Study On The Molecular Mechanism Of Marek’s Disease Virus-Encoded LORF9 In Viral Pathogenesis

Marek’s Disease(MD)is an immunosuppressive and neoplastic disease caused by Marek’s Disease Virus(MDV)infection in chickens.The genome size of MDV is about 177 kb,which consists of a long unique sequence(UL)region and a short unique sequence(US),as well as reverse repeats(TRL and IRL)at both ends of UL and reverse repeats(TRS and IRS)at both ends of US.Genes located in UL and US regions have high homology with other herpes viruses(HSV-1,VZV),but the functions of most genes are still unknown.Among them,LORF9 is a unique gene of MDV.Early studies have shown that the deletion of LORF9 in extra-strong MDV strains will reduce the pathogenicity of the virus,but the molecular mechanism of its involvement in the pathogenesis of MDV is still unclear.In order to further clarify the pathogenicity of LORF9 in other virulent strains,a mutant strain with LORF9 gene deletion and recovery was constructed by using Bacterial Artificial Chromosome technology(BAC)with very virulent strain Md5 as the parent strain.In order to explore the pathogenic effect of LORF9 gene-deleted strains in vivo,one-day-old SPF chickens were randomly divided into four groups,and 2000 PFU of virus was inoculated subcutaneously in the neck.The viral copy number of spleen DNA was detected by fluorescence quantitative PCR(q PCR)at 5,7,14 and 60 days after infection.The replication level of Md5BAC△LORF9 was significantly lower than that of Md5 BAC and Md5 BAC △ LORF9-Re.There was no significant difference between thymus and bursa of fabricius atrophy and uninfected group.After60 days’ death monitoring of all control groups and infected SPF chickens,it was found that the negative control group did not get sick,and the morbidity and mortality of Md5 BAC △ LORF9 group were 23.8% and 14.2%,while the morbidity and mortality of SPF chickens infected with Md5 BAC and Md5BAC△LORF9-Re were63.0% and 84.2%,respectively,and 44.4% and 84.2%,respectively.In order to further study the role of LORF9 in immune protection,one-day-old SPF chickens were inoculated with Md5 BAC △ LORF9 or CVI988/Rispens,and challenged with Md5 BAC five days after inoculation.The average weight of Md5 BAC group decreased significantly,but there was no significant difference between the average weight of Md5 BAC △ LORF9 and CVI988/Rispens vaccine groups and the negative group.In addition,Md5 BAC induced significant lymphocytic atrophy in unvaccinated chickens.Immune organ atrophy did not occur in chickens in Md5 BAC △ LORF9 or CVI988/ Rispens immune groups.During the 60-day pathogenicity monitoring,the negative control group did not get sick,while the incidence and mortality of SPF chickens in Md5 BAC infected group were 57.1% and42.8%,and the immune protection rates in Md5BAC△LORF9 and CVI988/Rispens immune groups were 94.4% and 93.7% respectively.The above results showed that MDV strain with LORF9 deletion had strong immune protection.In order to further explore the molecular mechanism of LORF9’ s involvement in the pathogenesis of MDV,the analysis of RACE experiments showed that LORF9 had only one transcript and no splice.Western blot and q PCR were used to detect the expression changes in vitro and in vivo.The results showed that the expression of LORF9 increased with the increase of virus infection time in vitro.The early expression level in vivo is high,indicating that LORF9 is an early expression protein.The experiment of cell localization and nuclear-cytoplasmic separation of LORF9 showed that LORF9 existed in both cytoplasm and nucleus.Transcriptome sequencing of chicken spleen on the fifth day of virus infection showed that Md5BAC△LORF9 infection could induce innate immune response.Further verification at the cellular level shows that it may has nothing to do with natural immunity.Epigenetic verification of LORF9 protein showed that LORF9 could not be phosphorylated by Us3,but it was a ubiquitinated protein,which could be ubiquitinated by K48 and K63,and MDV USP could inhibit the ubiquitination of LORF9.In this study,Md5 BAC △ LORF9 gene deletion strain and Md5 BAC △LORF9-Re recovery strain were successfully constructed,and the virus proliferation characteristics in vitro and pathogenicity in vivo were not affected after LORF9 gene deletion.The immune protection test showed that Md5 BAC △ LORF9 had similar immune protection to CVI988/Rispens.The functional analysis of LORF9 shows that LORF9 is an early expression protein with only one transcript in the nucleus.Transcript sequencing shows that the deletion of LORF9 will lead to the up-and-down regulation of most genes in the body,and arouse the changes of the body’s natural immune level and inflammatory factors.However,further studies show that LORF9 may not be involved in the regulation of type I interferon signaling pathway in natural immune response,LORF9 can be ubiquitinated by K48 and K63,and MDV USP can inhibit the ubiquitination of LORF9.This study provides a theoretical basis for clarifying the molecular mechanism of MDV,and also provides a new idea for the development of MDV gene deletion vaccine.