Rescue And Pathogenicity Study Of Tembusu Virus Mutant Strain RMM1775-ED?-N2S

Tembusu virus could cause a large decrease in egg production,accompanied by a decrease in appetite,motor disorder,high fever and other clinical symptoms,with a mortality rate of 5%~20%.Around 2010,the disease was widespread in Zhejiang,Shanghai and Jiangsu in the south of China,causing huge economic losses in the duck industry.So far there has not been effective treatment method,and vaccination is the main preventive and control measure against Tembusu.The pathogenicity of the strain FX2010 isolated from Feng Xian in Shanghai in 2010 was significantly different from the strain MM1775 early isolated in Malaysia.MM1775 was more pathogenic to mice than FX2010.Previous studies have shown that there are nine amino acid differences in the Domain I region(EDI)of E protein.In order to understand the relationship between the second amino acid in the EDI region of the Tembusu virus and the pathogenicity of the virus,a single point mutant plasmid was constructed using primer site-directed mutagenesis,that results in the second amino acid of EDI was changed from the original asparagine(N)of MM1775 strain to the serine(S)which is same as the FX2010 strain.The mutant plasmid and other three plasmids of MM1775 strain were used as templates to perform PCR amplification in vitro.Four segments of PCR product were obtained and then fused.A full-length cDNA was prepared and transcribed in vitro into RNA with infection effect.Then the RNA was used to transfect DF-1 cells and the single-point mutant virus rMM1775-EDI-N2 S was rescued.The mouse infection test showed that the rescued rMM1775-EDI-N2 S mutant virus still showed high pathogenicity which was same as the original virus rMM1775,indicating that the second amino acid in the EDI region of Tembusu virus was not the Amino acid sites that caused differences in pathogenicity between FX2010 and MM1775 in mice.The results of this study have a certain reference value for the further in-depth study of the pathogenicity and vaccine of the Tembusu virus.