| Bombyx mori nucleopolyhedrovirus(BmNPV)is threatening sericulture and causing serious economic losses every year.Clarifying the antiviral mechanism of silkworm has become an important but difficult point in current research.In the study of screening antiviral key genes or proteins,omics methods are commonly used to compare and analyze different resistant silkworms,but this method is difficult to focus and screen antiviral key factors,which also affects the research process of silkworm antiviral mechanism.In the early stage of this study,a mutant strain op50 derived from the p50 strain showed high sensitivity to BmNPV,which provided good material for more accurate screening of antiviral key factors.To date,with the wide application of metabolomics in silkworms,this technology has shown unique advantages in exploring the pathological state and metabolic reaction of the host,which is expected to open a new breakthrough in the identification of key antiviral factors.In this study,based on targeted metabolomics,the differences of 34 metabolites between op50 and its wild type responding to BmNPV infection were explored.The metabolic pathway enrichment analysis of the screened differential metabolites was carried out to clarify the key metabolites involved in virus resistance and study their functions in virus infection,aiming to provide new data support for elucidating the antiviral mechanism of silkworm.The main contents and results of this study are as follows:(1)Analysis of the resistance level to BmNPV in op50 and its wild type p50The synthesis or transport disorder of uric acid can cause the traits of oily silkworm.To preliminarily clarify the cause of op50 mutation,7 key genes involved in uric acid synthesis,transport,or accumulation in op50 and p50 were sequenced.It was found that the amino acid sequences of BmPNP and BmXDH involved in uric acid synthesis showed deletion and missense mutation.To analyze the resistance level of op50 and p50 to BmNPV.BV-e GFP was subcutaneously inoculated and ODV was orally fed to analyze the virus proliferation and survival rate in the two strains.The results showed that the BmNPV titer in op50 was significantly higher than that in p50,and its survival rate was also significantly lower than p50.It can be speculated that the mutation of op50 may be the reason for its sensitivity to BmNPV.(2)Metabolomic analysis of hemolymph after BmNPV infection in op50 and p50To identify the differential metabolites involved in responding to BmNPV infection in op50 and p50,34 metabolites in the hemolymph of op50 and p50 at 24,48 and 72 h after BmNPV infection were analyzed using UPLC-MS/MS.The discreteness of metabolomics data in different treatment groups was evaluated by PCA,and metabolomics data was verified using RT-q PCR.After comparing the significant differences of metabolites before and after virus infection,it was found that there were 4,25 and 17 metabolites in p50 and 8,11 and 22 metabolites in op50 after virus infection for 24,48 and 72 h,respectively.Further pathway enrichment analysis of these differential metabolites showed that the pathways involved in responding to BmNPV in the two silkworm strains were different at three time points,indicating that their response mechanisms were different.To screen the metabolic pathways involved in the response to BmNPV infection,these enriched pathways were functionally clustered,and the results were clustered into six candidate metabolic pathways,including fructose metabolism,glycolysis,sulfur metabolism,amino acid metabolism,purine metabolism and TCA cycle.(3)Identification of key metabolic pathways involved in responding to BmNPV infectionTo further screen the metabolic pathways related to BmNPV infection,the key metabolites in the six candidate metabolic pathways were selected for verification,including glucose in glycolysis and fructose metabolism,fumaric acid in TCA cycle,serine in amino acid metabolism,taurine in sulfur metabolism and inosine in uric acid metabolism.These metabolites were fed to op50 and p50,respectively.The expression of key enzyme genes in the metabolic pathway showed that these metabolites could activate the corresponding metabolic pathways,but only serine and inosine could significantly improve the survival rate of silkworm larvae.To further clarify the metabolic pathways involved in virus resistance,the regulatory relationship between candidate pathways was analyzed,and it was found that only inosine feeding significantly regulated other pathways of op50 and p50.Therefore,it is preliminarily clarified that inosine-mediated uric acid metabolism is a key pathway to improve the resistance of silkworm to BmNPV.(4)Mechanism analysis of inosine-mediated uric acid metabolism in BmNPV proliferationTo further clarify the function of inosine-mediated uric acid metabolism pathway in silkworm response to viral infection,inosine and febuxostat,uric acid synthesis inhibitor,were used to explore the pathway’s function in BmN cells.The results showed that inosine and febuxostat could activate and inhibit uric acid synthesis,respectively.Further analysis showed that inosine-mediated uric acid synthesis caused ROS accumulation and activated apoptosis,thereby inhibiting the proliferation of BmNPV.Additionally,the industrial strains Jingsong and Haoyue were selected to verify the above phenomenon.The results showed that compared with the control group,feeding with inosine not only significantly increased the survival rate of silkworms infected with BmNPV,but also never affect the body weight,whole cocoon weight and cocoon shell rate of silkworm larvae,indicating that the antiviral effect of uric acid metabolism was universal among different silkworm strains,which has certain application value for the prevention and control of viral diseases in sericulture. |
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