B. Mori Nucleopolyhedrovirus Research Articles

CRISPR/Cas9-mediated disruption of orf76 as an antiviral therapy against BmNPV in the transgenic silkworm

Viral diseases pose a significant threat to livestock husbandry and plant cultivation. CRISPR/Cas9-mediated targeted editing of viral genes offers a promising approach to antiviral therapy. The silkworm, Bombyx mori, is an economically important insect susceptible to infection by B. mori nucleopolyhedrovirus (BmNPV), and viral outbreaks cause severe economic losses to the sericulture industry. Here, we identified BmNPV orf76 as a viral late gene that is highly similar to Autographa californica multiple nucleopolyhedrovirus Ac93. The deletion of orf76 abolished BmNPV proliferation and hindered the production of infectious budded viruses. We generated a transgenic line, Cas9(+)/sgorf76(+), that did not affect the growth or development of the silkworm and demonstrated that the transgenic line Cas9(+)/sgorf76(+) efficiently cleaved orf76 at the sgorf76 site, resulting in large deletions at 120 h post-infection, with no observed off-target effects. Survival analyses revealed that the transgenic line Cas9(+)/sgorf76(+) exhibited significantly higher survival rates than the control lines Cas9(−)/sgorf76(−), regardless of the BmNPV inoculation dose. Additionally, the number of BmNPV DNA copies and the expression levels of viral genes were markedly inhibited in the transgenic line Cas9(+)/sgorf76(+) compared with the control line Cas9(−)/sgorf76(−). The results provide a promising target for Cas9-mediated antiviral therapy against BmNPV, and the findings provide new insights for baculovirus gene function studies and lepidopteran pest control.

Cloning and characterization of the histone variant gene H2A.Z in Bombyx mori.

H2A.Z, the most evolutionarily conserved variant of histone H2A, plays a pivotal role in chromatin remodeling and contributes significantly to gene transcription and genome stability. However, the role of H2A.Z in the silkworm (Bombyx mori) remains unclear. In this study, we cloned the BmH2A.Z from B. mori. The open reading frame of BmH2A.Z is 390 bp, encoding 129 amino acids, with a confirmed molecular weight of 13.4 kDa through prokaryotic expression analysis. Sequence analysis revealed that BmH2A.Z has a conserved H2A.Z domain and is closely related to the systemic evolution of other known H2A.Zs. The expression profile of BmH2A.Z at various developmental stages of the B. mori exhibited the highest expression level in the 1st instar, followed by the grain stage and the 2nd instar, and the lowest expression level in the moth. The highest transcript level of BmH2A.Z was observed in the head, with relatively lower levels detected in the blood than in the other tissues under consideration. In addition, the upregulation of BmH2A.Z resulted in the amplified expression of B. mori nucleopolyhedrovirus (BmNPV) genes, thus facilitating the proliferation of BmNPV. This study establishes a foundation for investigating the role of BmH2A.Z in B. mori and its participation in virus-host interactions.

Bombyx mori UFL1 facilitates BmNPV proliferation by regulating host cell apoptosis through PERK.

Ubiquitin-fold modifier 1 (UFM1) is attached to protein substrates through the sequential activity of an E1 (UBA5)-E2 (UFC1)-E3 (UFL1) cascade. UFL1 is the E3 ligase for UFMylation in vertebrates. However, there have been no studies on UFL1 in silkworm to date. In this study, we identified a UFL1 ortholog in Bombyx mori genome. Spatio-temporal expression profiles showed that BmUFL1 expression was high in the midgut, epidermis, and testis and in the pupa-adult stage. BmUFL1 knockdown inhibited B. mori nucleopolyhedrovirus (BmNPV)proliferation, while BmUFL1 overexpression promoted BmNPV proliferation. Mechanically, protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling and cell apoptosis are involved in BmUFL1-regulatedBmNPV proliferation. Overall, these results suggest that BmUFL1 facilitates BmNPV proliferation in silkworm.

Bombyx mori RPL13 participates in UV-induced DNA damage repair of B. mori nucleopolyhedrovirus through interaction with Bm65.

Ribosomal protein L13 (RPL13) is highly conserved in evolution. At present, the properties and functions of RPL13 have not been characterised in insects. In this study, Bombyx mori RPL13 (BmRPL13) was first found to be specifically recruited to the sites of ultraviolet (UV)-induced DNA damage and contributed to UV damage repair. Escherichia coli expressing BmRPL13 showed better resistance to UV radiation. After knocking down the expression of BmRPL13 in BmN cells, the repair speed of UV-damaged DNA slowed down. The further results showed that BmRPL13 interacted with B. mori nucleopolyhedrovirus (BmNPV) ORF65 (Bm65) protein to locate at the UV-induced DNA damage sites of BmNPV and helped repair UV-damaged viral DNA.

Role of serpin-25 in prophenoloxidase activation and expression of antimicrobial peptide genes in the silkworm Bombyx mori

Serine protease inhibitors (Serpins) are involved in diverse physiological and developmental processes, constituting one of the largest superfamilies of protease inhibitors. They are widely distributed across various organisms such as plants, animals, prokaryotes, and viruses. In insects, serpins exert their inhibitory effects on serine proteinase cascades, thereby regulating important pathways such as the Toll pathway and prophenoloxidase (proPO) in hemolymph. In this study, we cloned and analyzed the serpin-25 gene in Bombyx mori (Bmserpin-25). Bmserpin-25 mRNA was detected in all examined tissues, with high expression levels observed in the fat body, midgut, and epidermis. Upon injection of B. mori with four different microorganisms, including Gram-positive microbes (Micrococcus luteus), Gram-negative microbes (E. coli), fungi (Beauveria bassiana), and the B. mori nuclear polyhedrosis virus (BmNPV), the significant changes in Bmserpin-25 expression were observed specifically in response to E. coli and BmNPV treatments. Furthermore, the recombinant Bmserpin-25 was successfully expressed in Escherichia coli and purified for subsequent functional analysis. In vitro experiments showed that the recombinant Bmserpin-25 protein inhibited proPO activation but did not affect phenoloxidase activity. Additionally, injection of recombinant Bmserpin-25 protein into B. mori larvae resulted in a significant downregulation of antimicrobial peptide gene expression in the fat body. These findings emphasize the crucial role of serpin-25 in prophenoloxidase activation and the regulation of antimicrobial peptide gene expression in B. mori.

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.

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.

Single-nucleus sequencing of silkworm larval midgut reveals the immune escape strategy of BmNPV in the midgut during the late stage of infection

The midgut is an important barrier against microorganism invasion and proliferation, yet is the first tissue encountered when a baculovirus naturally invades the host. However, only limited knowledge is available how different midgut cell types contribute to the immune response and the clearance or promotion of viral infection. Here, single-nucleus RNA sequencing (snRNA seq) was employed to analyze the responses of various cell subpopulations in the silkworm larval midgut to B. mori nucleopolyhedrovirus (BmNPV) infection. We identified 22 distinct clusters representing enteroendocrine cells (EEs), enterocytes (ECs), intestinal stem cells (ISCs), Goblet cell-like and muscle cell types in the BmNPV-infected and uninfected silkworm larvae midgut at 72 h post infection. Further, our results revealed that the strategies for immune escape of BmNPV in the midgut at the late stage of infection include (1) inhibiting the response of antiviral pathways; (2) inhibiting the expression of antiviral host factors; (3) stimulating expression levels of genes promoting BmNPV replication. These findings suggest that the midgut, as the first line of defense against the invasion of the baculovirus, has dual characteristics of "resistance" and "tolerance". Our single-cell dataset reveals the diversity of silkworm larval midgut cells, and the transcriptome analysis provides insights into the interaction between host and virus infection at the single-cell level.

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.

Cytoskeleton Protein BmACT1 Is Potential for the Autophagic Function and Nuclear Localization of BmAtg4b in Bombyx mori.

Homologs of Autophagy-related (Atg) protein 4 are reported to cleave LC3 protein and facilitate autophagy occurrence differently in mammals, whereas their functions have not been investigated in insects. Three homologs, including BmAtg4a and its short form BmAtg4c as well as BmAtg4b, exist in Bombyx mori. Herein, the autophagic functions of BmAtg4a and BmAtg4b were investigated. qPCR detection found that BmAtg4a and BmAtg4b both peaked during larval-pupal metamorphosis when autophagy occurs robustly. Immunofluorescent staining showed that BmAtg4a was predominantly localized at the cytoplasm, while BmAtg4b had notable nuclear localization. Overexpression of BmAtg4a and BmAtg4b both slightly promoted basal autophagy but inhibited the autophagy induced by the infection of B. mori nucleopolyhedrovirus (BmNPV) and, thereby, its proliferation. In comparison, knockout of BmAtg4a or BmAtg4b significantly upregulated BmNPV-induced autophagy and its replication in BmN cells. Results of Co-immunoprecipitation associated with mass spectrum showed that the cytoskeleton protein B. mori actin A2 (BmACT2) and B. mori actin A1 (BmACT1) bound with BmAtg4a and BmAtg4b especially. Knockout of BmACT1 and BmACT2 inhibited BmAtg4b- and BmAtg4a-induced autophagy, respectively; moreover, knockout of BmACT1 reduced the ratio of cells with nuclear BmAtg4b. Of note, BmAtg4a and BmAtg4b had physical interaction, and they had an inhibitory effect on mutual autophagic function. In this work, we provide new insights into the autophagy machinery in insects as well as its function in the proliferation of BmNPV.

Reactive oxygen species–mediated phosphorylation of JNK is involved in the regulation of BmFerHCH on Bombyx mori nucleopolyhedrovirus proliferation

c-Jun N-terminal kinase (JNK) phosphorylation is widely observed during virus infection, modulating various aspects of the virus–host interaction. In our previous research, we have proved that B. mori ferritin heavy-chain homolog (BmFerHCH), an inhibitor of reactive oxygen species (ROS), facilitates B. mori nucleopolyhedrovirus (BmNPV) proliferation. However, one question remains: Which downstream signaling pathways does BmFerHCH regulate by inhibiting ROS? Here, we first determined that silencing BmFerHCH inhibits BmNPV proliferation, and this inhibition depends on ROS. Then, we substantiated that BmNPV infection activates the JNK signaling pathway. Interestingly, the JNK phosphorylation during BmNPV infection is activated by ROS. Further, we found that the enhanced nuclear translocation of phospho-JNK induced by BmNPV infection was dramatically reduced by pretreatment with the antioxidant N-acetylcysteine (NAC), whereas there was more detectable phospho-JNK in the cytoplasm. Next, we investigated how changes in BmFerHCH expression affect JNK phosphorylation. BmFerHCH overexpression suppressed the phosphorylation of JNK and nuclear translocation of phospho-JNK during BmNPV infection, whereas BmFerHCH knockdown facilitated phosphorylation of JNK and nuclear translocation of phospho-JNK. By measuring the viral load, we found the inhibitory effect of BmFerHCH knockdown on BmNPV infection depends on phosphorylated JNK. In addition, the JNK signaling pathway was involved in BmNPV-triggered apoptosis. Hence, we hypothesize that ROS-mediated JNK phosphorylation is involved in the regulation of BmFerHCH on BmNPV proliferation. These results elucidate the molecular mechanisms and signaling pathways of BmFerHCH-mediated response to BmNPV infection.

BmCH25H, a vertebrate interferon-stimulated gene(ISG) homolog, inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori.

Cholesterol-25-hydroxylase (CH25H) has been identified as an interferon-stimulated gene (ISG) in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol (25HC). However, invertebrates lack an antiviral system homologous to vertebrate interferons (IFNs) because the genomes of invertebrates do not encode IFN-like cytokines. Nevertheless, CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates. In this study, the Bombyx mori CH25H (BmCH25H) gene, of which the encoded protein has high homology with other lepidopteran species, was identified and located on chromosome 9. Interestingly, we found that the expression of BmCH25H was significantly upregulated in B.mori nucleopolyhedrovirus (BmNPV) -infected BmN cells and silkworm (B.mori) larvae at the early infection stage. The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC. More importantly, we demonstrated that during BmNPV infection, BmCH25H expression was increased through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, similar to the induction of ISGs following virus infection in vertebrates. This is the first report that CH25H has antiviral effects in insects; the study also elucidates the regulation of its expression and its mechanism of action.

Nuclear Factor Kappa B Promotes Ferritin Heavy Chain Expression in Bombyx mori in Response to B. mori Nucleopolyhedrovirus Infection.

Ferritin heavy chain (FerHCH) is a major component of ferritin and plays an important role in maintaining iron homeostasis and redox equilibrium. Our previous studies have demonstrated that the Bombyx mori ferritin heavy chain homolog (BmFerHCH) could respond to B. mori nucleopolyhedrovirus (BmNPV) infection. However, the mechanism by which BmNPV regulates the expression of BmFerHCH remains unclear. In this study, BmFerHCH increased after BmNPV infection and BmNPV infection enhanced nuclear factor kappa B (NF-κB) activity in BmN cells. An NF-κB inhibitor (PDTC) reduced the expression of the virus-induced BmFerHCH in BmN cells, and overexpression of BmRelish (NF-κB) increased the expression of virus-induced BmFerHCH in BmN cells. Furthermore, BmNPV infection enhanced BmFerHCH promoter activity. The potential NF-κB cis-regulatory elements (CREs) in the BmFerHCH promoter were screened by using the JASPAR CORE database, and two effective NF-κB CREs were identified using a dual luciferase reporting system and electrophoretic mobility shift assay (EMSA). BmRelish (NF-κB) bound to NF-κB CREs and promoted the transcription of BmFerHCH. Taken together, BmNPV promotes activation of BmRelish (NF-κB), and activated BmRelish (NF-κB) binds to NF-κB CREs of BmFerHCH promoter to enhance BmFerHCH expression. Our study provides a foundation for future research on the function of BmFerHCH in BmNPV infection.

Bombyx mori C-Type Lectin (BmIML-2) Inhibits the Proliferation of B. mori Nucleopolyhedrovirus (BmNPV) through Involvement in Apoptosis.

C-type lectins (CTLs) are widely distributed in mammals, insects, and plants, which act as pattern recognition receptors (PRRs) to recognize pathogens and initiate immune responses. In this study, we identified a C-type lectin gene called BmIML-2 from the silkworm Bombyx mori. Its open reading frame (ORF) encodes 314 amino acids, which contain dual tandem C-type lectin-like domain (CTLD). BmIML-2 is highly expressed in the fat body and is significantly induced at 24 h after BmNPV infection. Moreover, overexpression of BmIML-2 dramatically inhibited the proliferation of BmNPV, and knockdown assay via siRNA further validated the inhibition of BmIML-2 on viral proliferation. In addition, transcript level detection of apoptosis-related genes and observation of apoptosis bodies implied that overexpression of BmIML-2 promoted BmNPV-induced apoptosis. Immunofluorescence analysis indicated that BmIML-2 distributed throughout the cytoplasm and was slightly concentrated in the cell membrane. Taken together, our results suggest that BmIML-2 could inhibit in the proliferation of BmNPV by facilitating cell apoptosis.

Bombyx mori ferritin heavy-chain homolog facilitates BmNPV proliferation by inhibiting reactive oxygen species–mediated apoptosis

Ferritin heavy-chain homolog (FerHCH), an iron-binding protein, plays an important role in the host defense against oxidative stress and pathogen infections. In our previous research, Bombyx mori native ferritin had an interaction with B. mori nucleopolyhedrovirus (BmNPV). However, the underlying molecular mechanism of single ferritin homolog responses to BmNPV infection remains unclear. In this study, we found that BmNPV titer and B. mori FerHCH (BmFerHCH) expression were positively correlated with the ferric iron concentration. We performed RNA interference (RNAi) and overexpression experiments to investigate the effects of BmFerHCH on BmNPV proliferation. BmFerHCH knockdown suppressed BmNPV proliferation in vivo and in vitro, whereas BmFerHCH overexpression facilitated BmNPV proliferation. In addition, the oxidative stress level was increased significantly in BmN cells after budded virus infection, while BmFerHCH could neutralize the increased ROS production induced by BmNPV. Of note, we found that ROS was involved in BmNPV-induced apoptosis. Through inhibiting ROS, apoptosis was suppressed by BmFerHCH, whereas BmFerHCH knockdown facilitated apoptosis. Therefore, we hypothesize that BmFerHCH-mediated inhibition of virus-induced apoptosis depends on suppressing ROS accumulation and, thereby, facilitates virus replication. These results suggest that BmFerHCH plays an important role in facilitating BmNPV proliferation and modulating BmFerHCH is potential strategy for studying host–pathogen interactions.

Sirt5 Inhibits BmNPV Replication by Promoting a Relish-Mediated Antiviral Pathway in Bombyx mori.

Silent information regulators (Sirtuins) belong to the family of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases (HDACs) that have diverse functions in cells. Mammalian Sirtuins have seven isoforms (Sirt1–7) which have been found to play a role in viral replication. However, Sirtuin members of insects are very different from mammals, and the function of insect Sirtuins in regulating virus replication is unclear. The silkworm, Bombyx mori, as a model species of Lepidoptera, is also an important economical insect. B. mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects silkworms and causes serious losses in the sericulture industry. Here, we used the infection of the silkworm by BmNPV as a model to explore the effect of Sirtuins on virus replication. We initially knocked down all silkworm Sirtuins, and then infected with BmNPV to analyze its replication. Sirt2 and Sirt5 were found to have potential antiviral functions in the silkworm. We further confirmed the antiviral function of silkworm Sirt5 through its effects on viral titers during both knockdown and overexpression experiments. Additionally, Suramin, a Sirt5 inhibitor, was found to promote BmNPV replication. In terms of molecular mechanism, it was found that silkworm Sirt5 might promote the immune pathway mediated by Relish, thereby enhancing the host antiviral response. This study is the first to explore the role of Sirtuins in insect-virus interactions, providing new insights into the functional role of members of the insect Sirtuin family.

Deacetylation of HSC70-4 Promotes Bombyx mori Nucleopolyhedrovirus Proliferation via Proteasome-Mediated Nuclear Import.

Silkworm (Bombyx mori) is a model organism with great agricultural economic value that plays a crucial role in biological studies. B. mori nucleopolyhedrovirus (BmNPV) is a major viral pathogen found in silkworms, which leads to huge silk loss annually. In a recent lysine acetylome of silkworm infected with BmNPV, we focused on the heat shock cognate protein 70-4 (HSC70-4) lysine acetylation change due to the consequent nuclear accumulation and viral structure assembly. In this study, the genome replication, proliferation, and production of budded viruses (BVs) were arrested by HSP/HSC70 inhibitor treatment. However, HSC70-4 overexpression enhanced BmNPV reproduction. Furthermore, site-direct mutagenesis for acetylated mimic (K/Q) or deacetylated mimic (K/R) mutants of HSC70-4 demonstrated that lysine 77 (K77) deacetylation promotes HSC70-4 stability, viral DNA duplication, and HSC70-4 nuclear entry upon BmNPV challenge, and the nuclear propulsion of HSC70-4 after viral stimulus might be dependent on the interaction with the carboxyl terminus of HSC70-interacting protein (CHIP, an E3 ubiquitin ligase), followed by ubiquitin-proteasome system assistance. In this study, single lysine 77 deacetylation of HSC70-4 was deemed a part of the locomotive pathway for facilitating BmNPV proliferation and provided novel insights into the antiviral strategic development.

Advances in the Arms Race Between Silkworm and Baculovirus.

Insects are the largest group of animals. Nearly all organisms, including insects, have viral pathogens. An important domesticated economic insect is the silkworm moth Bombyx mori. B. mori nucleopolyhedrovirus (BmNPV) is a typical baculovirus and a primary silkworm pathogen. It causes major economic losses in sericulture. Baculoviruses are used in biological pest control and as a bioreactor. Silkworm and baculovirus comprise a well-established model of insect–virus interactions. Several recent studies have focused on this model and provided novel insights into viral infections and host defense. Here, we focus on baculovirus invasion, silkworm immune response, baculovirus evasion of host immunity, and enhancement of antiviral efficacy. We also discuss major issues remaining and future directions of research on silkworm antiviral immunity. Elucidation of the interaction between silkworm and baculovirus furnishes a theoretical basis for targeted pest control, enhanced pathogen resistance in economically important insects, and bioreactor improvement.

Identification of Peritrophins and Antiviral Effect of Bm01504 against BmNPV in the Silkworm, Bombyx mori.

The insect midgut secretes a semi-permeable, acellular peritrophic membrane (PM) that maintains intestinal structure, promotes digestion, and protects the midgut from food particles and pathogenic microorganisms. Peritrophin is an important PM protein (PMP) in the PM. Here, we identified 11 peritrophins with 1–16 chitin binding domains (CBDs) comprising 50–56 amino acid residues. Multiple CBDs in the same peritrophin clustered together, rather than by species. The CBD contained six highly conserved cysteine residues, with the key feature of amino acids between them being CX11-15CX5CX9-14CX11-12CX6-7C. Peritrophins with 2 and 4 CBDs (Bm09641 and Bm01504, respectively), and with 1, 8, and 16 CBDs (Bm11851, Bm00185, and Bm01491, respectively) were mainly expressed in the anterior midgut, and throughout the midgut, respectively. Survival rates of transgenic silkworms with Bm01504 overexpression (Bm01504-OE) and knockout (Bm01504-KO) infected with B. mori nucleopolyhedrovirus (BmNPV) were significantly higher and lower, whereas expression of the key viral gene, p10, were lower and higher, respectively, compared with wild type (WT). Therefore, Bm01504-OE and Bm01504-KO transgenic silkworms were more and less resistant, respectively, to BmNPV. Bm01504 plays important roles in resisting BmNPV invasion. We provide a new perspective for studying PM function, and reveal how the silkworm midgut resists invasive exogenous pathogenic microorganisms.

High-resolution analysis of baculovirus-induced host manipulation in the domestic silkworm, Bombyx mori.

Many parasites manipulate host behaviour to enhance their transmission. Baculoviruses induce enhanced locomotory activity (ELA) combined with subsequent climbing behaviour in lepidopteran larvae, which facilitates viral dispersal. However, the mechanisms underlying host manipulation system are largely unknown. Previously, larval locomotion during ELA was summarized as the distance travelled for a few minutes at several time points, which are unlikely to characterize ELA precisely, as ELA typically persists for several hours. In this study, we modified a recently developed method using time-lapse recording to characterize locomotion of Bombyx mori larvae infected with Bombyx mori nucleopolyhedrovirus (BmNPV) for 24 h at 3 s resolution. Our data showed that the locomotion of the mock-infected larvae was restricted to a small area, whereas the BmNPV-infected larvae exhibited a large locomotory area. These results indicate that BmNPV dysregulates the locomotory pattern of host larvae. Furthermore, both the mock- and BmNPV-infected larvae showed periodic cycles of movement and stationary behavior with a similar frequency, suggesting the physiological mechanisms that induce locomotion are unaffected by BmNPV infection. In contrast, the BmNPV-infected larvae exhibited fast and long-lasting locomotion compared with mock-infected larvae, which indicates that locomotory speed and duration are manipulated by BmNPV.