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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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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

The 871C silkworm strain exhibits a high level of resistance to Bombyx mori nucleopolyhedrovirus (BmNPV), making it a valuable variety for the sericulture industry. Understanding the underlying mechanism of its resistance holds great biological significance and economic value in addressing viral disease risks in sericulture. Initially, we infected the resistant strain 871C and its control strain 871 with BmNPV and conducted secondary infection experiments using the progeny occlusion bodies (OBs). As a result, a significant decrease in pathogenicity was observed. Electron microscopy analysis revealed that 871C produces progeny virions with defective DNA packaging, reducing virulence following BmNPV infection. Blood proteomic identification of the silkworm variety 871C and control 871 after BmNPV infection demonstrated the crucial role of the viral proteins P6.9 and VLF-1 in the production of defective viruses by impeding the proper encapsulation of viral DNA. Additionally, we discovered that BmHSP19.9 interacts with P6.9 and VLF-1 and that its expression is significantly upregulated after BmNPV infection. BmHSP19.9 exhibits strong antiviral activity, in part by preventing the entry of the proteins P6.9 and VLF-1 into the nucleus, thereby hindering viral nucleocapsid and viral DNA assembly. Our findings indicate that the antiviral silkworm strain 871C inhibits BmNPV proliferation by upregulating Bmhsp19.9 and impeding the nuclear localization of the viral proteins P6.9 and VLF-1, leading to the production of defective viral particles. This study offers a comprehensive analysis of the antiviral mechanism in silkworms from a viral perspective, providing a crucial theoretical foundation for future antiviral research and the breeding of resistant silkworm strains., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest to report regarding the present study., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Apoptosis is a cellular defense mechanism used for the elimination of host cells infected by viruses. Viruses have evolved corresponding inhibitors of apoptosis genes to promote their replication. Anti-apoptosis-related genes, involved in baculovirus proliferation, have been proposed but it is unclear whether these genes can be manipulated in gene therapy. We constructed a transgenic silkworm, using the CRISPR/Cas9 system to knock out the BmNPV inhibitor of apoptosis 2 (iap2). The sequencing results showed that all the sequences could edit the target site of BmNPV iap2 gene. There were no differences in economic traits and growth tests between the BmNPV iap2 knockout strain transgenic silkworm lines and the control groups. However, the mortality rate was significantly reduced, the median lethal dose (LD50) was about 100 times higher than the control group, and the onset time was prolonged by 1-2 days after knocking out BmNPV iap2. In addition, the expression levels of apoptotic-related genes Bmiap2, BmICE and BmDreed were significantly affected and the activity of caspase 9 was increased after BmNPV iap2 being edited in transgenic silkworm. These results demonstrated that gene editing BmNPV iap2 could significantly inhibit BmNPV replication and proliferation. This approach provides a new strategy for antiviral research., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down-regulation of BmPP2A expression. Furthermore, up-regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down-regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti-BmNPV mechanism, and a possible target for the breeding of silkworm-resistant strains., (© 2019 Institute of Zoology, Chinese Academy of Sciences.)

As an important insect immune response, apoptosis plays a critical role in the interaction between baculoviruses and insect hosts. Previous reports have identified inhibitor of apoptosis (IAP) proteins in both insects and baculoviruses, but the relationship between these proteins is still not clearly understood. Here, we found that insect IAP proteins were clustered with baculovirus IAP3, suggesting that the baculovirus iap3 gene might be derived from the Lepidoptera or Diptera. We demonstrated that Bombyx mori inhibitor of apoptosis (Bmiap) gene had an inhibitory effect on apoptosis in silkworm cells. Further analysis of the effects of Bmiap genes on the proliferation of B. mori nucleopolyhedrovirus (BmNPV) showed that both the Bmiap and BmNPV iap genes increased BmNPV proliferation after BmNPV infected silkworm cells. Our results also indicated that BmNPV IAP1 and IAP2 directly interacted with BmIAP in silkworm cells, implying that the Bmiap gene might be hijacked by BmNPV iap genes during BmNPV infection. Taken together, our results provide important insights into the functional relationships of iap genes, and improve our knowledge of apoptosis in baculoviruses and insect hosts., (© 2019 Institute of Zoology, Chinese Academy of Sciences.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens that causes severe economic losses to sericulture. Comparative transcriptomics analysis has been widely applied to explore the antiviral mechanism in resistant strains. Here, to identify genes involved in BmNPV infection, we identified differentially expressed genes (DEGs) and performed weighted gene co-expression network analysis (WGCNA) between two Bombyx mori strains: strain 871 (susceptible to BmNPV infection) and the near-isogenic strain 871C (resistant to BmNPV). Our results showed that 400 genes were associated with resistance in strain 871C, and 76 genes were related to susceptibility in strain 871. In addition, the correlation analysis of DEGs and WGCNA showed that 40 genes related to resistance were highly expressed in the resistant strain. Among them, gene BGIBMGA004291 was the most noticeable. We further identified the effect of gene BGIBMGA004291, which encoded a multiprotein bridge factor 2 (MBF2) family member (MBF2-10), on viral infection in cells. Our data suggested that MBF2-10 inhibited viral infection. Taken together, this study showed specific module trait correlations related to viral infection in strains 871 and 871C, and we identified a resistance-related gene. These findings suggested promising candidate genes with antiviral activity, aiding in the analysis of the antiviral molecular mechanisms in resistant strains., (© 2019 The Royal Entomological Society.)

Autophagy is a cell adaptive response that involves the process of microbial infections. Our previous study has indicated that Bombyx mori nucleopolyhedrovirus (BmNPV) infection triggers the complete autophagic process in BmN-SWU1 cells, which is beneficial to the viral infection. Autophagy-related (ATG) protein ATG13, as part of the ULK complex (a serine-threonine kinase complex composed of ULK1, ULK2, ATG13, ATG101, and FIP200), is the most upstream component of the autophagy pathway, and how it affects virus infections will improve our understanding of the interaction between the virus and the host. This study has determined that the overexpression of the BmAtg13 gene promotes the expression of viral genes and increases viral production in BmN-SWU1 cells, whereas knocking down the BmAtg13 gene suppresses BmNPV replication. Moreover, the BmAtg13 overexpression transgenic line contributed to viral replication and increased mortality rate of BmNPV infection. In contrast, the BmAtg13 knockout transgenic line reduced viral replication 96 h post-infection. Furthermore, BmATG13 directly interacted with viral protein BRO-B, forming a protein complex. Taken together, the findings of this study suggest that BmATG13 may be utilized by the BRO-B protein to promote BmNPV replication and proliferation, which, in turn, provides important insights into the mechanism that autophagy influences viral infection., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lysozymes is a ubiquitous immune effector that is widely distributed in both vertebrates and invertebrates. Previous reports have shown that lysozymes significantly inhibit viral infections in vertebrates. However, the antiviral effects of lysozymes in invertebrates remain unclear. Here, we investigated the role of lysozymes in Bombyx mori (B. mori) response to viral infection by overexpressing B. mori C-lysozyme (BmC-LZM) in larvae and cells. We found that BmC-LZM was up-regulated in cells in response to viral infection. Indeed, the overexpressing of BmC-LZM significantly inhibited viral replication in cells during late-stage infection. However, this effect was reversed by BmC-LZM mRNA. BmC-LZM was successfully overexpressed in B. mori strain 871 using Baculovirus Expression Vector System (BEVS). This overexpression markedly reduced viral proliferation and increased larval survival percentage. Thus, BmC-LZM inhibited viral replication both in vivo and in vitro, indicating that BmC-LZM is involved in the insect immune response to viral infection. Our results provide a basis for further applications of lysozymes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bombyx mori nuclear polyhedrosis virus (BmNPV) is an important pathogen of silkworms. Despite extensive studies in recent decades, the interaction between BmNPV and host cells is still not clearly understood. Autophagy is an intrinsic innate immune mechanism and it controls infection autonomously in virus-infected cells. In this study, we found that BmNPV infection could trigger autophagy, as demonstrated by the formation of autophagosomes, fluorescent Autophagy-related gene 8-Green Fluorescent Protein (ATG8-GFP) punctate, and lipidated ATG8. Meanwhile, autophagic flux increased significantly when monitored by the ATG8-GFP-Red Fluorescent Protein (RFP) autophagy tandem sensor and protein degradation of p62. In addition, almost all of the identified autophagy-related genes ( Atgs ) had been up-regulated post infection in mRNA levels. Then, we screened Atgs with the greatest fold-change during virus infection. Interestingly, all of the screened Atgs positively regulated the expression of virus genes. Further studies showed that Atg7 and Atg9 could contribute to the level of autophagy caused by viral infection. Our results demonstrated that BmNPV induced host cell autophagy to benefit its infection. These results offer insight into the complex interactions between virus and host cell, and viral pathogenesis., Competing Interests: The authors declare no conflict of interest.

Pathogen-inducible promoters have been studied extensively and widely used in resistance breeding and gene therapy. However, few reports have been published that explore the efficacy of Bombyx mori nucleopolyhedrovirus (BmNPV)-inducible promoters in antiviral research in the Bombyx mori (Lepidoptera). Here, we screened BmNPV promoters (VP1054, P33, Bm21, Bm122, 39K, P143, and P6.9) and found that the 39K promoter had the highest BmNPV-induced transcriptional activity by dual-luciferase reporter assays system. By 5' truncation analysis, two regions of 39K promoter were critical for optimal virus-inducible activity, indicated that they could serve as a candidate to produce synthetic pathogen-induced promoters. Furthermore, we enhanced the virus-inducible activity of BmNPV 39K promoter using a hybrid enhancer comprising hr3 and polh-up (designated as HP39K). Finally, we showed that RNAi regulated by HP39K promoter could significantly inhibit the proliferation of BmNPV in silkworm cells. Taken together, our results have practical value in antiviral research of silkworm and baculovirus expression system., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We have previously reported that baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) late expression factor 11 (lef-11) is associated with viral DNA replication and have demonstrated that it potentially interacts with itself; however, whether LEF-11 forms oligomers and the impact of LEF-11 oligomerization on viral function have not been substantiated. In this study, we first demonstrated that LEF-11 is capable of forming oligomers. Additionally, a series of analyses using BmNPV LEF-11 truncation mutants indicated that two distinct domains control LEF-11 oligomerization (aa 42-61 and aa 72-101). LEF-11 truncation constructs were inserted into a lef-11-knockout BmNPV bacmid, which was used to demonstrate that truncated LEF-11 lacking either oligomerization domain abrogates viral DNA replication. Finally, site-directed mutagenesis was used to determine that the conserved hydrophobic residues Y58&I59 (representing Y58 and I59), I85 and L88&L89 (representing L88 and L89) are required for LEF-11 oligomerization and viral DNA replication. Collectively, these data indicate that BmNPV LEF-11 oligomerization influences viral DNA replication.

We previously established and characterized two insect cell lines (BmN-SWU1 and BmN-SWU2) from Bombyx mori ovaries. Here, we examined their differential susceptibilities to Bombyx mori nucleopolyhedrovirus (BmNPV) despite having originated from the same tissue source. BmN-SWU1 cells were susceptible and supported high titers of BmNPV replication, while BmN-SWU2 cells were resistant to BmNPV infection. Subcellular localization analysis demonstrated that very few BmNPV particles could be imported into BmN-SWU2 cells. However, initiation of BmNPV DNA replication but not amplification was detected in BmN-SWU2 cells after transfection with vA4prm-VP39-EGFP bacmid DNA. BmNPV transcription assays showed that late and very late but not early viral genes apparently were blocked in BmNSWU2 cells by unknown mechanisms. Further syncytium formation assays demonstrated that the BmNPV envelope fusion protein GP64 could not mediate BmN-SWU2 host cell-cell membrane fusion. Taken together, these results indicate that these two cell lines represent optimal tools for investigating host-virus interactions and insect antiviral mechanisms.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF79 (Bm79) encodes an occlusion-derived virus (ODV)-specific envelope protein, which is a homologue of the per os infectivity factor 4 (PIF4) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To investigate the role of ORF79 in the BmNPV life cycle, a Bm79 knockout virus (vBm(Bm79KO)) was constructed through homologous recombination in Escherichia coli. Viral DNA replication, budded virus (BV) production and polyhedra formation were unaffected by the absence of BM79. However, results of the larval bioassay demonstrated that the Bm79 deletion resulted in a complete loss of per os infection. Immunofluorescence analysis showed that BM79 localized at the innernuclear membrane of infected cells through its N-terminal sorting motif (SM). Further bimolecular fluorescence protein complementation and co-immunoprecipitation assays demonstrated the interaction of BM79 with PIF1, PIF2, PIF3 and ODV-E66. Thus, BM79 plays an important role in per os infection and is associated with the viral PIF complex of BmNPV., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The baculovirus late expression factor 11 (LEF-11) has been reported to be involved in viral DNA replication and late/very late gene activation. In this study, serial N- and C-terminal truncations of Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 protein were fused with DsRed to investigate the nuclear localization signal by which LEF-11 enters the nucleus. Results show that 72-101 residues at the C-terminus are essential for BmNPV LEF-11 nuclear localization. Sequence alignment of this NLS from multiple LEF-11 homologs revealed high conservation in general. Site-directed mutation analysis showed that five basic residue clusters, namely, K(75)/R(76), H(81), K(83)/R(84), R(87) and K(100), were critical for the nuclear localization of BmNPV LEF-11. Co-IP analysis shows that LEF-11 binds directly to host importin α-3. Immunofluorescence analysis demonstrated that LEF-11 co-localizes with the immediate-early protein IE-1 at viral DNA replication sites in the nucleus. Further BiFC assays demonstrated the interaction of LEF-11 with LEF-3 and LEF-11 itself in the nucleus. Together, these results reveal a previous unknown mechanism for nuclear translocation of baculovirus LEF-11., (Copyright © 2014 Elsevier B.V. All rights reserved.)