Functional Characterization Of BmNPV-SOD In Interaction Between Baculovirus And Host

Baculoviruses are large double-stranded DNA viruses that are infectious pathogens of certain insect species.During the coevolution process of virus and host,complicated interactions between baculoviruses and their host insects have influenced both host insect,which promotes the up-grade of host defense mechanism and the evolution of virus attack strategy.The interaction between virus and host can be divided into two aspects.Firstly,the effect of baculoviruses on their insect hosts,including blocks the host cell cycle,induces host cell apoptosis,regulates host development and captures some host genes.Then,the effect of insect hosts on baculoviruses,including regulates baculovirus gene expression,influences viral replication and activates host autoimmunity.In this study,BmNPV-sod gene as the research object,four different recombinant bacmids were constructed by Red homologous recombinant technology and BmNPV Bac-to-Bac system.The effects of the BmNPV-sod deletion on virus replication,intracellular reactive oxygen species（ROS）levels,morphological changes in the BmN-SWU1 cells,the survival time of larvae and individual changes were detected from the cell level and larvae level respectively to explore the role of BmNPV-sod in the process of virus infection.The main results are as follows:1.Functional analysis of BmNPV-sod knockout in viral replication and apoptosisFour different recombinant bacmids were constructed by Red homologous recombination technology and BmNPV Bac-to-Bac system,namely,WT-BmNPV^+egfp,sod-ko-BmNPV^+egfp,sod-re-BmNPV⁺egfpegfp and sod-re-Bm^+egfp.BmN cells were transfected with four recombinant bacmids,the transfected supernatants were collected and used to infect BmN-SWU1 cells.Infected cells were monitored by fluorescence microscopy for enhanced green fluorescent protein（EGFP）expression.The result showed that there was no significant difference in intracellular EGFP expression at 24,48 and 72 h.p.t and secondary infection with the four different recombinant viruses exhibited no obvious differences.The transfection and infection experiment suggested that sod deletion did not affect BV production and sod gene is non-essential for viral replication.Both BmNPV-sod and the sod gene from the host could repair the defective of BmNPV-sod in knockout bacmid.In order to detect the activity of BmNPV-sod in the process of virus infection,four recombinant viruses were infected in BmN-SWU1 cells and analyzed the levels of intracellular ROS,the results indicated that the intracellular ROS level in BmN-SWU1cells infected with sod-ko-BmNPV^+egfp was significantly higher than that in cells infected with WT-BmNPV^+egfp,sod-re-BmNPV^+egfp,and sod-re-Bm^+egfp.Then,we analyzed intracellular SOD activity,the result suggested that SOD activity in the cells infected with sod-ko-BmNPV^+egfp was less than wild-type（wt）and repair recombinant viruses.In order to further explore the BmNPV-sod function,Hoechst 33258 and TUNEL staining were performed on BmN-SWU1 cells after infection with four recombinant viruses,the result showed that cell apoptosis occurred in all of the infected BmN cells at24,36,and 48 h.p.i.and BmNPV-sod may involed in cell apoptosis.In order to further confirm the incidence of apoptosis,caspase-3 activity was measured in BmN-SWU1cells after infection with the four recombinant viruses.Caspase-3 activity found to be higher in cells infected with sod-ko-BmNPV^+egfp compared to the other recombinant viruses.We also detected the relative expression levels of three apoptosis-related genes,suggested that caspase-3,-8,and-9,were upregulated when cells were infected with sod-ko-BmNPV^+egfp compared with wt and repair recombinant viruses.The above experimental results showed that BmNPV infection of BmN-SWU1 cells induced apoptosis in the host,and BmNPV-sod could inhibit the apoptosis in host cells.2.Functional analysis of Bm NPV-sod knockout in B.mori larvaeIn order to explore the influence of BmNPV-sod knockout in infection of B.mori larvae,the BVs were injected into newly molted fifth instar larvae at doses of 50,000,5,000,500,and 50 plaque forming unit（PFU）per larvae,and the LT₅₀ of the four recombinant viruses was analyzed and calculated.Interestingly,the LT₅₀ of the sod-ko-BmNPV^+egfp advanced at least 10.5-20.3 h compared with other recombinant viruses.Then,we also analyzed the the survival time of B.mori larvae after infection with four recombinant viruses,the result demonstrated that the survival time of B.mori larvae infected with 5,000,500,and 50 PFU of sod-ko-BmNPV^+egfp was lower than wt and repair recombinant viruses.The results implied that sod gene could delay larval death after virus infection.In addition,we detected SOD activity in the fat body after infection with the four recombinant viruses.SOD activities in larvae infected with 5,000,500,and 50 PFU of sod-ko-BmNPV^+egfp were significantly lower than after infection with other recombinant viruses.In order to explore the function of BmNPV-sod in infection of B.mori larvae,we observed the fat body of B.mori larvae after infection with the four recombinant viruses doses of 5,000 per larvae,it was found that the fat body showed disaggregation,detachment,and transformed into fragments after infection with sod-ko-BmNPV^+egfp.Hoechst 33258 staining also revealed that cell apoptosis had occurred more extensively in the fat body after infection with sod-ko-BmNPV^+egfp than with the other recombinant viruses.Three apoptosis-related genes in B.mori larvae fat body cells were determined by RT-qPCR,the result demonstrated that the relative expression levels of caspase-3,-8,and-9 were up-regulated and caspase-3 activity was significantly higher after infection with sod-ko-BmNPV^+egfp.3.Functional analysis of BmNPV-sod knockout in epidermis liquefactionIn addition,we observed and compared the morphological changes of the epidermis after infection with four recombinant viruses.Hematoxylin&Eosin（H&E）staining result showed that there was no significant difference of the epidermis observed after infection with the four recombinant viruses on the fourth day.With the increase of infection time,the epidermis was thinner after infection with sod-ko-BmNPV^+egfp than with wt and repair recombinant viruses on the fifth day.Then,we used fluorescent WGA-labeling（FITC-WGA,green）to label chitin.No evident changes observed in chitin on the fourth day after infection.However,the amount of chitin reduced on the fifth day after infection with sod-ko-BmNPV^+egfp.In order to explore the reasons for the differences in epidermis after infection with sod-ko-BmNPV^+egfp,we investigated the expressions of host liquefaction related genes BmNPV-chitinase and BmNPV-cathepsin.First,we determined the relative expression levels of BmNPV-chitinase and BmNPV-cathepsin in the B.mori larvae after infection with the four recombinant viruses on the fifth day by RT-qPCR.The results demonstrated that the relative expression levels of BmNPV-chitinase and BmNPV-cathepsin in the sod-ko-BmNPV^+egfp group were significantly higher than other three recombinant viruses.Western Blot result suggested that the protein expression levels of BmNPV-chitinase and BmNPV-cathepsin were higher in the larval fat body for the BmNPV-sod knockout recombinant virus.Then,BmN-SWU1 cells were used to analyze for expression changes of BmNPV-chitinase and BmNPV-cathepsin after infection with the four recombinant viruses.The relative expression levels of BmNPV-chitinase and BmNPV-cathepsin were greater after infection with sod-ko-BmNPV^+egfp,compared with wt and repair recombinant viruses.The above experimental results showed that BmNPV-sod knockout had an effect on the expression of Bm NPV-chitinase and BmNPV-cathepsin.Besides,BmNPV-chitinase and BmNPV-cathepsin were up regulated because of BmNPV-sod knockout both in BmN cells and B.mori larvae.Then above results provided a clue understanding about the finction of BmNPV-sod.