猪流行性腹泻病毒Nsp9突变毒株的构建及生物学特性分析OA

The nonstructural protein Nsp9 of porcine epidemic diarrhea virus(PEDV)plays an impor-tant role in viral replication,and its conserved cysteine residue at position 59 is involved in protein dimerization and RNA binding in vitro.This study aimed to investigate the impact of this site on PEDV replication capacity by constructing a recombinant virus with a mutation at the 59th amino acid of Nsp9 from cysteine(C)to alanine(A).Using CRISPR/Cas9-mediated reverse genetics technology and based on the backbone of a PEDV G2b epidemic strain,a recombinant virus was constructed with a mutation at the 59th amino acid of Nsp9.Through indirect immunofluorescence,Western-blot,and viral growth curve as-says,the infectivity,protein expression,and replication characteristics of the mutant virus were systematically analyzed to evaluate the impact of this mutation on PEDV replication capacity.The re-combinant virus rAH2012/12-Nsp9-C59A was successfully obtained.It could be stably passaged in Vero cells and effectively induced the expressed of PEDV N protein.Growth kinetics showed that the early replication trend of this recombinant mutant was generally consistent with that of the parental strain,while the viral titer in the later stages of infection was higher than that of the parental strain.This study revealed the impact of the cysteine mutation at position 59 of PEDV Nsp9 on viral replication in Vero cells,providing new insights for a deeper understanding of the function of PEDV Nsp9.