BmHSP19.9 targeting P6.9 and VLF-1 to mediate the formation of defective progeny viruses in the silkworm antiviral variety 871C.

The 871C silkworm strain exhibits a high level of resistance to Bombyx mori nucleopolyhedrovirus (BmNPV), making it a valuable variety for the sericulture industry. Understanding the underlying mechanism of its resistance holds great biological significance and economic value in addressing viral disease risks in sericulture. Initially, we infected the resistant strain 871C and its control strain 871 with BmNPV and conducted secondary infection experiments using the progeny occlusion bodies (OBs). As a result, a significant decrease in pathogenicity was observed. Electron microscopy analysis revealed that 871C produces progeny virions with defective DNA packaging, reducing virulence following BmNPV infection. Blood proteomic identification of the silkworm variety 871C and control 871 after BmNPV infection demonstrated the crucial role of the viral proteins P6.9 and VLF-1 in the production of defective viruses by impeding the proper encapsulation of viral DNA. Additionally, we discovered that BmHSP19.9 interacts with P6.9 and VLF-1 and that its expression is significantly upregulated after BmNPV infection. BmHSP19.9 exhibits strong antiviral activity, in part by preventing the entry of the proteins P6.9 and VLF-1 into the nucleus, thereby hindering viral nucleocapsid and viral DNA assembly. Our findings indicate that the antiviral silkworm strain 871C inhibits BmNPV proliferation by upregulating Bmhsp19.9 and impeding the nuclear localization of the viral proteins P6.9 and VLF-1, leading to the production of defective viral particles. This study offers a comprehensive analysis of the antiviral mechanism in silkworms from a viral perspective, providing a crucial theoretical foundation for future antiviral research and the breeding of resistant silkworm strains.

MicroRNA bmo-miR-31-5p inhibits apoptosis and promotes BmNPV proliferation by targeting the CYP9e2 gene of Bombyx mori.

BACKGROUND: Bombyx mori nuclear polyhedrosis virus (BmNPV), as a typical baculovirus, is the primary pathogen that infects the silkworm B. mori, a lepidopteran species. Owing to the high biological safety of BmNPV in infecting insects, it is commonly utilized as a biological insecticide for pest control. Apoptosis is important in the interaction between the host and pathogenic microorganisms. MicroRNAs (miRNAs) influence immune responses and promote stability of the immune system via apoptosis. Therefore, the study of apoptosis-related miRNA in silkworms during virus infection can not only provide support for standardizing the prevention and control of diseases and insect pests, but also reduce the economic losses to sericulture caused by the misuse of biological pesticides. RESULTS: Through transcriptome sequencing, we identified a miRNA, miR-31-5p, and demonstrated that it can inhibit apoptosis in silkworm cells and promote the proliferation of BmNPV in BmE-SWU1 cells. We identified a target gene of miR-31-5p, B. mori cytochrome P450 9e2 (BmCYP9e2), and demonstrated that it can promote apoptosis in silkworm cells and inhibit the proliferation of BmNPV. Moreover, we constructed transgenic silkworm strains with miR-31-5p knockout and confirmed that they can inhibit the proliferation of BmNPV. CONCLUSION: These data indicate that miR-31-5p may exert functions of inhibiting apoptosis and promoting virus proliferation by regulating BmCYP9e2. The findings demonstrate how miRNAs influence host cell apoptosis and how they are involved in the host immune system response to viruses, providing important insights into the applications of biological insecticides for pest control. © 2024 Society of Chemical Industry.

Establishment of a Secretory Protein-Inducible CRISPR/Cas9 System for Nosema bombycis in Insect Cells.

Gene editing techniques are widely and effectively used for the control of pathogens, but it is difficult to directly edit the genes of Microsporidia due to its unique spore wall structure. Innovative technologies and methods are urgently needed to break through this limitation of microsporidia therapies. Here, we establish a microsporidia-inducible gene editing system through core components of microsporidia secreted proteins, which could edit target genes after infection with microsporidia. We identified that Nosema bombycis NB29 is a secretory protein and found to interact with itself. The NB29-N3, which lacked the nuclear localization signal, was localized in the cytoplasm, and could be tracked into the nucleus after interacting with NB29-B. Furthermore, the gene editing system was constructed with the Cas9 protein expressed in fusion with the NB29-N3. The system could edit the exogenous gene EGFP and the endogenous gene BmRpn3 after overexpression of NB29 or infection with N. bombycis.

Bombyx moriSuppressor of Hairless is involved in the regulation of the silkworm cell cycle and endoreplication of the silk glands.

The Notch signaling pathway is important in cell cycle regulation and cell proliferation. The transcriptional repressor Suppressor of Hairless [Su(H)] is a molecular switch for downstream target genes of the Notch signaling pathway but the regulatory mechanism of the Su(H) gene in the cell cycle is unclear. We determined the function of the Notch signaling pathway and Bombyx mori Su(H) [BmSu(H)] in the regulation of the silkworm cell cycle. Inhibition of Notch signaling promoted the replication of DNA in silkworm gland cells and expression of the BmSu(H) gene was significantly reduced. Overexpression of the BmSu(H) gene inhibited DNA replication and cell proliferation of silkworm cells, whereas knockout of the BmSu(H) gene promoted DNA replication and cell proliferation. Knockout of the BmSu(H) in silkworms improved the efficiency of silk gland cell endoreplication and increased important economic traits. We demonstrated that BmSu(H) protein can directly bind to the promoters of BmCyclinA, BmCyclinE and BmCDK1 genes, inhibiting or promoting their transcription at the cell and individual level. This study identified molecular targets for genetic improvement of the silkworm and also provided insights into the regulatory mechanism of the cell cycle.

The Bombyx mori singed Gene Is Involved in the High-Temperature Resistance of Silkworms.

Temperature is an important factor in the growth, development, survival, and reproduction of organisms. The high-temperature resistance mechanism of insects may be significant for use in the prevention and control of insect pests. The silkworm, Bombyx mori, is an important Lepidoptera model species for studies on pest control in agriculture and forestry. We identified a gene in B. mori, the B. mori singed (Bmsn) gene, which is involved in the high-temperature resistance of silkworms. Sn proteins are highly conserved among species in many taxonomic groups. The overexpression of the Bmsn gene promoted the proliferation of silkworm cells, reduced oxidation, and reduced the accumulation of reactive oxygen species under stress. Interfering with the Bmsn gene had the opposite result. We constructed a transgenic B. mori strain that overexpressed the Bmsn gene. The physiological traits of the transgenic strain were significantly improved, and it had stronger high-temperature resistance. The Bmsn gene is involved in the process by which fat bodies respond to high-temperature stress. These findings provide insights into the mechanism of high-temperature resistance of insects and offer a new perspective on agricultural and forestry pest control.

BmATAD3A mediates mitochondrial ribosomal protein expression to maintain the mitochondrial energy metabolism of the silkworm, Bombyx mori.

ATAD3A is a mitochondrial membrane protein belonging to the ATPase family that contains the AAA+ domain. It is widely involved in mitochondrial metabolism, protein transport, cell growth, development and other important life processes. It has previously been reported that the deletion of ATAD3A causes growth and development defects in humans, mice and Caenorhabditis elegans. To delve into the mechanism underlying ATAD3A defects and their impact on development, we constructed a Bombyx mori ATAD3A (BmATAD3A) defect model in silkworm larvae. We aim to offer a reference for understanding ATAD3A genetic defects and elucidating the molecular regulatory mechanisms. The results showed that knockout of the BmATAD3A gene significantly affected the weight, survival rate, ATPase production and mitochondrial metabolism of individuals after 24 h of incubation. Combined metabolomics and transcriptomics analysis further demonstrated that BmATAD3A knockout inhibits amino acid biosynthesis through the regulation of mitochondrial ribosomal protein expression. Simultaneously, our findings indicate that BmATAD3A knockout impeded mitochondrial activity and ATPase synthesis and suppressed the mitochondrial oxidative phosphorylation pathway through B. mori mitochondrial ribosomal protein L11 (BmmRpL11). These results provide novel insights into the molecular mechanisms involved in the inhibition of development caused by ATAD3A deficiency, offering a potential direction for targeted therapy in diseases associated with abnormal ATAD3A expression.

BmNPV Bm60 is a key target gene used by a resistant strain of Bombyx mori to inhibit BmNPV proliferation.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes significant losses to the silkworm industry. Numerous antiviral genes and proteins have been identified by studying silkworm resistance to BmNPV. However, the molecular mechanism of silkworm resistance to BmNPV is unclear. We analyzed the differences between the susceptible strain 871 and a near-isogenic resistant strain 871C. The survival of strain 871C was significantly greater than that of 871 after oral and subcutaneous exposure to BmNPV. Strain 871C exhibited a nearly 10,000-fold higher LD50 for BmNPV compared to 871. BmNPV proliferation was significantly inhibited in all tested tissues of strain 871C using HE strain and fluorescence analysis. Strain 871C exhibited cellular resistance to BmNPV rather than peritrophic membrane or serum resistance. Strain 871C suppressed the expression of the viral early gene Bm60. This led to the inhibition of BmNPV DNA replication and late structural gene transcription based on the cascade regulation of baculovirus gene expression. Bm60 could also interact with the viral DNA binding protein and alkaline nuclease, as well as host proteins Methylcrotonoyl-CoA carboxylase subunit alpha, mucin-2-like protein, and 30 K-8. Overexpression of 30 K-8 significantly inhibited BmNPV proliferation. These results increase understanding of the molecular mechanism behind silkworm resistance to BmNPV and suggest targets for the breeding of resistant silkworm strains and the controlling pest of Lepidoptera.

Common strategies in silkworm disease resistance breeding research.

The silkworm, which is considered a model invertebrate organism, was the first insect used for silk production in human history and has been utilized extensively throughout its domestication. However, sericulture has been plagued by various pathogens that have  caused significant economic losses. To enhance the resistance of a host to its pathogens,numerous strategies have been developed. For instance, gene-editing techniques have been applied to a wide range of organisms, effectively solving a variety of experimental problems. This review focuses on several common silkworm pests and their pathogenic mechanisms, with a particular emphasis on breeding for disease resistance to control multiple types of silkworm diseases. The review also compares the advantages and disadvantages of transgenic technology and gene-editing systems. Finally, the paper provides a brief summary of current strategies used in breeding silkworm disease resistance, along with a discussion of the establishment of existing technologies and their future application prospects. © 2023 Society of Chemical Industry.

Apoptosis-related long non-coding RNA LINC5438 of Bombyx mori promotes the proliferation of BmNPV.

Apoptosis, as an important part of the immune response, is one of the core events in the host-virus interaction. Studies have shown that long non-coding RNAs (lncRNAs) play important roles in the process of cell apoptosis and pathophysiology. To investigate the apoptosis-related lncRNAs involved in Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworms, transcriptome sequencing was conducted based on silkworm cells infected with BmNPV before and after B. mori inhibitor of apoptosis (Bmiap) gene knockout. A total of 23 differentially expressed lncRNAs were identified as being associated with the mitochondrial apoptosis pathway. Moreover, we demonstrated that B. mori LINC5438 has the function of inhibiting apoptosis in silkworm cells. Overexpression of LINC5438 promoted the proliferation of BmNPV, while interference with LINC5438 inhibited its proliferation, indicating that LINC5438 plays an important role in BmNPV infection. Our results also showed that LINC5438 can regulate the expression of Bmiap, BmDronc, BmICE, and its predicted target gene BmAIF, suggesting that LINC5438 may function through the mitochondrial pathway. These findings provide important insights into the mechanisms of virus-host interaction and the applications of baculoviruses as biological insecticides.

Transcriptome analysis reveals that knocking out BmNPV iap2 induces apoptosis by inhibiting the oxidative phosphorylation pathway.

Apoptosis is essential for the normal growth, development, and immunity defense of living organisms, and its function and mechanisms have been intensively studied. When viral infection occurs, apoptosis is triggered, causing programmed death of the infected cells. Meanwhile, viruses have also evolved countermeasures to inhibit apoptosis in host cells. We previously constructed a transgenic silkworm line with significantly improved resistance to Bombyx mori nucleopolyhedrovirus (BmNPV) by knocking out the BmNPV inhibitor of apoptosis 2 (iap2) gene. However, the mechanism of how IAP2 induces apoptosis still needs to be further investigated. Here, the transcriptomes of Cas9(-)/sgiap2 (-) and Cas9(+)/sgiap2(+) strains were analyzed at 48 h after BmNPV infection, and a total of 709 differential genes were obtained. A KEGG analysis revealed that the differentially expressed genes were enriched in the oxidative phosphorylation, proteasome, and ribosome pathways. In the oxidative phosphorylation pathway, 41 differentially expressed genes were downregulated, and 12 of these genes were verified by qRT-PCR. More importantly, the knockout of BmNPV iap2 led to the inhibition of the oxidative phosphorylation pathway, followed by activated oxidative stress triggered apoptosis, thereby inhibiting the replication of BmNPV in vitro and vivo. The results provide a basis for the analysis of the initiation of apoptosis that can inhibit virus proliferation, and the study presents new ideas for the subsequent creation of resistant material.

Baculovirus LEF-11 interacts with BmIMPI to induce cell cycle arrest in the G2/M phase for viral replication.

Viruses arrest the host cell cycle and using multiple functions of host cells is an important approach for their replication. Baculovirus arrests infected insect cells at both the late S and G2/M phase, but the strategy employed by baculovirus is not clearly understood. Our research suggests that the Bombyx mori nucleopolyhedrovirus (BmNPV) could arrest the cell cycle in the G2/M phase to promote virus replication, and also that the viral protein LEF-11 could inhibit host cell proliferation and arrest the cell cycle by inhibiting the cell cycle checkpoint proteins BmCyclinB and BmCDK1. Furthermore, we found that LEF-11 interacts with BmIMPI to regulate cell proliferation, but not by direct interaction with BmCyclinB or BmCDK1. In addition, our findings showed that BmIMPI was important and necessary for LEF-11 induced cell cycle arrest in the G2/M phase. Moreover, BmIMPI was found to interact with BmCyclinB and BmCDK1, and down-regulate the expression of BmCyclinB and BmCDK1 to compromise the cell cycle and cell proliferation. Taken together, the data presented demonstrated that baculovirus LEF-11 regulates BmIMPI to inhibit host cell proliferation and provide a new insight into the molecular mechanisms employed by viruses to induce cell cycle arrest.

Identification of the Key Functional Domains of Bombyx mori Nucleopolyhedrovirus IE1 Protein.

The immediate early protein 1 (IE1) acts as a transcriptional activator and is essential for viral gene transcription and viral DNA replication. However, the key regulatory domains of IE1 remain poorly understood. Here, we analyzed the sequence characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) IE1 and identified the key functional domains of BmNPV IE1 by stepwise truncation. Our results showed that BmNPV IE1 was highly similar to Autographa californica nucleopolyhedrovirus (AcMNPV) IE1, but was less conserved with IE1 of other baculoviruses, the C-terminus of IE1 was more conserved than the N-terminus, and BmNPV IE1 was also necessary for BmNPV proliferation. Moreover, we found that IE1158-208 was a major nuclear localization element, and IE11-157 and IE1539-559 were minor nuclear localization elements, but the combination of these two minor elements was equally sufficient to fully mediate the nuclear entry of IE1. Meanwhile, IE11-258, IE1560-584, and the association of amino acids 258 and 259 were indispensable for the transactivation activity of BmNPV IE1. These results systematically resolve the functional domains of BmNPV IE1, which contribute to the understanding of the mechanism of baculovirus infection and provide a possibility to synthesize a small molecule IE1-truncated mutant as an agonist or antagonist.

BmCDK5 Affects Cell Proliferation and Cytoskeleton Morphology by Interacting with BmCNN in Bombyx mori.

The ordered cell cycle is important to the proliferation and differentiation of living organisms. Cyclin-dependent kinases (CDKs) perform regulatory functions in different phases of the cell cycle process to ensure order. We identified a homologous gene of the Cyclin-dependent kinase family, BmCDK5, in Bombyx mori. BmCDK5 contains the STKc_CDK5 domain. The BmCDK5 gene was highly expressed in S phase. Overexpression of the BmCDK5 gene accelerates the process of the cell cycle's mitotic period (M) and promotes cell proliferation; knocking out the BmCDK5 gene inhibited cell proliferation. Furthermore, we identified a protein, BmCNN, which can interact with BmCDK5 and represents the same express patterns as the BmCDK5 gene in the cell cycle phase and the spatial-temporal expression of B. mori. This study revealed that BmCDK5 and BmCNN play roles in promoting cell proliferation and regulating cytoskeleton morphology, but do not induce expression changes in microtubule protein. Therefore, our findings provide a new insight; the BmCDK5 gene has a regulatory effect on the cell cycle and proliferation of B. mori, which is presumably due to the interaction between BmCDK5 and BmCNN regulating changes in the cytoskeleton.

Analysis of Silver Nanoparticles for the Treatment and Prevention of Nucleopolyhedrovirus Affecting Bombyx mori.

Bombyx mori nucleopolyhedrovirus (BmNPV) causes major economic losses in sericulture. A number of agents have been employed to treat viral diseases. Silver nanoparticles (AgNPs) have wide applications in biomedical fields due to their unique properties. The anti-BmNPV effect of AgNPs has been evaluated, however, there are insufficient studies concerning its toxicity to other organisms and the environment. We chemically synthesized biocompatible BSA-AgNPs with a diameter range of 2-4 nm and characterized their physical properties. The toxicity of AgNPs towards cells and larvae with different concentrations was examined; the results indicated a biofriendly effect on cells and larvae within specific concentration ranges. The SEM observation of the surface of BmNPV after treatment with AgNPs suggested that AgNPs could destroy the polyhedral structure, and the same result was obtained by Coomassie blue staining. Further assays confirmed the weakened virulence of AgNPs-treated BmNPV toward cells and larvae. AgNPs also could effectively inhibit the replication of BmNPV in infected cells and larvae. In summary, our research provides valuable data for the further development of AgNPs as an antiviral drug for sericulture.

Stable transformation of fluorescent proteins into Nosema bombycis by electroporation.

BACKGROUND: Microsporidia are a group of intracellular parasitic eukaryotes, serious pathogens that cause widespread infection in humans, vertebrates, and invertebrates. Because microsporidia have a thick spore wall structure, the in vitro transformation, cell culture, and genetic operation technology of microsporidia are far behind that of other parasites. METHODS: In this study, according to an analysis of the life-cycle of microsporidia, Nosema bombycis, and different electro-transformation conditions, the transduction efficiency of introducing foreign genes into N. bombycis was systematically determined. RESULTS: We analyzed the direct electro-transformation of foreign genes into germinating N. bombycis using reporters under the regulation of different characteristic promoters. Furthermore, we systematically determined the efficiency of electro-transformation into N. bombycis under different electro-transformation conditions and different developmental stages through an analysis of the whole life-cycle of N. bombycis. These results revealed that foreign genes could be effectively introduced through a perforation voltage of 100 V pulsed for 15 ms during the period of N. bombycis sporeplasm proliferation. CONCLUSIONS: We present an effective method for electro-transformation of a plasmid encoding a fluorescent protein into N. bombycis, which provides new insight for establishing genetic modifications and potential applications in these intracellular parasites.

CRISPR/Cpf1 multiplex genome editing system increases silkworm tolerance to BmNPV.

The CRISPR/Cas9 genome editing technology is now widely used in insect studies, but the use of CRISPR can be further increased to improve insect genome engineering. We established a direct mutation at multiple loci in several genes simultaneously used by CRISPR/Cpf1 multiplex genome editing technology to target the BmNPV genome. We constructed a transgenic line that can target the BmNPV ie-1, gp64, and DNApoly genes simultaneously, and hybridized this line with an FnCpf1 transgenic line to obtain an FnCpf1 × gNPVM binary hybrid expression system and to activate the FnCpf1 gene editing system. We showed that the multiple gene editing system introduced deletions, mutations, and insertions at three target sites, and that it did not affect the economic traits of transgenic silkworm lines. The antiviral response of multiplexed genome editing lines increased significantly, and viral gene transcription and replication were significantly affected in the transgenic silkworm lines. This study provides innovative resistance materials for silkworm breeding and also provides a simplified platform for efficient insect multi genome engineering and genetic operation.

E2F4 regulates the cell cycle and DNA replication in the silkworm, Bombyx mori.

The E2F family of transcription factors is crucial for cell cycle progression and cell fate decisions. Although E2Fs have been widely studied in mammals, there have been few studies performed in insects. Here, we determined the function of E2F4 in the silkworm, Bombyx mori. We demonstrate that E2F proteins are highly conserved among species from lower animals to higher mammals. Overexpression of the BmE2F4 gene led to cell cycle arrest in the G1 phase, whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S phase. These results indicate that BmE2F4 is important in cell cycle regulation. We also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk gland. Furthermore, we identified a protein called Bm14-3-3ζ that can interact with BmE2F4 and allow it to localize in the nucleus. Overexpression of the Bm14-3-3ζ gene led to cell cycle arrest in the G1 phase, while knocking down the gene increased the proportion of cells in S phase. These findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation.

Bombyx mori Nucleopolyhedrovirus (BmNPV) Induces G2/M Arrest to Promote Viral Multiplication by Depleting BmCDK1.

Understanding virus-host interaction is very important for delineating the mechanism involved in viral replication and host resistance. Baculovirus, an insect virus, can cause S or G2/M phase arrest in insect cells. However, the roles and mechanism of Baculovirus-mediated S or G2/M phase arrest are not fully understood. Our results, obtained using flow cytometry (FCM), tubulin-labeling, BrdU-labeling, and CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS), showed that Bombyx mori nucleopolyhedrovirus (BmNPV) induced G2/M phase arrest and inhibited cellular DNA replication as well as cell proliferation in BmN-SWU1 cells. We found that BmNPV induced G2/M arrest to support its replication and proliferation by reducing the expression of BmCDK1 and BmCyclin B. Co-immunoprecipitation assays confirmed that BmNPV IAP1 interacted with BmCDK1. BmNPV iap1 was involved in the process of BmNPV-induced G2/M arrest by reducing the content of BmCDK1. Taken together, our results improve the understanding of the virus-host interaction network, and provide a potential target gene that connects apoptosis and the cell cycle.

MicroRNA-6498-5p Inhibits Nosema bombycis Proliferation by Downregulating BmPLPP2 in Bombyx mori.

As microRNAs (miRNAs) are important expression regulators of coding RNA, it is important to characterize their role in the interaction between hosts and pathogens. To obtain a comprehensive understanding of the miRNA alternation in Bombyx mori (B. mori) infected with Nosema bombycis (N. bombycis), RNA sequencing and stem-loop qPCR were conducted to screen and identify the significantly differentially expressed miRNAs (DEmiRNAs). A total of 17 such miRNAs were identified in response to N. bombycis infection, among which miR6498-5p efficiently inhibited the proliferation of N. bombycis in BmE-SWU1 (BmE) cells by downregulating pyridoxal phosphate phosphatase 2 (BmPLPP2). In addition, a fluorescence in situ hybridization (FISH) assay showed that miR6498-5p was located in the cytoplasm of BmE cells, while it was not found in the schizonts of N. bombycis. Further investigation of the effect of BmPLPP2 on the proliferation of schizonts found that the positive factor BmPLPP2 could facilitate N. bombycis completing its life cycle in cells by overexpression and RNAi of BmPLPP2. Our findings offer multiple new insights into the role of miRNAs in the interaction between hosts and microsporidia.