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A reverse genetics platform for foot-and-mouth disease virus (FMDV) is an indispensable tool for studying the pathogenic mechanism, protein function, and vaccine development. However, the conventional method of constructing infectious clones of FMDV is usually laborious, time-consuming, and costly. Objective To establish a new reverse genetics platform for rapid rescue of FMDV based on infectious subgenomic amplicons (ISA), which can avoid in vitro ligation and bacterial cloning. Methods The whole gene of FMDV O/GDLeiZh/2020 strain was divided into five overlapping fragments and then individually amplified by high-fidelity PCR. The T7 promoter sequence was added to the 5′-end gene and the poly(A) tail was introduced at the 3′-end. At the same time, the poly(C) sequence and molecular markers were introduced by fusion PCR. Two large fragments covering the whole gene of FMDV were obtained by multiple rounds of fusion PCR amplification and co-transfected into BSR/T7 cells expressing T7 RNA polymerase. The cell supernatant was collected 72 h post-transfection. The rescued virus was identified and characterized by RT-PCR, indirect immunofluorescence, electron microscopy, plaque assay, and one-step growth curve assay. Results The typical cytopathic effect of FMDV was observed 60 h post-transfection. Sequencing, immunofluorescence, and electron microscopy collectively confirmed that infectious FMDV was successfully rescued. Furthermore, one-step growth curve and plaque assays demonstrated that the rescued virus retained replication kinetics and biological characteristics comparable to those of the wild-type virus. Conclusion This study successfully establishes a new method for rapid and efficient rescue of FMDV based on ISA, which will lay a solid foundation for further improving FMDV rescue technology and rapidly expanding its application in the future.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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