Molecular Characteristics And The Function Against White Spot Syndrome Virus Infection Of NELF-B And NELF-E In Cherax Quadricarinatus

White spot syndrome virus(WSS V)has a wide range of hosts,strong infectivity and high fatality rate,which has caused huge economic losses to the global shrimp breeding industry.In view of the fact that the pathogenesis of WSS V and the antiviral immune mechanism of the host are still not fully understood,so far no effective method has been found to cure WSSV NELF plays an important role in regulating the expression of genes related to cell cycle control,proliferation,metabolism,DNA repair and cell response to stimulation and stress.At the same time,the transcription extension of NELF is also a key link in the gene expression of pathogenic virus.However,whether NELF has antiviral function in invertebrates has not been reported.In this study,the cDNA sequences of negative elongation factors B and E(named CqNELF-B and CqNELF-E)were cloned from Cherax quadricarinatus.The partial sequence of CqNELF-B cDNA was 2107 bp,including ORF 1743 bp,5'-UTR 202 bp and 162 bp 3'-UTR,encoding 580 amino acid residues and contains a COBRA-1 domain.The full length sequence of CqNELF-E cDNA is 1753 bp,including ORF 816 bp and 3'-UTR 744 bp,encoding 271 amino acid residues,including a homeobox associated leucin zipper(HALZ)and RNA recognition motif(RRM).Multiple sequence alignment and phylogenetic tree construction showed that CqNELF-B and CqNELF-E had high homology with shrimp and crab crustaceans.The results of tissue distribution showed that CqNELF-B and CqNELF-E were expressed in different tissues,the expression of CqNELF-B and CqNELF-E were the highest in muscle,but low in gill,heart and intestine.In addition,WSSV infection test showed that the mRNA transcription level of CqNELF-B was significantly up-regulated at 6 h after WSSV infection,and similarly,the mRNA transcription level of CqNELF-E was significantly up-regulated at 6 h and 12 h after WSSV infection.Furthermore,RNA interference assay showed that the transcription levels of IE1 and VP28 of WSSV were significantly up-regulated at 3 h,6 h and 12 h after knocking down CqNELF-B and CqNELF-E,respectively,indicating that CqNELF-B and CqNELF-E have potential functions in antiviral immunity.Considering the role of CqNELF-B and CqNELF-E in regulating gene transcription,we performed double luciferase reporter gene detection,and found that the overexpression of CqNELF-B and CqNELF-E genes in SF9 cells significantly inhibited the initiation efficiency of extremely early gene promoters(WSV051,IE1 and WSV083)of WSSV.In conclusion,CqNELF-B and CqNELF-E play an important role in the immune defense of WSSV in red claw crayfish,and provide a potential drug target for the treatment of crustacean diseases caused by WSSV.