Nuclear polyhedrosis virus (NPV), a typical baculovirus, is a major pathogen infecting lepidopteran insects and serves as an eco-friendly biopesticide with high biological safety. Studying the interactions between Bombyx mori and baculovirus infection in lepidopteran models will provide valuable insights for improving pest control efficacy while mitigating economic losses in sericulture caused by pesticide overuse. Lysozyme, a widely distributed antibacterial enzyme in both vertebrates and invertebrates, functions as a classic immune effector. Although multiple lysozymes have been identified in B. mori, their specific roles in combating Bombyx mori nucleopolyhedrovirus (BmNPV) remain uncharacterized. In this study, we characterized the seven known lysozymes in B. mori and confirmed that all members exhibit conserved structural features typical of the lysozyme family. Interestingly, BmC-LZM-like4 is atypical among lysozyme genes, as it is intronless and contains an extended C-terminal sequence. Following BmNPV infection, the expression levels of BmC-LZM-like2 and BmC-LZM-like4 were significantly upregulated. Functional validation through RNA interference (RNAi) and overexpression assays demonstrated that both BmC-LZM-like2 and BmC-LZM-like4 suppress BmNPV proliferation in vitro and in vivo, suggesting their critical roles in the antiviral response. Furthermore, we investigated the potential regulatory mechanisms governing the expression of these lysozymes. Our findings indicate that the Dorsal-mediated signaling pathway may be involved in modulating the expression of BmC-LZM-like2 and BmC-LZM-like4. These data indicate that B. mori lysozyme family members BmC-LZM-like2 and BmC-LZM-like4 may be transcriptionally activated via the Dorsal-mediated signaling pathway, thereby exerting antiviral effects against baculovirus proliferation.

Diapause hormone (DH) is an important endocrine substance capable of influencing diapause in Lepidoptera moths that is encoded by the neuropeptide hormone DH-PBAN gene. Imidazole derivative KK-42 is a synthetic insect growth regulator that can affect diapause in Lepidoptera moths, and appears to have an opposite physiological function to DH. To test the hypothesis that KK-42 may be operating through DH to affect diapause, here, we used two Lepidoptera species Bombyx mori L. and Antheraea pernyi that enter egg and pupal diapause, respectively, through examining whether KK-42 can influence DH-PBAN and some associated mRNA expression. We found that the protein sequences of DH-PBAN in insects were highly variable, although the PRXamide C-terminus was conserved. We also found that KK-42 induced significant up-regulation and prolonged expression duration of DH-PBAN in both A. pernyi and B. mori pupae, as well as in trimolter larvae of B. mori that were induced by the application of KK-42 from the normal tetramolter larvae. In addition, KK-42 can significantly upregulate glutamic acid decarboxylase (GAD) expression in B. mori in transcriptome data. Our findings suggested that KK-42 influences diapause by upregulating GAD expression, promoting DH accumulation to prolong the secretion time of DH-PBAN.

Silk proteins are natural biopolymers with outstanding mechanical properties and diverse biological functions. However, the extraction and processing of silk from cocoons inevitably alter their molecular weight (Mw) distributions, profoundly affecting physicochemical properties, bioactivity, and processability. In this review, we first outline the molecular structures and characteristics of silk fibroin (SF) and silk sericin (SS). We then systematically summarize current strategies for the regulation, characterization, and fractionation of silk protein Mw. Subsequently, we discuss the critical roles of Mw in the fabrication of silk-based materials, including particles, fibers, hydrogels, and micropatterned constructs, as well as its impact on key material properties such as mechanical performance and biodegradability. Furthermore, we highlight the Mw-dependent biological activities of silk proteins, including mitogenic, antioxidant, and immunomodulatory effects. Finally, we analyze challenges and perspectives, aiming to promote a Mw-oriented design paradigm that integrates the processing, structure, properties, and biological functions of silk protein materials.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a persistent threat to sericulture and a classic model for insect-reovirus interactions. Here, we investigated the role of Bombyx mori ribosomal protein L40 (BmRPL40) in virus-host interactions using this model. We found that BmCPV infection significantly downregulated host BmRPL40 expression. Both knockdown and overexpression experiments further demonstrated that BmRPL40 expression levels were positively correlated with viral replication. Moreover, BmRPL40 overexpression suppressed the IMD and Toll pathways, thereby impairing host immunity and facilitating viral replication. Accordingly, upon BmCPV infection, the host downregulates BmRPL40 to counteract its immunosuppressive effects and enhance antiviral defense. Collectively, these findings reveal a mechanism by which the silkworm modulates viral replication and activates host defense through BmRPL40 expression, offering critical insights into virus-host interactions at the translational level.

Delivering protein therapeutics to the brain through nanocarriers requires overcoming both the blood-brain barrier (BBB) and intracellular lysosomal degradation. Here, we report a silk-based supramolecular protein delivery platform that addresses these dual physiological barriers. Two phenolic compounds are grafted onto silk sericin (SS), a biocompatible and bioactive natural protein, yielding phenolic SS capable of assembling with protein cargos via supramolecular interactions. To facilitate BBB penetration, the iRGD peptide is incorporated to enable transcytosis via the bystander effect. Phenolic modification alters the ratio of amino to carboxyl groups on SS, thereby tuning its isoelectric point. In acidic lysosomes, the nanocomplex undergoes a surface charge reversal from negative to positive, promoting lysosomal escape and cytosolic release of catalase. In parallel, phenolic SS exhibits intrinsic anti-inflammatory activity, repolarizing activated microglia toward an anti-inflammatory phenotype. In APP/PS1 transgenic mice, systemic administration of the nanocomplex reduces oxidative stress and neuroinflammation, leading to significant improvements in cognitive function, compared to a non-charge-reversal control. Collectively, this strategy provides a versatile and translatable framework for engineering protein-based nanocarriers to deliver protein therapeutics for neurodegenerative disease treatments.

Antimicrobial peptides (AMPs) are critical components of the innate immune system in insects, but their regulation and contribution to antiviral defence remain poorly understood. In this study, we investigated the expression, antiviral activity and regulation of Bombyx mori Lebocin-3 (BmLEB3) during infection with Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). BmLEB3 expression was markedly induced following BmCPV infection in the silkworms. RNAi-mediated knockdown of BmLEB3 increased the transcriptional level of viral marker gene vp1 in BmN cells, whereas overexpression led to the inhibition of viral replication in BmN cells. Mechanistically, BmCPV induced the expression of the pattern-recognition receptor BmPGRP-S1 and the transcription factor BmRelish, and silencing of either gene reduced BmLEB3 expression, suggesting that immune deficiency (IMD)-associated signalling contributes to BmLEB3 regulation. Moreover, BmLEB3 was induced by 20-hydroxyecdysone (20E) treatment, further strengthening its antiviral effect. Molecular docking analysis revealed a potential interaction between 20E and BmPGRP-S1, indicating a possible hormonal modulation of IMD-associated AMP expression. Collectively, our results identify BmLEB3 as a virus-responsive AMP and suggest that its expression is modulated by IMD-associated signalling and hormonal cues during BmCPV infection in B. mori. These findings provide insight into the molecular regulation of antiviral immune responses in the silkworm and provide a basis for improving disease resistance in sericulture against microbial pathogens.

Autophagy is a critical cellular process that regulates host-virus interactions, yet its functional dynamics in silkworms during infection with Bombyx mori Nucleopolyhedrovirus (BmNPV) are not fully understood. This study explored the key role of the autophagy-related gene Atg5 in silkworms during BmNPV infection. The temporal expression pattern of Atg5 showed a significant increase during the early infection stage (12-24 h) and a significant decrease at later stages (48-72 h) in both BmN cells and larval tissues. RNAi-mediated silencing of Atg5 significantly reduced VP39 expression, accompanied by decreased mitochondrial damage and improved host survival. In contrast, overexpression of Atg5 significantly increased VP39 expression and viral DNA loads in BmN cells, confirming its positive regulatory effect on viral replication. Furthermore, a successfully constructed recombinant baculovirus with stable Atg5 expression caused significantly higher VP39 expression and viral DNA accumulation in the silkworms following infection compared to the control. Collectively, these findings demonstrate the pro-viral role of Atg5 during BmNPV infection by promoting autophagy-mediated viral replication. This study enhances our understanding of the molecular mechanisms behind autophagy-virus interactions in B. mori and provides a potential target for developing antiviral strategies in sericulture.

Ribosomal protein L11 (RPL11) plays important roles in ribosome biogenesis and stress responses, yet its antiviral function remains poorly understood. This study characterized BmRPL11 and its role in Bombyx mori nucleopolyhedrovirus (BmNPV) infection. BmRPL11 is evolutionarily conserved and shares high sequence homology and protein structure with RPL11 from other species. Tissue expression profiling showed predominant BmRPL11 expression in silk glands and the fat body, with upregulation after BmNPV infection. Functional assays demonstrated that BmRPL11 knockdown enhanced, whereas overexpression suppressed, BmNPV proliferation in BmN cells and silkworm larvae. Mechanistically, BmNPV infection induced nuclear export of BmRPL11 and was associated with enhanced apoptosis, while autophagy-related genes were not markedly affected by BmRPL11 knockdown. IP-MS identified candidate BmRPL11-interacting proteins during infection, including RPL5, whereas a canonical murine double minute 2 (MDM2) homologue was not detected. These findings identify BmRPL11 as an antiviral factor in silkworms, potentially acting through an apoptotic rather than an autophagic pathway.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a double-stranded RNA reovirus, causes significant losses in sericulture; however, the host factors that support its replication remain poorly understood. Here, we identify Tudor staphylococcal nuclease (TSN) as a critical pro-viral host factor in silkworms. TSN is a multifunctional, evolutionarily conserved protein involved in transcription and RNA processing. BmTSN expression was increased markedly in BmCPV-infected midgut tissues. RNAi-mediated knockdown in BmN cells suppresses viral replication, whereas overexpression enhances it. Mechanistically, BmTSN drives a metabolic shift by enhancing lipid accumulation; knockdown of BmTSN reduces the expression of BmSREBP1 and other lipid-regulatory genes. Disruption of either BmTSN or BmSREBP1 reduces BmCPV replication. In a reciprocal effect, RNAi-mediated knockdown of BmSREBP1 also reduces BmTSN expression. Furthermore, BmSREBP1 transcriptionally activates BmTSN expression by binding to the BmTSN promoter, suggesting a regulatory loop between these factors. Together, these findings reveal a virus-induced BmTSN-BmSREBP1 axis that links host lipid metabolism to BmCPV replication. This work identifies BmTSN as a key host-dependency factor and provides new insights into virus-host interactions in insects.

The silkworm, Bombyx mori, is an economically important insect severely impacted by Bombyx mori nucleopolyhedrovirus (BmNPV), a double-stranded DNA virus that causes substantial losses to the sericulture industry. To elucidate the mechanisms of BmNPV infection and replication, we performed transcriptome sequencing of BmN cells infected with a recombinant BmNPV at 12 and 24 h post infection (hpi). A total of 1136 differentially expressed genes (DEGs) were identified in the 12 hpi group, including 789 up-regulated and 347 down-regulated genes, while 5191 DEGs were detected at 24 hpi, including 2102 up-regulated and 3089 down-regulated genes. Functional annotation via GO and KEGG analyses highlighted the ECM-receptor interaction pathway as particularly significant. Furthermore, RT-qPCR results demonstrated that Map3k12 inhibits BmNPV replication. These findings lay the groundwork for further investigation into the molecular mechanisms of BmNPV infection and can be utilized for breeding dominant genes conferring resistance to nucleopolyhedrovirus in the silkworm.

Bombyx mori is an important economic insect, and its production efficiency is seriously threatened by B. mori nucleopolyhedrovirus (BmNPV). A deeper understanding of host factors exploited during viral infection is crucial for developing disease-resistant silkworm strains. In the current study, we explored the function of Spase12, a subunit of the signal peptidase complex, in the silkworm's antiviral defense. The tissue expression profile showed that Spase12 is primarily expressed in the midgut and ovaries, and its expression was significantly upregulated in BmN cells and midgut tissues following BmNPV infection. Additionally, bioinformatics analyses predicted that Spase12 is a two-pass endoplasmic reticulum membrane protein, consistent with its role in protein processing and secretion. Moreover, RNAi-mediated knockdown of Spase12 significantly suppressed BmNPV replication, as evidenced by decreased VP39 expression. On the other hand, overexpression of Spase12 in BmN cells and in vivo experiments using a recombinant baculovirus system confirmed increased viral proliferation and DNA load in the midgut of infected silkworm larvae. Together, these findings established a pro-viral role for Spase12 during BmNPV infection, likely hijacked by the virus to facilitate its replication. This study offers novel insights into host-virus interactions in silkworms and highlights Spase12 as a potential molecular target for breeding BmNPV-resistant B. mori strains.

Insects detect pathogens through their germ-line encoded pattern recognition receptors (PRRs). Among these, β-1,3-glucan recognition protein (βGRP) is a crucial PRR that specifically identifies pathogenic microorganisms and triggers innate immune signaling cascades. However, it remains unclear whether βGRP can detect viruses and protect the host from viral threats. In this study, using high-throughput sequencing technology, we observed a significant suppression of βGRP-3 in Bombyx mori during infection with the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). Moreover, overexpression of βGRP-3 in BmN cell lines resulted in a reduction of BmCPV proliferation, whereas knockdown of βGRP-3 in BmN cells promoted BmCPV proliferation. These findings suggest that the βGRP family functions not only as anti-bacterial, antifungal, and anti-yeast PRRs but also as protectors against various harmful viruses in insects.

The structural integrity of viral envelopes is a critical determinant of infectivity for enveloped viruses, directly influencing vector stability, functional accuracy of surface-displayed epitopes, and preservation of native conformational states required for membrane protein studies. However, conventional purification methods often disrupt envelope integrity and cause envelope proteins to lose their activity. Here, we systematically compared discontinuous, continuous, and optimized continuous sucrose density gradient centrifugation protocols for purifying Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Through cryo-EM, we demonstrated that our optimized continuous sucrose gradient protocol significantly increased the proportion of AcMNPV budded virions with intact envelopes from 36% to 81%, while preserving the metastable prefusion conformation of the fusion protein GP64. This advancement should prove useful for structural studies of viral envelope proteins and may enhance applications in gene therapy and vaccine development utilizing enveloped viruses.

As obligatory intracellular parasites, viruses must rely on metabolic reprogramming of host cells to meet their replication needs. Baculovirus is an important biopesticide and a vector for the preparation of biological products. In addition, one of its representative species, Bombyx mori nucleopolyhedrovirus (BmNPV-Baculoviridae), also causes huge losses to the insect industry. In our previous study, amino acid metabolism has been found to play a crucial role in the BmNPV infection process. However, the mechanisms by which BmNPV reprograms host amino acid metabolism remains unclear. In fact, current insights in the importance of amino acid metabolism are limited to the impact of glutamine on viral infection. Therefore, unraveling the mechanism of amino acid metabolism reprogramming induced by baculovirus would advance this field of research to a great extent. In this study, targeted metabolomics revealed that the preferred amino acids of BmNPV budded virus (BV) include arginine, lysine, proline, isoleucine, histidine and others. In addition, most of the viral amino acids were found to be increased in the hemolymph of BmNPV infected silkworms at the later stage of infection, especially arginine, valine, phenylalanine and others. Furthermore, the importance of arginine for BmNPV proliferation was validated. Next, we confirmed that the expression of the arginine transporter Slc7a6 was strongly induced by BmNPV infection and that Slc7a6 could promote arginine uptake to support BmNPV proliferation in host cells. Moreover, using Slc7a6 knockout cells which eliminate extracellular arginine uptake, we confirmed that BmNPV could induce mitochondrial autophagy, thereby supplementing intracellular arginine and providing necessary amino acids for BmNPV proliferation. Overall, these findings support a model in which baculovirus (BmNPV) enhances the uptake of exogenous amino acids by inducing the expression of amino acid transporters and activating autophagy of organelles to maintain intracellular amino acid levels, thereby facilitating virus proliferation.

Although it is reported that the protein tyrosine phosphatase gene of baculovirus (group I nucleopolyhedrovirus) can induce enhanced locomotory activity (ELA) in caterpillars, our understanding of the host factors that are involved in the regulation of the behavioral change is still limited. Previously, single-nucleus RNA sequencing (snRNA-seq) was used to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte, muscle cell types and other unannotated cells in the silkworm larvae brains. Analysis of viral transcriptomes in each brain cell subset revealed that all brain cells could be infected by Bombyx mori nucleopolyhedrovirus (BmNPV) at 96 hours post infection but infection occurred at low levels. Furthermore, we found that chemosensory protein 3 (CSP3), encoding a small secreted protein that is possibly implicated in the transport of semiochemicals, was significantly up-regulated after BmNPV infection in most of the brain cell clusters. Knockdown of BmCSP3 resulted in significantly reduced ELA in BmNPV-infected silkworm larvae. In parallel, targeted metabolomics revealed significant shifts in the abundance of specific lipids and neurotransmitters. Subsequently, structural modeling and molecular dynamics experiments indicated that CSP3 has a large hydrophobic pocket that manifests significant flexibility and likely can accommodate divergent ligand structures or mixtures of them, including known neurotransmitters of the brain and (lyso)glycerophospholipids from larval head samples. In vitro binding assays have confirmed the interaction of several neurotransmitters and an eicosanoid to purified BmCSP3 protein. Our study provides insights into the regulation of insect behavior following analysis of viral infection at the single-cell transcriptome level and reveals an unexpected function for CSP proteins in the insect brain.

It is generally believed that no functional antiviral pathway exists in insects, that is homologous to the interferon signaling system. However, we have previously identified an interferon-stimulated gene (ISG) homologous gene, BmCH25H, in the silkworm and revealed that BmCH25H relied on its hydroxylase activity as an antiviral effector. Therefore, we speculate that there may be additional ISG homologous genes in the silkworm genome, some of which may play an antiviral role. In this study, based on knowledge on ISGs in mammals, 135 ISG homologous genes were identified in the silkworm genome using gene homology sequence alignment and conserved domain matching methods. Among these ISG homologous genes in the silkworm, we conducted in-depth research on an important immunological transcription factor, nuclear factor interleukin 3 regulated (NFIL3). Our results found that BmNFIL3 could inhibit the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV). Furthermore, we confirmed that the NFκ-B-related transcription factor BmRelish was regulated by BmNFIL3 and that its induction after BmNPV infection was mediated by BmNFIL3. More importantly, we demonstrated that BmNFIL3 relied on BmRelish for its anti-BmNPV effects. This study represents the first systematic identification of ISG homologous genes in invertebrates and also constitutes the first report that NFIL3 has antiviral effects in insects. These findings will provide new perspectives for the further understanding of antiviral immunity in insects.

The relationship among the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), its predator Serangium japonicum (Coleoptera: Coccinellidae), and its parasitoid Encarsia formosa (Hymenoptera: Aphelinidae) can be defined as intraguild predation (IGP). IGP represents an interaction that combines elements of both predation and competition between two species. In our previous study, predators (S. japonicum) often indirectly reduce the number of parasitoids (E. formosa) by consuming shared food resources, such as B. tabaci. To suppress whitefly outbreaks, a combination of parasitoids and predators is often released together using a mixed-species approach. However, the impact of the parasitic behavior of parasitoids on the feeding behavior of specific predators, such as S. japonicum, has not yet been well-studied. We conducted a laboratory study to examine how the parasitoid E. formosa affects the predation rates and performance of S. japonicum. Results indicate that S. japonicum shows a preference for non-parasitized whiteflies over parasitized ones, regardless of the availability of choices. Furthermore, we found a correlation between the preference for non-parasitized whiteflies and the duration of parasitization. As the developmental days of the parasitoid mummies increased, the food intake of the predatory group decreased significantly. Additionally, as the parasitoid mummy develops, S. japonicum spends more time exploring and probing the mummy before deciding whether to consume it. In summary, our study suggests that S. japonicum exhibits a significant preference for consuming non-parasitized whiteflies. Therefore, it is important to coordinate the release timing of S. japonicum and E. formosa in the biological control of B. tabaci.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogen that causes significant financial losses to the sericulture industry. Currently, no effective methods exist to avoid or treat this pathogen, underscoring the urgent need for novel BmNPV control tactics. This study assessed the antiviral activity of Cidofovir against BmNPV in the BmN cell line and silkworms. The cytotoxicity test revealed that 4 mg/mL Cidofovir was the optimal concentration to use for silkworms. Western blotting and qRT-PCR results showed a significant reduction of viral protein VP39 in BmN cells, fat body, and midgut tissues. Additionally, BmNPV DNA load was significantly lower in samples treated with Cidofovir, indicating effective suppression of viral replication. Gene expression analysis demonstrated significant upregulation of Relish, STAT, and Ago2 genes in both BmN cells and silkworm tissues after Cidofovir treatment, along with increased expression of antimicrobial peptides Cec-A and Cec-B. Enhanced activities of key immune and antioxidant enzymes, including phenoloxidase, superoxide dismutase, and catalase, were observed in the hemolymph of Cidofovir-treated larvae, suggesting an overall strengthening of host defense mechanisms. Additionally, the 16S rRNA sequencing suggests that both viral infection and Cidofovir treatment alter the composition of gut microbiota in silkworms, with some unique taxa emerging in response to each condition. Together, these findings demonstrate that Cidofovir not only suppresses BmNPV proliferation but also activates multiple innate immune responses, emphasizing Cidofovir's potential as a promising antiviral agent for controlling BmNPV infection in silkworms.

The silk industry is facing serious threats from various pathogens, with Bombyx mori Nucleopolyhedrovirus (BmNPV) and Cytoplasmic polyhedrosis virus (BmCPV) causing considerable annual losses. Unfortunately, conventional control measures are insufficient to deal with the problem of viral diseases. Hence, substitute control strategies are immediately needed, and to achieve that, a deeper understanding of the immune mechanisms of the silkworm against viral pathogenesis is crucial. The current study focuses on evaluating the antiviral potential of BmMBF2 in limiting BmNPV and BmCPV infections. The tissue expression profile of silkworms revealed that BmMBF2 was predominantly expressed in the midgut, and infections with BmNPV/BmCPV resulted in the upregulation of this gene. RNAi-mediated downregulation of BmMBF2 significantly promoted the proliferation of the viruses, while overexpression of BmMBF2 significantly suppressed the replication of BmNPV and BmCPV. Additionally, the 20-hydroxyecdysone (20E) treatment leads to the upregulation of the BmMBF2 and a reduction in virus proliferation. Furthermore, the expression of the BmMBF2 gene through recombinant baculovirus system inhibited viral replication and reduced the DNA load of BmNPV in the silkworms. These results provide valuable insights into the antiviral potential of BmMBF2 in the silkworm, which could aid in the development of novel approaches to counter viral infections.