Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest.

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Study on anti-BmNPV mechanism of branched-chain amino acid aminotransferases in silkworm.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important virus that threatens sericulture industry. At present, there is no effective treatment for BmNPV infection in silkworms, and lncRNA plays an important role in biological immune response and host-virus interaction, but there are relatively few studies in silkworms. In this study, the four midgut tissue samples of the resistance strain NB (NB) and susceptible strain 306 (306) and the NB and 306 continuously infected with BmNPV for 96 h are used for whole transcriptome sequencing to analyze the differences in the genetic background of NB and 306 and the differences after inoculation of BmNPV, and the significantly different mRNA, miRNA and lnRNA between NB and 306 after BmNPV inoculation were screened. By comparing NB and 306, 2651 significantly different mRNAs, 57 significantly different miRNAs and 198 significantly different lncRNAs were screened. By comparing NB and 306 after BmNPV inoculation, 2684 significantly different mRNAs, 39 significantly different miRNAs and 125 significantly different lncRNAs were screened. According to the significantly different mRNA, miRNA and lncRNA screened from NB and 306 and NB and 306 after virus inoculation, the mRNA-miRNA-lncRNA regulatory network was constructed before and after virus inoculation, and the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo_chr7_8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo_chr7_8305 regulates BmBCAT. The whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. The BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis may play a potential role in the interaction between silkworms and BmNPV. These results provide new insights into the interaction mechanism between silkworms and BmNPV.

Comparative analysis of the intestinal flora of BmNPV-resistant and BmNPV-sensitive silkworm varieties.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a very common and infectious virus that affects silkworms and hinders silk production. To investigate the intestinal flora of BmNPV-resistant and BmNPV-sensitive silkworm varieties, 16 S rDNA high-throughput sequencing was performed. The results of the cluster analysis showed that the intestinal flora of the resistant silkworm variety was more abundant than that of the sensitive silkworm variety. This was found even when infection with BmNPV caused a sharp decline in the number of intestinal floral species in both resistant and sensitive silkworm varieties. The abundances of the intestinal flora, including Aureimonas, Ileibacterium, Peptostreptococcus, Pseudomonas, Enterococcus, and Halomonas, in the resistant variety were considerably greater after infection with BmNPV than those in the sensitive variety. After infection with BmNPV, four kinds of important intestinal bacteria, namely, f_Saccharimonadaceae, Peptostreptococcus, Aureirmonas, and f_Rhizobiaceae, were found in the resistant silkworm variety. In the sensitive silkworm variety, only Faecalibaculum was an important intestinal bacterium. The differential or important bacteria mentioned above might be involved in immunoreaction or antiviral activities, especially in the intestines of BmNPV-resistant silkworms. By conducting a functional enrichment analysis, we found that BmNPV infection did not change the abundance of important functional components of the intestinal flora in resistant or sensitive silkworm varieties. However, some functional factors, such as the biosynthesis, transport, and catabolism of secondary metabolites (e.g., terpenoids and polyketides) and lipid transport and metabolism, were more important in the resistant silkworm variety than in the sensitive variety; thus, these factors may increase the resistance of the host to BmNPV. To summarize, we found significant differences in the composition, abundance, and function of the intestinal flora between resistant and sensitive silkworm varieties, especially after infection with BmNPV, which might be closely related to the resistance of resistant silkworm varieties to BmNPV.

BmNPV p35 regulates apoptosis in Bombyx mori via a novel target of interaction with the BmVDAC2-BmRACK1 complex.

Voltage-dependent anion channel 2 (VDAC2) is an important channel protein that plays a crucial role in the host response to viral infection. The receptor for activated C kinase 1 (RACK1) is also a key host factor involved in viral replication. Our previous research revealed that Bombyx mori VDAC2 (BmVDAC2) and B. mori RACK1 (BmRACK1) may interact with Bombyx mori nucleopolyhedrovirus (BmNPV), though the specific molecular mechanism remains unclear. In this study, the interaction between BmVDAC2 and BmRACK1 in the mitochondria was determined by various methods. We found that BmNPV p35 interacts directly with BmVDAC2 rather than BmRACK1. BmNPV infection significantly reduced the expression of BmVDAC2, and activated the mitochondrial apoptosis pathway. Overexpression of BmVDAC2 in BmN cells inhibited BmNPV-induced cytochrome c (cyto c) release, decrease in mitochondrial membrane potential as well as apoptosis. Additionally, the inhibition of cyto c release by BmVDAC2 requires the involvement of BmRACK1 and protein kinase C. Interestingly, overexpression of p35 inhibited cyto c release during mitochondrial apoptosis in a RACK1 and VDAC2-dependent manner. Even the mutant p35, which loses Caspase inhibitory activity, could still bind to VDAC2 and inhibit cyto c release. In summary, our results indicated that BmNPV p35 interacts with the VDAC2-RACK1 complex to regulate apoptosis by inhibiting cyto c release. These findings confirm the interaction between BmVDAC2 and BmRACK1, the interaction between p35 and the VDAC2-RACK1 complex, and a novel target that BmNPV p35 regulates apoptosis in Bombyx mori via interaction with the BmVDAC2-BmRACK1 complex. The result provide an initial exploration of the function of this interaction in the BmNPV-induced mitochondrial apoptosis pathway.

A CRM1-dependent nuclear export signal in Autographa californica multiple nucleopolyhedrovirus Ac93 is important for the formation of intranuclear microvesicles.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is highly conserved in all sequenced baculovirus genomes, and it plays important roles in both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. In this study, we characterized a cellular CRM1-dependent nuclear export signal (NES) of AcMNPV Ac93. Bioinformatic analysis revealed that AcMNPV Ac93 may contain an NES at amino acids 115-125. Green fluorescent protein (GFP) fused to the NES (GFP:NES) of AcMNPV Ac93 is localized to the cytoplasm of transfected cells. Multiple point mutation analysis demonstrated that NES is important for the nuclear export of GFP:NES. Bimolecular fluorescence complementation experiments and co-immunoprecipitation assays confirmed that Ac93 interacts with Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits cellular CRM1-dependent nuclear export of GFP:NES. To determine whether the NES in AcMNPV Ac93 is important for the formation of intranuclear microvesicles, an ac93-null AcMNPV bacmid was constructed; the wild-type and NES-mutated Ac93 were reinserted into the ac93-null AcMNPV bacmid. Immunofluorescence analysis showed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in infected cells, while the construct containing point mutations at residues 123 and 125 of Ac93 resulted in a defect in budded virus production and the abolishment of intranuclear microvesicles. Together, these data demonstrate that Ac93 contains a functional NES, which is required for the production of progeny viruses and the formation of intranuclear microvesicles.IMPORTANCEAutographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is important for the formation of intranuclear microvesicles. However, how the baculovirus manipulates Ac93 for the formation of intranuclear microvesicles is unclear. In this study, we identified a nuclear export signal (NES) at amino acids 115-125 of AcMNPV Ac93. Our results showed that the NES is required for the interaction between Ac93 and Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits the nuclear export of green fluorescent protein fused to the NES. Our analysis revealed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in AcMNPV-infected cells. Together, our results indicate that Ac93 participates in the formation of intranuclear microvesicles via the Ac93 NES-mediated CRM1 pathway.

Transcriptome analysis indicates the mechanisms of BmNPV resistance in Bombyx mori midgut.

Bombyx mori nucleopolyhedrovirus (BmNPV) caused serious economic losses in sericulture. Analyzing the molecular mechanism of silkworms (B. mori) resistance to BmNPV is of great significance for the prevention and control of silkworm virus diseases and the biological control of agricultural lepidopteran pests. In order to clarify the defense mechanisms of silkworms against BmNPV, we constructed a near isogenic line BC8 with high resistance to BmNPV through the highly BmNPV-resistant strain NB and the highly BmNPV-susceptible strain 306. In this study, RNA-Seq technique was used to analyze the transcriptome level differences in the midgut of BC8 and 306 following BmNPV infection. A total of 1350 DEGs were identified. Clustering analysis showed that these genes could be divided into 8 clusters with different expression patterns. Functional annotations based on GO and KEGG analysis indicated that they were involved in various metabolism pathways. Finally, 32 BmNPV defense responsive genes were screened. They were involved in metabolism, reactive oxygen species (ROS), signal transduction and immune response, and insect hormones. The further verification shows that HSP70 should participate in resistance responses of anti-BmNPV. These findings have paved the way in further functional characterization of candidate genes and subsequently can be used in breeding of BmNPV resistance dominant silkworms.

The Bombyx mori G protein ß subunit 1 (BmGNß1) gene inhibits BmNPV infection.

G protein ß subunit 1 (GNß1) has several functions, including cell growth regulation, the control of second messenger levels, and ion channel switching. Previous transcriptome analyses in our laboratory have shown that BmGNß1 transcription is reduced following infection with Bombyx mori nucleopolyhedrovirus (BmNPV), but it is unknown what role this gene may have in the host response to BmNPV infection. In this study, the BmGNß1 gene was cloned using the RACE method. After BmNPV infection, BmGNß1 was downregulated in Baiyu strains in tissues such as the hemolymph and midgut. Indirect immunofluorescence showed that BmGNß1 was localized to the cytoplasm. We further constructed a BmGNß1-pIZ/V5-His-mCherry overexpression plasmid and designed siRNA to evaluate the role of BmGNß1 in host response to infection. The results showed that BmGNß1 overexpression inhibited BmNPV proliferation, while knockdown of BmGNß1 was correlated with increased BmNPV proliferation. The siRNA-mediated reduction of BmGNß1 was correlated with an increase in BmNPV infection of BmN cells, increased BmNPV vp39 transcription, and reduced survival time of BmNPV-infected B. mori. Overexpression of BmGNß1 in BmN cells was also correlated with apoptosis and a modification in transcript levels of genes involved in host response to BmNPV infection (PI3K, AKT, Bmp53, BmFOXO, Caspase-1, Bmp21, BmPKN and BmCREB), suggesting that BmGNß1 may influence the apoptotic host response of infected B. mori through the PI3K-AKT pathway. This study provides potential targets and theoretical support for breeding BmNPV-resistant silkworm varieties.

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.

Bombyx mori triose-phosphate transporter protein inhibits Bombyx mori nucleopolyhedrovirus infection by reducing the cell glycolysis pathway.

Bombyx mori triose-phosphate transporter protein (BmTPT) is a member of the solute carrier (SLC) family. Its main function is to transport triose phosphate between intracellular and extracellular. In this study, BmTPT was cloned and characterised from the fat body of the silkworm Bombyx mori, resulting in an open reading frame (ORF) with a full length of 936 bp, which can encode 311 amino acid residues and has eight transmembrane structural domains. BmTPT was distributed throughout the cell and deposited the most in the nucleus, and is expressed in all tissues of Bombyx mori. Bombyx mori nucleopolyhedrovirus (BmNPV) infection significantly up-regulated BmTPT expression in immune tissue fat bodies. In addition, overexpression of BmTPT significantly inhibited BmNPV infection and markedly reduced the expression of enzymes related to the cellular glycolytic pathway; on the contrary, down-regulation of BmTPT expression by RNA interference resulted in robust replication of BmNPV and a significant increase in the expression of enzymes related to the cellular glycolytic pathway. This is the first report that BmTPT has antiviral effect in silkworm, and also could result in a lack of energy and raw materials for BmNPV replication and infection through down-regulation of the cellular glycolytic pathway.

Single-Nucleus Sequencing of Silkworm Larval Brain Reveals the Key Role of Lysozyme in the Antiviral Immune Response in Brain Hemocytes.

INTRODUCTION: The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm (Bombyx mori) is an economically important insect and a lepidopteran model species. The diversity of cell types in the silkworm brain, and how these cell subsets produce an immune response to virus infection, remains largely unknown. METHODS: Single-nucleus RNA sequencing (snRNA-seq), bioinformatics analysis, RNAi, and other methods were mainly used to analyze the cell types and gene functions of the silkworm brain. RESULTS: We used snRNA-seq to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte-like cell, and muscle cell types in the B. mori nucleopolyhedrovirus (BmNPV)-infected and BmNPV-uninfected silkworm larvae brain at the late stage of infection. Further, we found that the cell subset that exerts an antiviral function in the silkworm larvae brain corresponds to hemocytes. Specifically, antimicrobial peptides were significantly induced by BmNPV infection in the hemocytes, especially lysozyme, exerting antiviral effects. CONCLUSION: Our single-cell dataset reveals the diversity of silkworm larvae brain cells, and the transcriptome analysis provides insights into the immune response following virus infection at the single-cell level.

Compatibility of the fungus Beauveria bassiana and Trichoplusia ni SNPV against the cabbage looper Trichoplusia ni: crop plant matters.

BACKGROUND: Microbial insecticides are an important weapon in insect pest management, but their use is still relatively limited. One approach for increasing their efficacy and use could be to combine different pathogens to increase pest mortality. However, little is known about whether increasing pathogen diversity will improve pest management. Here, we investigated the compatibility of two pathogens for the management of the cabbage looper, Trichoplusia ni, T. ni nucleopolyhedrovirus (TniSNPV) and the entomopathogenic fungus Beauveria bassiana, on two crops, tomato and broccoli. The pathogens were applied to individual plants using ultra low volume sprays, alone or in combination, either synchronously or asynchronously. Healthy third-instar T. ni larvae were introduced to the plants before application and collected by destructive sampling 24 h after the last pathogen application. RESULTS: Combined applications did not result in an increase in larval mortality compared to TniSNPV alone, although mortality was generally high. B. bassiana was considerably less effective on broccoli compared to tomato. In both the combined treatments, virus-induced mortality was approximately 50% lower when applied together with the fungus, while fungus-induced mortality was not affected by the virus, even when the virus was introduced 24 h before the fungus. CONCLUSION: While our results suggest that applying this combination of entomopathogens would not be beneficial for pest management, this study illustrates the need to consider the target crop as an important driver of the efficacy of both single and mixed pathogen applications in the field. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Bmo-miR-6498-5p suppresses Bombyx mori nucleopolyhedrovirus infection by down-regulating BmPLPP2 to modulate pyridoxal phosphate content in B. mori.

The RNA interference pathway mediated by microRNAs (miRNAs) is one of the methods to defend against viruses in insects. Recent studies showed that miRNAs participate in viral infection by binding to target genes to regulate their expression. Here, we found that the Bombyx mori miRNA, miR-6498-5p was down-regulated, whereas its predicted target gene pyridoxal phosphate phosphatase PHOSPHO2 (BmPLPP2) was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Both in vivo and in vitro experiments showed that miR-6498-5p targets BmPLPP2 and suppresses its expression. Furthermore, we found miR-6498-5p inhibits BmNPV genomic DNA (gDNA) replication, whereas BmPLPP2 promotes BmNPV gDNA replication. As a pyridoxal phosphate (PLP) phosphatase (PLPP), the overexpression of BmPLPP2 results in a reduction of PLP content, whereas the knockdown of BmPLPP2 leads to an increase in PLP content. In addition, exogenous PLP suppresses the replication of BmNPV gDNA; in contrast, the PLP inhibitor 4-deoxypyridoxine facilitates BmNPV gDNA replication. Taken together, we concluded that miR-6498-5p has a potential anti-BmNPV role by down-regulating BmPLPP2 to modulate PLP content, but BmNPV induces miR-6498-5p down-regulation to promote its proliferation. Our findings provide valuable insights into the role of host miRNA in B. mori-BmNPV interaction. Furthermore, the identification of the antiviral molecule PLP offers a novel perspective on strategies for preventing and managing viral infection in sericulture.

Studying the role of Bombyx mori molybdenum cofactor sulfurase in Bombyx mori nucleopolyhedrovirus infection.

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture.

Multifaceted interactions between host ESCRT-III and budded virus-related proteins involved in entry and egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

Ras3 in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus.

Bombyx mori ras protein3 (BmRas3) is a small molecular protein in the GTPase superfamily, which has the activity of binding guanosine nucleotides and GTP enzymes. It acts as a molecular switch by coupling extracellular signal to different cellular response through the conversion between Ras-GTP conformation and Ras-GDP conformation, thus regulating signal pathways responsible for cell growth, migration, adhesion, survival and differentiation. However, few studies have been done on Ras3 in silkworm, and its function and mechanism are unclear. In this study, we found that the overexpression of BmRas3 inhibited the infection of BmNPV(B. mori nucleopolyhedrovirus), while knockdown of BmRas3 could promote the infection of BmNPV. In addition, after the BmRas3 in silkworm larvae was knockdown, the anti-BmNPV ability of silkworm decreased and the survival rate of silkworm was affected. Additionly in the cells with BmRas3 overexpression, the transcription level of BmMapkk6 、BmP38、BmJNK、BmERK1/2 and BmERK5 were significantly increased after BmNPV infection, and the transcript levels of BmMapkk6、BmP38、BmJNK、BmERK1/2 and BmERK5 were also inhibited to varying degrees This is the first report on the antiviral effect of BmRas3 in silkworm, which provides a new direction for further study on the anti-BmNPV mechanism of silkworm and screening and cultivation of anti-BmNPV silkworm strain.

Glucose-regulated protein 94 facilitates the proliferation of the Bombyx mori nucleopolyhedrovirus via inhibiting apoptosis.

Glucose regulatory protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family, that plays an important role in secreted protein folding. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. Previous studies showed that HSP90 members promote BmNPV replication in silkworm, but the function of BmGRP94 in BmNPV infection and proliferation is still not understood. In this study, we investigated the interplay between BmGRP94 and BmNPV infection in silkworm. We first identified a single gene of BmGRP94 in the Bombyx mori genome, which encodes a polypeptide with 810 amino acids in length. Spatio-temporal expression profiles showed that BmGRP94 was highly expressed in hemocytes and midgut, and was significantly induced by BmNPV infection. Furthermore, overexpression of BmGRP94 facilitates viral proliferation, while BmGRP94 inhibition evidently decreased BmNPV proliferation in BmN cells and in silkworm midgut. Mechanistically, BmGRP94 inhibition triggers ER stress, as judged by increased expression of PERK/ATF4/ERO1, H2O2 production, and ER calcium efflux, which promotes cell apoptosis to restrict BmNPV replication in silkworm. These results suggest that BmGRP94 plays an important role in facilitating BmNPV proliferation, and provides a potential molecular target for BmNPV prevention.

The DEAD/H-box helicase DHX9 contributes to suppression of Bombyx mori nucleopolyhedrovirus propagation in B. mori cells.

Antiviral immune responses are mainly triggered through the recognition of virus-derived nucleic acids by host-specific pattern recognition receptors (PRRs). Here, we identified and characterized homologs of human PRRs for virus-derived DNA in Bombyx mori upon infection with a nucleopolyhedrovirus (NPV), a member of the family Baculoviridae. We found that progeny virus production of B. mori NPV was promoted in B. mori cells silenced with B. mori homolog of DEAD/H box polypeptide 9 gene (Bm-DHX9), but not in cells silenced with the other examined genes. Silencing of Bm-DHX9 expression has no effect on apoptosis induction, one of the major antiviral responses in B. mori cells. We also showed that Bm-DHX9 has the ability to bind DNA containing unmethylated C-phosphate-G-motif, which are characteristic of microbial pathogens and contained in the NPV genome with high frequency. Our findings suggest that Bm-DHX9 has the potential for sensing NPV-derived DNA to induce antiviral immune responses.

A gut-isolated Enterococcus strain (HcM7) triggers the expression of antimicrobial peptides that aid resistance to nucleopolyhedrovirus infection of Hyphantria cunea larvae.

BACKGROUND: Commensal microorganisms are widely distributed in insect gut tissues and play important roles in host nutrition, metabolism, reproductive regulation, and especially immune functioning and tolerance to pathogens. Consequently, gut microbiota represent a promising resource for the development of microbial-based products for pest control and management. However, the interactions among host immunity, entomopathogen infections, and gut microbiota remain poorly understood for many arthropod pests. RESULTS: We previously isolated an Enterococcus strain (HcM7) from Hyphantria cunea larvae guts that increased the survival rates of larvae challenged with nucleopolyhedrovirus (NPV). Here, we further investigated whether this Enterococcus strain stimulates a protective immune response against NPV proliferation. Infection bioassays demonstrated that re-introduction of the HcM7 strain to germfree larvae preactivated the expression of several antimicrobial peptides (particularly H. cunea gloverin 1, HcGlv1), resulting in the significant repression of virus replication in host guts and hemolymph, and consequently improved host survivorship after NPV infection. Furthermore, silencing of the HcGlv1 gene by RNA interference markedly enhanced the deleterious effects of NPV infection, revealing a role of this gut symbiont-induced gene in host defenses against pathogenic infections. CONCLUSION: These results show that some gut microorganisms can stimulate host immune systems, thereby contributing to resistance to entomopathogens. Furthermore, HcM7, as a functional symbiotic bacteria of H. cunea larvae, may be a potential target for increasing the effectiveness of biocontrol agents against this devastating pest. © 2023 Society of Chemical Industry.

Uric acid metabolism promotes apoptosis against Bombyx mori nucleopolyhedrovirus in silkworm, Bombyx mori.

The white epidermis of silkworms is due to the accumulation of uric acid crystals. Abnormal silkworm uric acid metabolism decreases uric acid production, leading to a transparent or translucent phenotype. The oily silkworm op50 is a mutant strain with a highly transparent epidermis derived from the p50 strain. It shows more susceptibility to Bombyx mori nucleopolyhedrovirus (BmNPV) infection than the wild type; however, the underlying mechanism is unknown. This study analysed the changes in 34 metabolites in p50 and op50 at different times following BmNPV infection based on comparative metabolomics. The differential metabolites were mainly clustered in six metabolic pathways. Of these, the uric acid pathway was identified as critical for resistance in silkworms, as feeding with inosine significantly enhanced larval resistance compared to other metabolites and modulated other metabolic pathways. Additionally, the increased level of resistance to BmNPV in inosine-fed silkworms was associated with the regulation of apoptosis, which is mediated by the reactive oxygen species produced during uric acid synthesis. Furthermore, feeding the industrial strain Jingsong (JS) with inosine significantly increased the level of larval resistance to BmNPV, indicating its potential application in controlling the virus in sericulture. These results lay the foundation for clarifying the resistance mechanism of silkworms to BmNPV and provide new strategies and methods for the biological control of pests.

Bombyx mori nucleopolyhedrovirus induces BmFABP1 downregulation to promote viral proliferation.

Fatty acid binding proteins (FABPs) play an important role as endogenous cytoprotectants. However, studies on FABPs in invertebrates are scarce. Previously, we discovered Bombyx mori fatty acid binding protein 1 (BmFABP1) through co-immunoprecipitation. Here, we cloned and identified BmFABP1 from BmN cells. The results of immunofluorescence indicated that BmFABP1 was localized in the cytoplasm. The tissue expression profile of silkworms showed that BmFABP1 was expressed in all tissues except hemocytes. The expression level of BmFABP1 gradually decreases in BmN cells and B. mori larvae after infection with B. mori nucleopolyhedrovirus (BmNPV). Upregulation of BmFABP1 expression through overexpression or WY14643 treatment significantly inhibited the replication of BmNPV, while downregulation of BmFABP1 expression by RNA interference promoted the replication of BmNPV. The same results were obtained in experiments on silkworm larvae. These results suggest that BmNPV induces BmFABP1 downregulation to promote its proliferation and that BmFABP1 has a potential anti-BmNPV role. This is the first report on the antiviral effect of BmFABP1 in silkworms and provides new insights into the study of the FABP protein family. Also, it is important to study BmNPV resistance in silkworms to breed transgenic silkworms with BmNPV resistance.