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Abstract: All lepidopteran baculovirus genomes sequenced encode a homolog of the Bombyx mori nucleopolyhedrovirus orf61 gene (Bm61). To determine the role of Bm61 in the baculoviral life cycle, we constructed a Bm61 knockout virus and characterized it in cells. We observed that the Bm61 deletion bacmid led to a defect in production of infectious budded virus (BV). Quantitative PCR analysis of BV in the media culturing the transfected cell indicated that BV was not produced due to Bm61 deletion. Electron microscope analysis showed that in the knockout of Bm61, nucleocapsids were not transported from the nucleus to the cytoplasm. From these results we concluded that BM61 is required in the BV pathway for the egress of nucleocapsids from the nucleus to the cytoplasm. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. • At present, there have been studies on small RNA regulation by using sensitive silkworm varieties, but no studies have conducted whole transcriptome analysis by comparing resistant and sensitive silkworm strans inoculated with virus. • The mRNA-miRNA-lncRNA ceRNA regulatory network was constructed, from which the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened, and 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. • From the perspective of lncRNA to explain the significantly different genes, our research results may provide new clues to the molecular mechanism that reveals the interaction between silkworm and virus. [ABSTRACT FROM AUTHOR]

Diagram illustrating the potential mechanism of BmNPV infection in the 306 strain. The three-color modules represent distinct signaling pathways: NF-kB (depicted in purple), MAPK (illustrated in green), and JAK/STAT (highlighted in pink). Genes outlined in red signify upregulation, while those outlined in green denote downregulation. Genes outlined in black represent expression levels of uncertainty. Illustrations were generated using BioRender. [Display omitted] • Integration of third-generation and second-generation high-throughput sequencing technologies for precise identification and analysis of alternative splicing (AS) patterns. • Identification of five pivotal genes (Dsclp, LOC692903, LOC101743583, LOC101742498, LOC101743809) associated with the response to BmNPV infection. • Discovery of novel transcripts, expanding the current understanding beyond what is available in SilkBase. The silkworm, Bombyx mori, stands out as one of the few economically valuable insects within the realm of model organisms. However, Bombyx mori nucleopolyhedrovirus (BmNPV) poses a significant threat, decreasing the quality and quantity of silkworm cocoons. Over the past few decades, a multitude of researchers has delved into the mechanisms that underlie silkworm resistance to BmNPV, employing diverse methodologies and approaching the problem from various angles. Despite this extensive research, the role of alternative splicing (AS) in the silkworm's response to BmNPV infection has been largely unexplored. This study leveraged both third-generation (Oxford Nanopore Technologies) and second-generation (Illumina) high-throughput sequencing technologies to meticulously identify and analyze AS patterns in the context of BmNPV response, utilizing two distinct silkworm strains—the susceptible strain 306 and the resistant strain NB. Consequently, we identified five crucial genes (Dsclp , LOC692903, LOC101743583, LOC101742498, LOC101743809) that are linked to the response to BmNPV infection through AS and differential expression. Additionally, a thorough comparative analysis was conducted on their diverse transcriptomic expression profiles, including alternative polyadenylation, simple sequence repeats, and transcription factors. [ABSTRACT FROM AUTHOR]

Interacting with cell surface attachment factors or receptors is the first step for virus infection. Glycans cover a thick layer on eukaryotic cells and are potential targets of various viruses. Bombyx mori nuclear polyhedrosis viruses (BmNPV) is a baculovirus that causes huge economic loss to the sericulture industry but the mechanism of infection is unclear. Looking for potential host receptors for the virus is an important task. In this study, we investigated the role of glycosaminoglycan (GAG) modifications, including heparan sulfate (HS) and chondroitin sulfate (CS), during BmNPV infection. Enzymatic removal of cell surface HS and CS effectively inhibited BmNPV infection and replication. Exogenous HS and CS can directly bind to BmNPV virion in solution and act as neutralizers for viral infection. Furthermore, the expression of enzymes involved in GAG biosynthesis was upregulated in the BmNPV susceptible silkworm after virus administration, but down-regulated in the resistant strain after virus treatment, suggesting that BmNPV was able to utilize host cell machinery to promote the biosynthesis of GAGs. This study demonstrated HS and CS as important attachment factors that facilitate the viral entry process, and targeting HS and CS can be an effective means of inhibiting BmNPV infection. • BmNPV infection was suppressed after cell surface heparin sulfate and chondroitin sulfate were reduced. • Heparan sulfate and chondroitin sulfate can directly bind to BmNPV in solution. • GAG biosynthesis was promoted in the susceptible silkworm but suppressed in the resistant strain upon BmNPV treatment. [ABSTRACT FROM AUTHOR]

Interacting with cell surface attachment factors or receptors is the first step for virus infection. Glycans cover a thick layer on eukaryotic cells and are potential targets of various viruses. Bombyx mori nuclear polyhedrosis viruses (BmNPV) is a baculovirus that causes huge economic loss to the sericulture industry but the mechanism of infection is unclear. Looking for potential host receptors for the virus is an important task. In this study, we investigated the role of glycosaminoglycan (GAG) modifications, including heparan sulfate (HS) and chondroitin sulfate (CS), during BmNPV infection. Enzymatic removal of cell surface HS and CS effectively inhibited BmNPV infection and replication. Exogenous HS and CS can directly bind to BmNPV virion in solution and act as neutralizers for viral infection. Furthermore, the expression of enzymes involved in GAG biosynthesis was upregulated in the BmNPV susceptible silkworm after virus administration, but down-regulated in the resistant strain after virus treatment, suggesting that BmNPV was able to utilize host cell machinery to promote the biosynthesis of GAGs. This study demonstrated HS and CS as important attachment factors that facilitate the viral entry process, and targeting HS and CS can be an effective means of inhibiting BmNPV infection. • BmNPV infection was suppressed after cell surface heparin sulfate and chondroitin sulfate were reduced. • Heparan sulfate and chondroitin sulfate can directly bind to BmNPV in solution. • GAG biosynthesis was promoted in the susceptible silkworm but suppressed in the resistant strain upon BmNPV treatment. [ABSTRACT FROM AUTHOR]

Baculoviruses have been developed as long-term and environmentally friendly biopesticides. However, solar ultraviolet radiation can reduce the activity of baculovirus. Radiation sensitive 23 (Rad23) can recognize DNA damage and is involved with nucleotide excision repair (NER). In the current study, BmRad23 was accumulated mainly within the nucleus. Host cell reactivation (HCR) assays have shown that BmRad23 significantly facilitated the expression of UV-damaged mCherry reporter gene. Reverse transcription quantitative PCR (RT-qPCR) result showed that the mRNA expression level of BmRad23 was increased in (Bombyx mori nuclear polyhedrovirus, BmNPV) BmNPV-infected BmN cells. However, the expression of BmRad23 was increased significantly when BmNPV budded viruses (BVs) or BmN cells were irradiated with UV light. Overexpression of BmRad23 promoted the mRNA levels of two UV-induced DNA damage repair genes which were from Bombyx mori and BmNPV, respectively. Meanwhile, the overexpression of BmRad23 in BmN cells was conducive to the proliferation of BmNPV and UV-damaged BmNPV. The recombinant BmNPV BVs expressing BmRad23 showed stronger resistance to UV radiation than the control virus. In conclusion, the results revealed that BmRad23 contributed to the proliferation of BmNPV and the repair of UV-damaged BmNPV, which would provide a reference for the development of efficient baculovirus pesticides against UV radiation. Unlabelled Image • BmRad23 was conducive to the repair of UV-damaged report gene. • The mRNA expression of BmRad23 was significantly increased if B. mori cells or BmNPV BVs were irradiated with UV light. • BmRad23 was conducive to the proliferation of BmNPV and the repair of UV-damaged BmNPV. [ABSTRACT FROM AUTHOR]

The silkworm Bombyx mori ( B. mori ), a lepidopteran model organism, has become an important model for molecular biology researches with its genome completely sequenced. Silkworms confront different types of virus diseases, mainly including those caused by Bombyx mori nucleopolyhedrovirus (BmNPV), Bombyx mori densovirus type 1 (BmDNV-1), Bombyx mori bidesovirus (BmBDV) which was termed as Bombyx mori densovirus type 2 (BmDNV-2) or Bombyx mori parvo-like virus (BmPLV) before in sericulture. B. mori offers excellent models to study the molecular mechanisms of insect innate immune responses to viruses. A variety of molecules and pathways have been identified to be involved in the immune responses in the silkworm to viruses, such as the antimicrobial peptides, prophenoloxidase-activating system, apoptosis, ROS, small RNA and related molecules. Here in this review, we summarize the current research advances in molecules involved in silkworm anti-virus pathways. Moreover, taking BmNPV as an example, we proposed a schematic model of molecules and pathways involved in silkworm immune responses against virus infection. We hope this review can facilitate further study of antiviral mechanisms in silkworm, and provide a reference for virus diseases in other organisms. [ABSTRACT FROM AUTHOR]

It is unclear how, or to what extent, baculovirus DNA that has been damaged by ultraviolet (UV) light is repaired during infection and replication. In our previous study, expression of Bombyx mori nucleopolyhedrovirus (BmNPV) ORF Bm65, a homolog of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac79, correlated with decreased inactivation of virus by UV irradiation. In the current study, we accumulated more evidence pointing to a role for Bm65 in repair of UV-induced DNA damage. The localization of Bm65 was studied using enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bm65 expression plasmid. The results indicate that Bm65-EGFP accumulates in the nucleus. A host cell reactivation assay showed that Bm65 significantly increased the expression of UV-damaged mCherry reporter gene. An assay measuring cyclobutane pyrimidine dimers (CPDs) in UV-irradiated BmN cells found that CPD quantity was decreased in cells transfected with a Bm65 expression plasmid. We also showed that after UVC treatment, the viability of Bm65-transfected cells was higher than that of egfp-transfected cells. These results suggest that Bm65 may be involved in the repair of baculovirus DNA that has been damaged by UV light. [ABSTRACT FROM AUTHOR]

[Display omitted] Solar ultraviolet radiation (UV) can cause DNA damage in microorganisms. Flap endonuclease 1 (FEN1) is a structure-specific nuclease and plays important roles in DNA replication and repair. At present, the properties and functions of FEN1 have not been characterized in detail in invertebrates such as Bombyx mori. In this study, Bombyx mori FEN1 (BmFEN1) was expressed in E. coli, and was shown to have nuclease activity that nonspecifically cleaved DNA in vitro. However, inside the cell, BmFEN1 did not cleave DNA randomly. Truncated BmFEN1 missing the nuclear localization signal (346–380 aa) still had the nuclease activity, but was no longer precisely localized to the sites of UV-induced DNA damage. It was further found that BmFEN1 favored the faster repair of UV-damaged DNA. The present study will provide a reference for further understanding the functions of BmFEN1 and UV-induced DNA damage repair mechanisms in insects. [ABSTRACT FROM AUTHOR]

[Display omitted] • Trends of metabolite levels in silkworm hemolymph upon BmNPV treatment were revealed. • BmNPV reprogrammed metabolic pathways involving trehalose, glutamine, and tryptophan. • Tryptophan inhibited BmNPV replication while glutamine promoted it. • Trehalose and riboflavin affected BmNPV replication in a complex manner. Elucidating the mechanism of infection of Bombyx mori nuclear polyhedrosis virus (BmNPV) and host antiviral response remains a major scientific task in sericulture. Virus invasion causes a series of antiviral immune responses in the host, and successful infection leads to massive changes in the host's physiological and biochemical state. Current research mainly focuses on silkworm genes and proteins associated with viral infection and resistance, but little is known regarding the host metabolic pathways that the virus utilizes for optimal replication. In this work, key metabolites involved in viral infection were identified, including trehalose, riboflavin, tryptophan, tyrosine, and phenylalanine. The genes associated with metabolite biosynthesis and catabolism were analyzed, and their expression levels were found to be largely consistent with their respective metabolite levels before and after viral treatment in both strains. The screened metabolites were further investigated for their roles in viral replication using exogenous metabolite addition into the culture medium. The results showed that tryptophan effectively inhibited BmNPV replication, while glutamine promoted viral replication in a dose-dependent manner. Trehalose and riboflavin exhibited a complex effect on BmNPV replication. This study outlines the critical metabolites and metabolic pathways required for BmNPV to proliferate and infect the host, indicting the potential of metabolite-based treatment for viral inhibition. [ABSTRACT FROM AUTHOR]

Abstract: Orf91 (Bm91) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes a predicted 105-amino-acid protein, but its function remains unknown. In the current study, 5′-RACE revealed that the transcription initiation site of Bm91 was – 12 nucleotides upstream of the start codon ATG, transcription of Bm91 was detected from 12 to 96h postinfection (p.i.) and Bm91 protein was detected from 24 to 96h p.i. in BmNPV-infected BmN cells. Furthermore, Western blot analysis revealed that Bm91 was in occlusion-derived virus (ODV) but not in budded virus (BV). To investigate the role of Bm91 in baculovirus life cycle, a Bm91-knockout virus was constructed by bacmid recombination in E. coli. Fluorescence and light microscopy showed that the production of BV and occlusion bodies (OBs) in Bm91-deficient-virus-infected BmN cells were similar to those in wild-type-virus-infected ones. Bioassay results showed that genetic deletion of Bm91 did not significantly affect BmNPV infectivity, but extended the median lethal time (LT50). Taken together, these results indicate that Bm91 is not essential for viral propagation in vitro, but absence of the gene may affect the virulence of ODVs in silkworm larvae. [Copyright &y& Elsevier]

• Baculovirus proliferation inhibition was observed in BmNPV-resistant silkworm strains. • Virus gene transcription was significantly inhibited in resistant silkworm tissues. • Insect innate immunity responses contribute to BmNPV resistance at varying levels. • Antiviral activity of BmSOCS2 on BmNPV proliferation inhibition evidenced in vitro. The resistance of silkworm to infection by Bombyx mori nuclear polyhedrosis virus (BmNPV) is a main focus of sericultural research. Previously, a BmNPV-resistant strain, NB, was identified among a collection of Chinese silkworm strains in our lab. To better understand the molecular mechanism of NB strain resistance, the patterns of host immune response gene transcription in resistant (NB) and susceptible (306) strains were examined. Quantative real-time PCR (qRT-PCR) revealed that multiple insect innate immune signaling pathways (Toll, Imd and JAK/STAT) were strongly activated upon infection with BmNPV. Notably, Suppressor of cytokine signaling 2 (BmSOCS2) mRNA expression was significantly up-regulated in midgut tissues of the resistant NB strain, suggesting that the BmSOCS2 gene product may be involved in host immune defense against BmNPV infection. A significant inhibition of BmNPV replication was also observed in BmN cells transfected with a vector encoding BmSOCS2. The results suggest that BmSOCS2 is a key gene involved in the resistance of the NB silkworm strain to BmNPV infection. [ABSTRACT FROM AUTHOR]