dilution. With the same RNA samples, RT-PCR of satCMV was performed using degenerate primers satCMV-int-5F (5-GCGGTGTGGGWTAMCTCC-3, W=A/T, M=C/A) and satCMV-int-3R (5-GCYTCTCCCTGYGTGCG-3, Y=T/C), which were designed based on the alignment of 173 reported satCMV in GenBank. About 220 bp PCR product was detected. The purified fragment was cloned into pMD18-T (Takara) and sequenced using universal primers M13F for two individual clones. Through the BLAST analysis on NCBI website, sequences of 223 bp PCR product shared highest similarity with satCMV. This partial satCMV sequence from cabbage in Tai'an city was submitted to GenBank obtaining accession number KY747451 as TA-ca isolate. It shared 95.52% identity with satCMV China-5 isolate (AJ236905) and 95.07 % identity with satCMV FFQT1-3 isolate (HQ283395). To our knowledge, this is the first report of CMV naturally infecting celery cabbage in Shandong province of China. In addition, this is the first report of CMV-associated satRNA naturally infecting celery cabbage in China. Since satCMV can adjust symptoms caused by CMV, it is necessary to clone the full length genome of satCMV TA-ca isolate and test its effect on symptoms caused by CMV in the near future. It will provide substantial material and data for management of disease caused by CMV.