| The silkworm(Bombyx mori)is kind of Lepidoptera insect with important economic values,which is faced with serious disease threats.BmNPV is the worst pathogen to sericulture that often brings economic losses to sericultural production.The virus is highly infectious and difficult to control,and it is often outbreaks in countries with B.mori breeding,causing enormous economic losses.The traditional disinfection method can prevent the disease to some extent,however it is of great limitations.Therefore,studying the infection mechanism of BmNPV and the resistance mechanism of host B.mori as well as breeding the B.mori varieties finally is one of the urgent subjects for current sericultural technological research workers.The repercussion study between BmNPV and the host has already made many achievements,and a number of genes that participate in the interaction of B.mori and virus and have anti-virus functions have been discovered,but it is still unknown about the infection process of BmNPV.In the process of infection,which passages of the host B.mori participate in the antivirus activity? How does the virus hijack the host cells for its own reproduction? These issues still awaits a further study.In this study,we identified the differences in the resistance level and tissue infection of BmNPV between 871 and 871C;The invasion and infection of BmNPV between different resistant strains were analyzed;and the period of blocking of BmNPV in resistant lines was determined;the differential expression genes of 871 and 871 C were analyzed by comparing transcriptome analysis;through genetic cloning,sequence analysis,functional validation,and finally we get BmCTL-S5 with significant antiviral activity.1.Identification on the antiviral differences of 871 and 871CThrough the single head quantitative method of oral food,the half lethal dose(LD50)of 871 and 871 C for BmNPV infection were determined to be 3 x 103 PDV/head and 2.5 x 107 PDV/head respectively,,871 C had significant resistance to BmNPV compared with 871.Analysis of the survival curve of silkworm(Bombyx mori)by oral feeding(2 × 106 PDV/ head),the death peak of 871 and 871 C are respectively on the 5th day and the 8th day indicating that the death of 871 C is delayed after being infected..According to the survival curve analysis of subcutaneous injection(2×104 BV/head),871 completely died on the 8th day while 871 C died a little after being infected.By the histological,immunohistochemical and microscopic observation,we found that the nuclear expansion phenomenon appeared when the 871 midgut was infected for 24 hours,the cell nucleus coloring shallowed at 48 h,and nuclear exfoliation phenomenon appeared at 96 h.No significant change was found in 871 C after infection BmNPV.The blood was observed after oral infection with BmNPV,72 hours after infection,we could observe the multi polyhedrosis in the haemocytes of 871,96 h p.i.the haemocytes are filled with polyhedrosis,and the blood contains free polyhedrons.There was no significant change in the resistant strain.These results indicate that the resistant strain 871 C has a mechanism for resistance to BmNPV.2.Analysis of the infection processes of BmNPV in 871 and 871 C and Difference of tissue infectionThe expression of BmNPV vp39 gene was detected by qRT-PCR,for identify the infection process of BmNPV in 871 and 871 C.The results showed that the first to detect the expression of vp39 in midgut after oral infection of 6h;12h expression was detected in blood cells and fat body cells;the expression of 24 h tissue silkworm head and trachea;48h expression in epidermal cells;72 h expression was detected in the silk gland and genital gland.These results indicated that BmNPV was initially infected in the midgut of Bombyx mori,then enters the blood and fat body for secondary infection,and then infects silkworm head,epidermis and tracheal cells,finally BmNPV infects silkworm glands and gonads.There was no difference in the infection process of resistant strain 871 C and susceptible strain 871 in BmNPV,but the expression level of vp39 was significantly lower in 871 C at the same time point.qRT-PCR was used to detect the expression of vp39 gene in 871 and 871 C tissues,and found that there were significant differences in the same tissue between different strains.The infection of 871 C to BmNPV was significantly lower than that of susceptible strain BmNPV(871).The comparison on the different tissues of the same strain showed that the infection degree of blood and fat body was highest,which was followed by the midgut,head,epidermis and trachea,and the infection rate of silk gland and gonad was lowest.The results showed that the difference of tissue infection was confirmed by the analysis of the tracer virus with EGFP fluorescent label,which was consistent with the qRT-PCR results.3.Analysis of the difference of BmNPV in the infection key pointsThere are two stages of primary infection and systemic infection in baculovirus infection.In the process of BmNPV infection in host silkworm,the initial infection of the virus into the midgut and the duplication in the midgut is critical to the systemic infection,and the BmNPV invasion in haemocyte influences the proliferation activity of virus directly.Through oral administration and subcutaneous injection,we detected the infection of midgut and hemolymph during BmNPV infection.It showed that after treatment with fluorescent whitening agent(FB28 0.05%),the death time of 871 and 871 C being infected by BmNPV was 1-2 days earlier than that of control group with a significant increase in mortality.These results indicated that the membrane had an important role in defense against BmNPV infection;But the two strains of BmNPV sensitivity changes did not appear differences,indicating that the barrier effect of the membrane is not the main factor affecting the resistance difference between 871 and 871 C.For BmNPV,after oral infection with 6h,the number of DNA copies and the expression of vp39 in the midgut were detected and analyzed,the results showed that there was no significant difference between the two groups,indicating that there was no difference between the two groups in the invasion of BmNPV cells.Detection of virulence after incubation with BV showed that the serum of 871 and 871 C had no significant effect on virus virulence.After subcutaneous injection of 24 h,there was no significant difference in BV entering the haemocytes of 871 and 871 C.Results of DNA replication and gene expression levels of BmNPV in midgut cells and blood cells,suggesting that BmNPV can invade both cells during infection,but then BmNPV in the infected cells in the proliferation of differences,suggesting that the resistant strain 871 C has the mechanism of intracellular inhibition of viral replication and proliferation.4.Analysis of BmNPV blocked period in resistant silkwormHaemolymphs of 871 and 871 C were collected at 3h,6h,12 h,24h and 48 h post infected by BmNPV,RNA was extracted for reverse transcription to obtain cDNA.The BmNPV genes expression in host cells was analyzed by RT-PCR by using BmNPV ie-1(immediate early gene),gp64(early gene),vp39 and lef-3(late gene),and p10(very late gene)primers.Results showed that ie-1,gp64 and vp39 could be expressed in 871 and 871 C for less than 6 hours,and the expression level of these genes were not significantly different in the two silkworm strains,which indicates that the BmNPV was not affected at the beginning of infection.The expression levels of these genes in the two differentiated lines were significantly different after 12 h p.i.and were up-regulated in 871,downregulated in 871 C.At the same time,ie-1,vp39 and p10 were detected in the fat body and midgut of the two silkworm lines,and the results were consistent with those in the haemocytes.Together the results indicate that BmNPV was inhibited in the early stage of infection,hinting that there must be some antiviral mechanisms or molecules existed in 871 C,that inhibit the expression of some early genes of the virus,thus leading to the virus replication and proliferation was blocked.5.Comparison transcriptome analysis of BmNPV-host interaction and resistance mechanismsThe above experiments indicated that the difference in proliferation of BmNPV between 871 and 871 C was mainly caused by intracellular effects.In order to comprehensively analyze the differential mechanism of 871 and 871 C anti-BmNPV,we selected BmNPV not infected 12 h,12h after infection(pre-viral DNA pre-replication stage),24h(DNA replication stage),48h(BV formation stage)of silkworm midgut as the transcriptome sequencing material.A total of 8 libraries and 37.18 Gb data were obtained by using Hiseq2500 platform,1031 differentially expressed genes(DEGs)were obtained by comparative transcriptome analysis.The BmNPV infection resulted in significant changes in 749 and 381 genes in susceptible strain 871 and resistant strain 871 C,respectively.BmNPV infection could induce the whole genome expression of resistant strains.The change of susceptible strain 871 was significantly higher than that of resistant strain 871 C.GO analysis and KEGG analysis showed that the differential gene mainly focused on the transport and metabolism of amino acids,the transport of carbohydrates Metabolism and signal transduction.BmNPV infection caused Toll,IMD,PPO pathway and pattern recognition and other multiple innate immune pathway changes.Resistant silkworm lines had significant differences in pattern recognition,immune pathways,apoptosis,effector molecules,antiviral related genes and other antiviral pathways and susceptible strains.The comparative analysis DEGs of 871 and 871 C uninfected showed that 143 and 177 DEGs was highly expressed in 871 and 871 C respectively.It was found that the expression level of BGIBMGA003660 not only in 871 C was 5.25 times than that of 871(871_C v.s.871C_C)through the construction of Venn diagram analysis,the gene expression was up-regulated after BmNPV infected,and 871 C was always higher than 871 expression level about twice.These results suggest that BGIBMGA3660 may have a role in inhibiting the proliferation of BmNPV.6.Cloning and expression of BmCTL-S5 and analysis of its antiviral functionIn order to further verify that the candidate gene BGIBMGA003660 has the effect of inhibiting the proliferation of BmNPV,cell level experiments were used to verify the function.We successfully cloned BGIBMGA003660(BmCTL-S5)gene using cDNA from 871 C midgut.Bioinformatics analysis showed that the nucleotide sequence contains 612 bp,encoding 209 aa,the molecular weight of the amino acid sequence is 22854.78,the isoelectric point(pI)is 5.66;it contains a conserved motif of EPQ(Glu-Pro-Gln),and a CRD conserved domain of 162 aa,The C-terminal containing 21 aa signal peptide.Secondary structure analysis shows that BmCTL-S5 may have two ?-super-helices and seven ?-folds.Multiple sequence alignment analysis shows that BmCLT-S5 had high homology with other insect C-type Lectin,which had high conservation at both ends.The subcellular localization analysis shows that BmCLT-S5 was evenly distributed in the cytoplasm.The expression level of BmCLT-S5 gene was higher in the resistant strain(871C)than that in the susceptible strain(871)by RT-PCR,and the expression of BmCTLS5 was up-regulated after BmNPV infection.Over expression vector fused with Flag tag and Cas9 knockout vector were constructed successfully.The overexpression vector was transiently transfected into BmN-SWU1 cells and subjected to viral infection experiments,it was found that the fluorescence signal of virus was not expressed in the cells overexpressing BmCLT-S5;The stable cells were screened by Zecoin antibiotics,and then the infection experiments were carried out.By RT-PCR,cell infection rate and Western blotting,it was found that the level of gene expression,DNA replication and virus proliferation were inhibited.We successfully constructed BmCLT-S5 Cas-9 knockout vector Cas9-CTL.Transient transfection and sequencing analysis showed that the knockout vector could significantly modify the BmCLT-S5 gene to cause nucleic acid sequence deletion and point mutation,and the transient transfection efficiency was 38%.The results showed that the proliferation of BmNPV in the experimental group,which transfected with Cas9-CTL,was significantly higher than that in the control group.These results indicated that BmCLT-S5 could significantly inhibit the replication and proliferation of BmNPV. |
PDF Full Text Request