The Effect Of Japanese Encephalitis Virus SL-? Region And NS5 Protein 644 Locus On Viral Pathogenecity

Japanese encephalitis(JE)is an insect-borne zoonotic disease caused by the Japanese encephalitis virus(JEV),with approximately 67 000 cases of infection and 20 000 deaths each year,which have important public health significance.Pigs usually serve as storage hosts before JEV infects humans.After infection,pregnant sows suffer from miscarriage,stillbirths,fetal malformations,and mummified fetuses;boars have orchitis,reproductive disorders,and have a huge impact on the livestock husbandry industry.The 3' non-coding region of flavivirus plays a very important role in the virus life cycle.The study of the RNA elements formed in the non-coding regions of other members of the Flaviviridae family(eg dengue virus,West Nile virus,etc.)revealed the important functions of the SL-? structure and the 3'SL structure in virus replication and the significant impact in lesion and mouse pathogenicity.In addition,flavivirus NS5 protein also plays an important role in virus replication and antagonizing host innate immunity.The RNA-dependent RNA polymerase(RdRp)region of the NS5 protein can specifically recognize the 3' end of the viral genome to initiate the synthesis of the virus.However,the Japanese encephalitis virus is less studied in this regard.Based on the sequence analysis of Japanese encephalitis virus and the comparison of the RNA secondary structure model of the 3' non-coding region,this study will discuss the impact of base mutation of RNA secondary structure element SL-? and the 644 site mutation of NS5 protein in the RdRp region on the replication and pathogenicity of the Japanese encephalitis virus.Using the reverse genetic system to construct a full-length Japanese encephalitis virus infectious clone with a mutated gene,we obtained infective viral RNA by in vitro transcription.After transfected to cells,we rescued the recombinant virus and compared viruses carrying different mutated genes in proliferation,Viral Replication on BHK-21 Cells and Pathogenicity in Mice.This study found that when the 248 th nucleotide in the 3' non-coding region of Japanese encephalitis virus is changed from thymine to cytosine,its genomic 258 th nucleotide will also change from adenine to guanine at the same time;There is a mutation from guanine to adenine in the natural nucleotide at the 254 th nucleotide,but guanine is the main one in nature.The mutated base causes the top loop of the SL-? structure to shrink,while the side loop increases;The replication ability of the recombinant virus which SL-? has mutated gene was significantly up-regulated in BHK-21 cells;SL-? with mutated gene cooperates with NS5 protein with the 644 amino acids(Asn?Thr)in virus replication and in the early period of virus replication,this cooperation will produce a large number of negative viral RNA strands,which greatly enhances the replication of the virus.The recombinant virus with mutated gene in SL-? structure has a remarkable increase in replication ability in cells which also produces cytopathic,however the peak release of the virus was delayed,and the virulence of mice decreased significantly.We complement the study of RNA element function in the 3' non-coding region of Japanese encephalitis virus and provides a new perspective for the future research of yellow virus.This study provides new ideas for the development of novel drugs using flavivirus NS5 protein as a drug target.