Bombyx mori Nucleopolyhedrovirus Hijacks Multivesicular Body as an Alternative Envelopment Platform for Budded Virus Egress.

Baculovirus budded virus (BV) acquires its envelope and viral membrane fusion proteins from the plasma membrane (PM) of the host cell during the budding process. However, this classical BV egress pathway has been questioned because an intracellularly localized membrane fusion protein, SPΔnGP64 (glycoprotein 64 [GP64] lacking the signal peptide [SP] n region), was assembled into the envelope to generate infective BVs in our recent studies. Here, we identify an additional pathway for Bombyx mori nucleopolyhedrovirus (BmNPV) BV assembly and release that differs, in part, from the currently accepted model for the egress pathway of baculovirus. Electron microscopy showed that during infection, BmNPV-infected cells contained many newly formed multivesicular body (MVB)-like compartments that included mature virions at 30 h postinfection (p.i.). Immunoelectron microscopy demonstrated that the MVBs contained CD63, an MVB endosome marker, and GP64, a BmNPV fusion glycoprotein. MVB fusion with the PM and the release of mature virions, together with naked nucleocapsids, were observed at the cell surface. Furthermore, MVB egress mediated the translocation of SPΔnGP64 to the PM, which induced cell-cell fusion until 36 h p.i. This BV egress pathway can be partially inhibited by U18666A incubation and RNA interference targeting MVB biogenesis genes. Our findings indicate that BmNPV BVs are enveloped and released through MVBs via the cellular exosomal pathway, which is a subordinate BV egress pathway that produces virions with relatively inferior infectivity. This scenario has significant implications for the elucidation of the BmNPV BV envelopment pathway. IMPORTANCE BmNPV is a severe pathogen that infects mainly Bombyx mori, a domesticated insect of economic importance, and accounts for approximately 15% of economic losses in sericulture. BV production plays a key role in systemic BmNPV infection of larvae. Despite the progress made in the functional gene studies of BmNPV, BmNPV BV egress is ill-understood. This study reports a previously unreported MVB envelopment pathway in BmNPV BV egress. To our knowledge, this is the first report of a baculovirus using dual BV egress pathways. This specific BV egress mechanism explains the cause of the non-PM-localized SPΔnGP64-rescued gp64-null bacmid infectivity, elucidating the reason underlying the retention of SP by BmNPV GP64. The data obtained elucidate an alternate molecular mechanism of baculovirus BV egress.

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.

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

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

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

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

Characterization of the UDP-glycosyltransferase UGT33D1 in silkworm Bombyx mori.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are important metabolizing enzymes functioning by adding a sugar moiety to a small lipophilic substrate molecule and play critical roles in drug/toxin metabolism for all realms of life. In this study, the silkworm Bombyx mori UGT33D1 gene was characterized in detail. UGT33D1 was found localized in the endoplasmic reticulum (ER) compartment just like other animal UGTs and was mainly expressed in the silkworm midgut. We first reported that UGT33D1 was important to BmNPV infection, as silencing UGT33D1 inhibited the BmNPV infection in silkworm BmN cells, while overexpressing the gene promoted viral infection. The molecular pathways regulated by UGT33D1 were analysed via transcriptome sequencing upon UGT33D1 knockdown, highlighting the important role of the gene in maintaining a balanced oxidoreductive state of the organism. In addition, proteins that physically interact with UGT33D1 were identified through immunoprecipitation and mass spectrometry analysis, which includes tubulin, elongation factor, certain ribosomal proteins, histone proteins and zinc finger proteins that had been previously reported for human UGT-interacting proteins. This study provided preliminary but important functional information on UGT33D1 and is hoped to trigger deeper investigations into silkworm UGTs and their functional mechanisms.

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-regulated BmNPV proliferation. Overall, these results suggest that BmUFL1 facilitates BmNPV proliferation in silkworm.

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.

Comprehensive transcriptome sequencing of silkworm Midguts: Uncovering extensive isoform diversity and alternative splicing in BmNPV-Sensitive and BmNPV-resistant strains.

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.

An electrochemical immunosensor based on hybrid self-assembled monolayers for rapid detection of Bombyx mori nucleopolyhedrovirus.

Bombyx mori nucleopolyhedrovirus (BmNPV) is highly contagious and poses a serious threat to sericulture production. Because there are currently no effective treatments for BmNPV, a rapid and simple detection method is urgently needed. This paper describes an electrochemical immunosensor for the detection of BmNPV. The immunosensor was fabricated by covalently immobilizing anti-BmNPV, a biorecognition element, onto the surface of the working gold electrode via 11-mercaptoundecanoic acid (MUA)/ß-mercaptoethanol (ME) hybrid self-assembled monolayers. Electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) were used to characterize the electrochemical performance and morphology of the immunosensor, respectively. Under optimum conditions, the developed immunosensor exhibited a linear response to BmNPV polyhedrin in the range of 1 × 102-1 × 108 fg/mL, with a low detection limit of 14.54 fg/mL. The immunosensor also exhibited remarkable repeatability, reproducibility, specificity, accuracy, and regeneration. Normal silkworm blood was mixed with BmNPV polyhedrin and analyzed quantitatively using this sensor, and the recovery was 92.31 %-100.61 %. Additionally, the sensor was used to analyze silkworm blood samples at different time points after BmNPV infection, and an obvious antigen signal was detected at 12 h post infection. Although this result agreed with that provided by the conventional polymerase chain reaction (PCR) method, the electroanalysis method established in this study was simpler, shorter in detection period, and lower in material cost. Furthermore, this innovative electrochemical immunosensor, developed for the ultra-sensitive and rapid detection of BmNPV, can be used for the early detection of virus-infected silkworms.

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

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

Development of a rapid visual detection technology for BmNPV based on CRISPR/Cas13a system.

Pathogenic microorganism of silkworm are important factors that threaten the high-quality development of sericulture. Among them, Bombyx mori nucleopolyhedrovirus (BmNPV) caused diseases often lead to frequent outbreaks and high mortality, resulting in huge losses to sericultural industry. Current molecular detection methods for BmNPV require expensive equipment and sikilled technical personnel. As a result, the most commonly detection method for silkworm egg production enterprises involves observing the presence of polyhedra under a microscope. However, this method has low accuracy and sensitivity. There is an urgent need to develop a new detection technology with high sensitivity, high specificity, and applicability for silkworm farms, silkworm egg production enterprises and quarantine departments. In this study, we successfully established the CRISPR/Cas13a BmNPV visualized detection technology by combining Recombinase Polymerase Amplification (RPA) technology and CRISPR/Cas13a system. This technology is based on microplate lateral, flow test strips and portable fluorescence detector. The detection sensitivity can reach up to 1 copies/µL for positive standard plasmid and 1 fg/µL for BmNPV genome in 30-45 min, demonstrating high sensitivity. By detecting silkworm tissues infected with different pathogens, we determined that CRISPR/Cas13a detection technology has good specificity. In summary, the newly established nucleic acid detection technology for BmNPV is characterized by high sensitivity, high specificity, low cost and convenience for visualization. It can be applied in field detection and silkworm egg quality monitory system.

Characterization of a silkworm UFM1 homolog in regulating Bombyx mori unfolded protein response and nucleopolyhedrovirus replication.

The Ubiquitin (Ub)-like molecules is essential for animal development and the physiopathology of multiple tissues in the vertebrate. Ubiquitin-fold modifier 1 (UFM1) is one of the newly-identified UBL, which is covalently attached to its substrates through the orchestrated action of a dedicated enzymatic cascade. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. However, there are no studies on UFMylation and the effect of UFMylation on BmNPV replication in silkworm. In this study, we identified BmUFM1 in the B. mori genome. Spatio-Temporal expression profiles showed that BmUFM1 expression was highly in hemocytes and response to various pathogenic stimuli. Furthermore, BmUFM1 is involved in the regulation of ER stress induced Unfolded Protein Response (UPR) and knockdown of BmUFM1 inhibited BmNPV replication. Overall, these results suggest that BmUFM1 plays an important role in facilitating BmNPV proliferation in silkworm. Our findings advance the understanding of UFM1's conjugation machinery, and also provides a potentially molecular target for BmNPV prevention and silkworm breeding.

BmNPV Orf 65 (Bm65) Is Identified as an Endonuclease Directly Facilitating UV-Induced DNA Damage Repair.

Baculoviruses have been used as biopesticides for the control of Lepidoptera larvae. However, solar UV radiation reduces the activity of baculovirus. In this study, an UV endonuclease, Bm65, was found encoded in the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV). Bm65 (the ortholog of AcMNPV orf79) was guided by a key nuclear localization signal to enter the nucleus and accumulated at UV-induced DNA damage sites. Subsequent results further showed that Bm65-mediated DNA damage repair was not the only UV damage repair pathway of BmNPV. BmNPV also used host DNA repair proteins to repair UV-induced DNA damage. In summary, these results revealed that Bm65 was very important in UV-induced DNA damage repair of BmNPV, and BmNPV repaired UV-damaged DNA through a variety of ways. IMPORTANCE Baculovirus biopesticides are environmentally friendly insecticides and specifically infect invertebrates. UV radiation from the sunlight greatly reduces the activity of baculovirus biopesticides. However, the molecular mechanisms of most baculoviruses to repair UV-induced DNA damage remain unclear. Nucleotide excision repair (NER) is a major DNA repair pathway that removes UV-induced DNA lesions. At present, there are few reports about the nucleotide excision repair pathway in viruses. Here, we showed for the first time that the baculovirus Bm65 endonuclease actually cleaved UV-damaged DNA. Meanwhile, we found that BmNPV used both viral-encoded enzymes and host DNA damage repair proteins to reverse UV-induced DNA damage. These results will provide a reference for the research of UV damage repair of other viruses.

LincRNA_XR209691.3 could promote Bombyx mori nucleopolyhedrovirus replication by interacting with BmHSP70.

Long non-coding RNAs (lncRNAs), a class of transcripts exceeding 200 nucleotides and lacking protein coding potential, have been proven to play important roles in viral infection and host immunity. Bombyx mori nucleopolyhedrovirus (BmNPV) is an important pathogen, which causes the silkworm disease and leads to a huge challenge to the sericultural industry. At present, research on the roles of insect lncRNAs in host-virus interaction are relatively few. In this study, we explored the function of lincRNA_XR209691.3 that was significantly up-regulated in the silkworm fat body upon BmNPV infection. Firstly, the subcellular localization experiment confirmed that lincRNA_XR209691.3 was present in both the nucleus and cytoplasm. Enhancing the expression of lincRNA_XR209691.3 in BmN cells could promote the proliferation of BmNPV, while inhibition of lincRNA_XR209691.3 by RNA interference suppresses the proliferation of BmNPV. Combining RNA pull-down and mass spectrometry, we identified the host and BmNPV proteins that could interact with lincRNA_XR209691.3. Next, by using truncation experiment and RNA immunoprecipitation (RIP) assay, it was found that lincRNA_XR209691.3 could bind to the Actin domain of BmHSP70. Subsequently, overexpression of lncRNA_XR209691.3 in BmN cells promoted the expression of BmHSP70, while knockdown of BmHsp70 suppressed the replication of BmNPV. Based on the above results, it is speculated that lincRNA_XR209691.3 could promote the proliferation of BmNPV through interaction with BmHSP70, possibly by improving the stability of BmHSP70 and thereby enhancing the expression of BmHSP70. Our results shed light on the lncRNA function in insect-pathogen interactions and provide a new clue to elucidate the molecular mechanism of BmNPV infection.

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

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

Label-free proteomics analysis on the envelope of budded viruses of Bombyx mori nucleopolyhedrovirus harboring differential localized GP64.

Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 is the key membrane fusion protein that mediates budded virus (BV) infection. We recently reported that BmNPV GP64's n-region of signal peptide (SP) blocked the SP-cleavage and mediated GP64 localization on the plasma membrane (PM); n-region (SP∆nGP64) absence caused GP64 intracellular localization, however, SP∆nGP64 was still incorporated into virion to generate BVs with lower infectivity. To better understand the biogenesis of the envelope of BmNPV BV, we conducted a label-free ESI mass spectrometry analysis of the envelope of purified BVs harboring PM localized GP64 or intracellular localized SP∆nGP64. The results indicated that 31 viral proteins were identified on the envelope, among which 15 were reported in other viruses. The other 16 proteins were first reported in BmNPV BV, including the BmNPV-specific protein BRO-A and proteins associated with vesicle transportation. Six proteins with significant intensity differences were detected in virions with differential localized GP64, and five specific proteins were identified in virions with GP64. Meanwhile, we identified 81 host proteins on the envelope, and seven lipoproteins were first identified in baculovirus virion; other 74 proteins are involved in the cytoskeleton, DNA-binding, vesicle transport, etc. In the meantime, eight and five specific host proteins were, respectively, identified in GP64 and SP∆nGP64's virions. The two virions shared 68 common host proteins, and 8 proteins were identified on their envelopes with a significant difference. This study provides new insight into the protein composition of BmNPV BV and a clue for further investigation of the budding mechanism of BmNPV.

Expression and localization of Bombyx mori nucleopolyhedrovirus GP37.

Mitochondria play an essential role in intracellular energy metabolism. This study described the involvement of Bombyx mori nucleopolyhedrovirus (BmNPV) GP37 (BmGP37) in host mitochondria. Herein, the proteins associated with host mitochondria isolated from BmNPV-infected or mock-infected cells by two-dimensional gel electrophoresis were compared. One mitochondria-associated protein in virus-infected cells was identified as BmGP37 by liquid chromatography-mass spectrometry analysis. Furthermore, the BmGP37 antibodies were generated, which could react specifically with BmGP37 in the BmNPV-infected BmN cells. Western blot experiments showed that BmGP37 was expressed at 18 h post-infection and was verified as a mitochondria-associated protein. Immunofluorescence analysis demonstrated that BmGP37 localized to the host mitochondria during BmNPV infection. Furthermore, western blot analysis revealed that BmGP37 is a novel component protein of the occlusion-derived virus (ODV) of BmNPV. The present results indicated that BmGP37 is one of the ODV-associated proteins and may have important roles in host mitochondria during BmNPV infection.

The development of single-chain antibody anchored on the BmE cell membrane to inhibit BmNPV infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) poses a significant threat to sericulture production, and traditional sanitation practices remain the main strategy for controlling BmNPV infection. Although RNAi targeting BmNPV genes engineered into transgenic silkworms has shown to be a promising approach in reducing viral infection, it cannot block viral entry into host cells. Therefore, there is an urgent need to develop new effective prevention and control measures. In this study, we screened a monoclonal antibody 6C5 that potently neutralizes BmNPV infection by clamping the internal fusion loop of the BmNPVglycoprotein64 (GP64). Furthermore, we cloned the VH and VL fragments of mAb-6C5 from the hybridoma cell, and the eukaryotic expression vector of scFv6C5 was constructed to anchor the antibody on the cell membrane. The GP64 fusion loop antibody-expressing cells exhibited a reduced capacity for BmNPV infection. The results from our study provide a novel BmNPV control strategy and lay the foundation for the future development of transgenic silkworms with improved antiviral efficacy.

Apoptosis-related long non-coding RNA LINC5438 of Bombyx mori promotes the proliferation of BmNPV.

Apoptosis, as an important part of the immune response, is one of the core events in the host-virus interaction. Studies have shown that long non-coding RNAs (lncRNAs) play important roles in the process of cell apoptosis and pathophysiology. To investigate the apoptosis-related lncRNAs involved in Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworms, transcriptome sequencing was conducted based on silkworm cells infected with BmNPV before and after B. mori inhibitor of apoptosis (Bmiap) gene knockout. A total of 23 differentially expressed lncRNAs were identified as being associated with the mitochondrial apoptosis pathway. Moreover, we demonstrated that B. mori LINC5438 has the function of inhibiting apoptosis in silkworm cells. Overexpression of LINC5438 promoted the proliferation of BmNPV, while interference with LINC5438 inhibited its proliferation, indicating that LINC5438 plays an important role in BmNPV infection. Our results also showed that LINC5438 can regulate the expression of Bmiap, BmDronc, BmICE, and its predicted target gene BmAIF, suggesting that LINC5438 may function through the mitochondrial pathway. These findings provide important insights into the mechanisms of virus-host interaction and the applications of baculoviruses as biological insecticides.

The Bmtret1 Gene Family and Its Potential Role in Response to BmNPV Stress in Bombyx mori.

Trehalose is a non-reducing disaccharide and participates in physiological activities such as organ formation, energy metabolism, and stress resistance in insects. The Bmtret1 gene family is mainly involved in in the sugar metabolism of silkworm. In the present study, phylogenetic analysis divided 21 Bmtret1 orthologs into three clades. These genes are equally distributed on the nine chromosomes. The cis-elements in the promoter regions of Bmtret1s indicated the possible function of Bmtret1s in response to hormones and environmental stimulus. The qPCR analysis showed the significantly different expression levels of Bmtret1s in different tissues and organs, indicating possible functional divergence. In addition, most Bmtret1s showed disturbed expression levels in response to silkworm nuclear polyhedrosis virus (BmNPV) stresses. Our results provide a clue for further functional dissection of the Tret1s in Bombyx mori and implicate them as potential regulators of antiviral responses.