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[Objective] A genetically engineered virus strainrGS15-∆2 with deletion of the residues at the positions 519–565 and 628–747 of the non-structural protein 2 (NSP2) had been rescued based on the PRRSV/GSWW/2015 infectious clone. This study aims to construct and rescue an engineered virus strain with the deletion of three sites in NSP2 based onrGS15-∆2. [Methods] Based on the infectious clones ofrGS15-∆2, two recombinant plasmids with the deletion of three sites were constructed by fusion PCR. Specifically, the dominant epitope at the amino acid site 323–364 or 372–433 of NSP2 was further deleted on the basis ofrGS15-∆2. The recombinant plasmids were linearized and mixed with liposome, which were transfected into Marc-145 cells for virus rescue. The growth characteristics of the engineered virus strains were analyzed by electron microscopy, immunofluorescence assay (IFA), virus titer determination, and growth curve establishment. [Results] The engineered virus strainsrGS15-∆3-1 andrGS15-∆3-2 were rescued successfully. Virions with the diameter from 50 nm to 80 nm can be observed under an electron microscope. The results of IFA confirmed the expression of PRRSV N protein by the rescued virus strains and the parent strain GS15. Furthermore, the rescued viruses were cultured with Marc-145 cells for 40 passages, and the deletion regions were confirmed to be stable by RT-PCR and sequencing. The titers ofrGS15-∆3-1 andrGS15-∆3-2 were 2.00×106.0 TCID50/mL and 2.25×105.8 TCID50/mL, respectively, which had differences from that of the parent strain (P < 0.05). The growth curves showed that the rescued viruses had lower replication levels than the parent strain, and they reached the peak titers 24 h later than the parent strain. [Conclusion] We characterized the growth of the viruses with the deletion of multiple sites in NSP2 of PRRSV. The findings laid a foundation for the development of novel PRRSV-labeled vaccines and provided a new strategy for the prevention and control of porcine reproductive and respiratory syndrome.
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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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