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Extrachromosomal circular DNAs (eccDNAs) has been found to be widespread and functional in various organisms. However, comparative analyses of pre- and post-infection of virus are rarely known. Herein, we investigated the changes in expression patterns of eccDNA following infection with Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and explore the role of eccDNA in viral infection. Circle-seq was used to analyze eccDNAs in the midgut of BmCPV-infected and BmCPV-uninfected silkworms. A total of 5508 eccDNAs were identified, with sizes varying from 72 bp to 17 kb. Most of eccDNAs are between 100 to 1000 bp in size. EccDNA abundance in BmCPV-infected silkworms was significantly higher than in BmCPV-uninfected silkworms. GO and KEGG analysis of genes carried by eccDNAs reveals that most are involved in microtubule motor activity, phosphatidic acid binding, cAMP signaling pathway, and pancreatic secretion signaling pathways. Several eccDNAs contain sequences of the transcription factor SOX6, sem-2, sp8b, or Foxa2. Association analysis of eccDNA-mRNA/miRNA/circRNA revealed that some highly expressed genes are transcribed from relevant sequences of eccDNA and the transcription of protein coding genes influenced the frequency of eccDNA. BmCPV infection resulted in changes in the expression levels of six miRNAs, but no known miRNAs with altered expression levels due to changes in eccDNA abundance were identified. Moreover, it was found that 1287 and 924 sequences representing back-spliced junctions of circRNAs were shared by the junctions of eccDNAs in the BmCPV-infected and uninfected silkworms, respectively, and some eccDNAs loci were shared by circRNAs on Chromosomes 2, 7, 11, 14, and 24, suggesting some eccDNAs may exert its function by being transcribed into circRNAs. These findings suggest that BmCPV infection alter the expression pattern of eccDNAs, leading to changes in RNA transcription levels, which may play roles in regulating BmCPV replication. In the future, further experiments are needed to verify the association between eccDNA-mRNA/miRNA/circRNA and its function in BmCPV infection. [ABSTRACT FROM AUTHOR]

The gut is not only used by insects as an organ for the digestion of food and absorption of nutrients but also as an important barrier against the invasion and proliferation of pathogenic microorganisms. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an insect‐specific virus, predominantly colonizes the midgut epithelial cells of the silkworm, thereby jeopardizing its normal growth. However, there is limited knowledge of the cellular immune responses to viral infection and whether the infection is promoted or inhibited by different types of cells in the silkworm midgut. In this study, we used single‐nucleus RNA sequencing to identify representative enteroendocrine cells, enterocytes, and muscle cell types in the silkworm midgut. In addition, by analyzing the transcriptional profiles of various subpopulations in the infected and uninfected groups, we found that BmCPV infection suppresses the response of the antiviral pathways and induces the expression of BmHSP70, which plays a role in promoting BmCPV replication. However, certain immune genes in the midgut of the silkworm, such as BmLebocin3, were induced upon viral infection, and downregulation of BmLEB3 using RNA interference promoted BmCPV replication in the midgut of B. mori. These results suggest that viral immune evasion and active host resistance coexist in BmCPV‐infected silkworms. We reveal the richness of cellular diversity in the midgut of B. mori larvae by single‐nucleus RNA sequencing analysis and provide new insights into the complex interactions between the host and the virus at the single‐cell level. [ABSTRACT FROM AUTHOR]

ABSTRACT Bombyx mori cypovirus (BmCPV), a member of the Reoviridae family, is a well-established research model for double-stranded RNA (dsRNA) viruses with segmented genomes. Despite its small genome size, the coding potential of BmCPV remains largely unexplored. In this study, we identified a novel small open reading frame within the S10 dsRNA genome, encoding a small viral peptide (VSP59) with 59 amino acid residues. Functional characterization revealed that VSP59 acts as a negative regulator of viral replication. VSP59 predominantly localizes to the cytoplasm, where it interacts with prohibitin 2 (PHB2), an inner membrane mitophagy receptor. This interaction targets mitochondria and triggers caspase 3-dependent apoptosis. Transient expression of vsp59 in BmN cells suppressed viral replication, an effect that was reversed by silencing PHB2 expression. Moreover, recombinant BmCPV with a mutated vsp59 exhibited reduced replication. Our findings demonstrate that VSP59 interacts with PHB2 on mitochondria, inducing apoptosis and thereby diminishing viral replication. This study expands our understanding of the genetic information encoded by the BmCPV genome and highlights the role of novel small peptides in host-virus interactions.IMPORTANCEA novel small open reading frame (sORF) from the viral genome was identified and characterized. The sORF could encode a small viral peptide (VSP59) that targeted mitochondria and induced prohibitin 2-related apoptosis, further attenuating Bombyx mori cypovirus replication.

Growth factors play a critical role in wound healing, and finding a suitable biosustained-release system has always been a research hotspot. Bombyx mori cypovirus (BmCPV) is an insect virus, which produces polyhedra that encapsulate progeny virions. In this study, we found that the viral structural protein VP7 encoded by the BmCPV genomic dsRNAs S7 segment can interact with polyhedrin (Polh) encoded by the BmCPV genomic dsRNAs S10 segment. We also confirmed that the amino acid sequence at position 331-360 (VP7-tag) of VP7 is needed to interact with Polh. We found that VP7-tag can be used as an immobilization signal to direct the incorporation of foreign proteins into polyhedra. Furthermore, we constructed polyhedra (bFGF-polyhedra) containing basic fibroblast growth factor (bFGF) using a baculovirus expression system co-expressing Polh and bFGF-VP7 (fusion of VP7-tag to C-terminus of bFGF). We found that bFGF-VP7 embedded into polyhedra was difficult to degrade in the natural environment, and bFGF-VP7 was continuously released from the polyhedra, enhancing cell proliferation and migration. The animal model was used to assess the effect of bFGF-polyhedra spray on the healing of full-thickness wounds. bFGF-polyhedra promoted the expression of TGF-β1, α-SMA, and PCNA, inhibited the expression of proinflammatory factors NF-κB and COX-2, promoted the proliferation and differentiation of fibroblasts, enhanced collagen production and epidermal regeneration, and improved wound healing. These results indicated that bFGF-polyhedra has a promising potential for accelerating wound healing. [Display omitted] • Residues 331-360 at VP7's N-terminus interact with Polh, signaling protein embedding &supporting polyhedron development. • Few studies on polyhedron as a sustained-release system exist, and polyhedra-protected internal proteins represent a novel protein storage strategy. • Polyhedrin microcrystals encapsulating exogenous proteins promote skin wound healing, offering a new solution for acute wound treatment. [ABSTRACT FROM AUTHOR]

[Display omitted] • The role of lncRNA was explored in the process of BmCPV infection • Linc20486 is beneficial for BmCPV replication in vivo and in vitro. • Linc20486 could inhibite the transcription of AGO2 and Dicers to promote BmCPV replication. The silkworm holds pivotal economic importance, serving not only as a primary source of silk but also as a prominent model organism in scientific research. Nonetheless, silkworm farming remains vulnerable to diverse factors, with viral infections posing the gravest threat to the sericulture industry. Among these, the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a member of the Reoviridae family and the cytoplasmic polyhedrosis virus genus, emerges as a significant pathogen in silkworm production. BmCPV infection primarily induces midgut sepsis in silkworms, spreads rapidly, and can inflict substantial economic losses on sericulture production. Presently, effective strategies for preventing and treating BmCPV infections are lacking. Long non-coding RNA (lncRNA) constitutes a class of RNA molecules with transcripts exceeding 200 nt, playing a crucial role in mediating the interplay between pathogens and host cells. Investigation through high-throughput technology has unveiled that BmCPV infection markedly upregulates the expression of Linc20486. This observation suggests potential involvement of Linc20486 in regulating virus replication. Indeed, as anticipated, knockdown of Linc20486 in cells profoundly impedes BmCPV replication, whereas overexpression significantly enhances virus propagation. To probe into the mechanism underlying Linc20486 ′s impact on virus replication, its effects on autophagy, innate immunity, and RNAi-related pathways were scrutinized. The findings revealed that Linc20486 exerts significant influence on the expression of RNAi pathway-related genes, such as Dicer1, Dicer2 and AGO2. This discovery holds promise for unveiling novel avenues to comprehend and combat BmCPV infections in silkworms. [ABSTRACT FROM AUTHOR]

To date, some DNA viruses and single-stranded RNA viruses have been found to generate circRNAs. However, the reports on circRNAs produced by double-stranded RNA viruses are very limited. In this study, Bombyx mori cypovirus (BmCPV), a typical double-stranded RNA virus belonging to the Reoviridae, was demonstrated to generate viral circRNAs (vcircRNAs) and a vcircRNA_000048 whose sequence corresponds with the region 164–1245 nt on the BmCPV genomic dsRNA S5 segment (GQ294468.1) was validated by PCR, Sanger sequencing, reverse transcription-rolling circle amplification, and Northern blotting. Furthermore, we verified that vcircRNA_000048 translates a micropeptide vsp21 with 21 amino acid residues in an IRES-dependent manner, and vsp21 attenuates the viral replication. These findings provided a novel clue to understanding the regulation of viral multiplication and interaction of reovirus with the host. [ABSTRACT FROM AUTHOR]

Continuous outbreaks of viral diseases in humans facilitates a need for the rapid development of viral test kits and vaccines. These require expression systems to produce a pure and high yield of target viral proteins. We utilized a baculovirus–silkworm expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. First, we had to develop a strategy for constructing a recombinant baculovirus for RBD expression. For this, the coding region of the Bombyx mori cypovirus (BmCPV) polyhedron was assembled with the Bombyx mori nuclear polyhedrosis virus (BmNPV) promoter. We demonstrated that the recombinant baculovirus has the ability to form polyhedrons within host silkworm cells. In addition, the encapsulated BVs are able to infect silkworms by ingestion and induce foreign protein expression. In this way, we utilized this novel system to obtain a high yield of the target foreign protein, the RBD of the SARS-CoV-2 S protein. However, the viral infection rate of our recombinant BV needs to be improved. Our study shed light on developing a highly efficient expression system for the production of antigens and subsequent immunoassays and vaccines. [ABSTRACT FROM AUTHOR]

β-Arrestin 2 is known to be a widely distributed adaptor protein in mammals but its function has never been reported in Lepidoptera insects. Herein, the β - Arrestin 2 (BmArrestin 2) gene from silkworm was cloned and characterized. The spatiotemporal expression level of BmArrestin 2 was highest in the gonads at the 3rd day of 5th instar, whereas the highest and lowest abundance of BmArrestin 2 were identified in the tracheal and testis, respectively. BmArrestin 2 is mainly distributed in the cytoplasm. Furthermore, in BmN cells，overexpression of BmArrestin 2 promoted Bombyx mori nucleopolyhedrovirus (BmNPV) and B. mori cytoplasmic polyhedrosis virus (BmCPV) replication as the increment of the concentration of plasmid transfection, whereas silencing the gene with specific siRNA inhibited viral replication. Replication of BmNPV and BmCPV also was weakened using BmArrestin 2 antiserum as the increment of the concentration. Immunofluorescent staining revealed the invasion of recombinant BmNPV or BmCPV was decreased after blocking endogenous BmArrestin 2. On the other hand, BmArrestin 2 co-localizes with recombinant BmNPV and BmCPV virions in BmN cells. These results suggest that BmArrestin 2 may represent a novel target for antiviral strategies, as it is an adaptor protein that plays a key role in virus replication. [ABSTRACT FROM AUTHOR]

Circular DNAs derived from single-stranded RNA viruses play important roles in counteracting viral infection. However, whether double-stranded RNA viruses generate functional circular DNAs is still unknown. Using circDNA sequencing, divergent PCR, DNA in situ hybridization and rolling circular amplification, we presently confirmed that in silkworm, Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a double-stranded RNA virus belonging to cypovirus, is prone to produce a BmCPV-derived circular DNA termed as vcDNA-S7. We have also found that vcDNA-S7 formation is mediated by endogenous reverse transcriptase (RT), and the proliferation of BmCPV can be inhibited by vcDNA-S7 in vitro and in vivo. Moreover, we have discovered that the silkworm RNAi immune pathway is activated by vcDNA-S7, while viral small interfering RNAs (vsiRNAs) derived from transcribed RNA by vcDNA-S7 can be detected by small RNA deep sequencing. These results suggest that BmCPV-derived vcDNA-S7, mediated by RT, can serve as a template for the biogenesis of antiviral siRNAs, which may lead to the repression of BmCPV infection. To our knowledge, this is the first demonstration that a circular DNA, produced by double stranded RNA viruses, is capable of regulating virus infection. [ABSTRACT FROM AUTHOR]

Circular DNAs derived from single-stranded RNA viruses play important roles in counteracting viral infection. However, whether double-stranded RNA viruses generate functional circular DNAs is still unknown. Using circDNA sequencing, divergent PCR, DNA in situ hybridization and rolling circular amplification, we presently confirmed that in silkworm, Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a double-stranded RNA virus belonging to cypovirus, is prone to produce a BmCPV-derived circular DNA termed as vcDNA-S7. We have also found that vcDNA-S7 formation is mediated by endogenous reverse transcriptase (RT), and the proliferation of BmCPV can be inhibited by vcDNA-S7 in vitro and in vivo. Moreover, we have discovered that the silkworm RNAi immune pathway is activated by vcDNA-S7, while viral small interfering RNAs (vsiRNAs) derived from transcribed RNA by vcDNA-S7 can be detected by small RNA deep sequencing. These results suggest that BmCPV-derived vcDNA-S7, mediated by RT, can serve as a template for the biogenesis of antiviral siRNAs, which may lead to the repression of BmCPV infection. To our knowledge, this is the first demonstration that a circular DNA, produced by double stranded RNA viruses, is capable of regulating virus infection.

Bombyx mori cypovirus (BmCPV), a member of the family Reoviridae, is a model of Cypovirus, has a 10 segmented double-stranded RNA genome. However, so far, only one viral small peptide vSP27 with negative regulation on viral infection was identified; the mechanisms underlying host-BmCPV interaction are still unknown. Here, we identified that vSP27 was translated from a BmCPV derived circular RNA (circRNA-vSP27). Subsequently, results showed that vSP27 induced generation of ROS activated the NF-κB signaling pathway, induced the expression of antimicrobial peptides, and suppressed BmCPV infection. On the other hand, we identified a nuclear protein Akirin that could hijack vSP27, positively regulate the NF-κB pathway, and lead to inhibiting the viral infection. Altogether, our data suggested that BmCPV derived circRNA-vSP27 with small peptide translation activity may be employed by the host immunity in defense against the BmCPV infection. [ABSTRACT FROM AUTHOR]

Continuous outbreaks of viral diseases in humans facilitates a need for the rapid development of viral test kits and vaccines. These require expression systems to produce a pure and high yield of target viral proteins. We utilized a baculovirus–silkworm expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. First, we had to develop a strategy for constructing a recombinant baculovirus for RBD expression. For this, the coding region of the Bombyx mori cypovirus (BmCPV) polyhedron was assembled with the Bombyx mori nuclear polyhedrosis virus (BmNPV) promoter. We demonstrated that the recombinant baculovirus has the ability to form polyhedrons within host silkworm cells. In addition, the encapsulated BVs are able to infect silkworms by ingestion and induce foreign protein expression. In this way, we utilized this novel system to obtain a high yield of the target foreign protein, the RBD of the SARS-CoV-2 S protein. However, the viral infection rate of our recombinant BV needs to be improved. Our study shed light on developing a highly efficient expression system for the production of antigens and subsequent immunoassays and vaccines.

Mulberry silkworm, Bombyx mori L. is afflicted especially by microorganisms causing silkworm diseases. These diseases are caused by viruses (BmNPV, BmIFV, BmDNVs, BmCPV); bacteria (Bacillus thuringiensis, Staphylococcus spp., Streptococcus spp.); fungi (Beauveria bassiana , Nomuraea rileyi, Aspergillus flavus); microsporidians (Nosema bombycis , Vairimorpha spp., Pleistophora spp., Thelohania spp. and Microsporidium spp.). Silkworm diseases cause severe crop losses to the farmers as there are no curative measures for their management; only general prophylactic measures are readily available for preventing the incidence and spread of the diseases. Most of the external symptoms of silkworm diseases are atypical and except for a few, they could not be identified by tell-tale features. Disease diagnosis/detection plays a pivotal role in designing prevention strategies to offset the cocoon crop losses. Traditional light microscopy and histochemical methods are not practical; besides being not-so-sensitive; especially non-occluded viruses which possibly could not be detected; and non-specific. More sensitive and specific assays employed to detect/diagnose silkworm viral and microsporidian diseases are based on antigen-antibody (serological) reactions. The immunoassays developed for these purposes include precipitin, fluorescent antibody, immunoblotting, latex agglutination tests; enzyme-linked immunosorbent (ELISA) and dipstick immunoassays. These tests detect silkworm viruses and microsporidians at very low concentrations with high specificity in silkworm tissue and faecal matter. Silkworm larval rearings at a young age are often certified as disease free for further rearing and cocoon production utilizing several variants of latex agglutination, ELISA and dipstick immunoassays. This chapter presents methods for isolation, propagation, purification of silkworm pathogens and protocols for different immunodiagnostic methods. [ABSTRACT FROM AUTHOR]

[Objective]The resistance classification to Bombyx mori cytoplasmic polyhediosis virtis(BmCPV) of the silkworm varieties from Yunnan sericultural areas were investigat in order to breed resistant variety and study key gene.[Method]On the basis of previous work, we selected 58 silkworm varieties to investigate the infection rate of BmCPV.The same concentration of BmCPV suspension liquid were fed on the 3rd instais larva, 7 days after inoculation, determining infection amount through microscopy and the data were analyzed by using analysis of variance, multiple comparison, and combination of clustering analysis.[Result]The results showed that the infection rate among different varieties were different significance(P <0.01), we obtained 4 variety classifications by K-Mean clustering analysis method, high resistant varieties were variety 10 and variety 14, high susceptible varieties were variety 23. We obtained 2 categories and 4 variety classifications by the method of system clustering tree, high resistant varieties had 5, high susceptible varieties had 10. [Conclusion] The resistant ability of 58 silkworm varieties to BmCPV from Yunnan seric ultural areas were divided into 4 levels according to the method of system clustering, high resistant varieties accounted for 9 % , high susceptible varieties accounted for 17 % , intemiediate levels of sensibility and resistance in 34 % and 40 % respectively. [ABSTRACT FROM AUTHOR]

Abstract Bombyx mori cypovirus (BmCPV) is one of the most infectious pathogen in sericulture and a member of the family Reoviridae. It specifically infects the midgut of silkworm. The BmCPV genome consists of 10 dsRNAs segments (S1–S10), which have generally been assumed to be monocistronic. In this study, a small open reading frame encoding the peptide S5-sORF, containing 27 amino acid residues, was predicted in a region of the negative (−) strand of BmCPV segment S5. An immunofluorescence assay detected S5-sORF in the cytoplasm and nuclei of BmCPV-infected cells, and it was also detected in the virion with western blotting, suggesting that S5-sORF may be assembled into the BmCPV virion. Viral gene expression was inhibited by overexpressed S5-sORF, and viral multiplication was dose-dependently suppressed by the S5-sORF peptide. A viable recombinant virus, BmCPV-S5-sORFmut, in which the start codon (ATG) of S5-sORF was mutated to a stop codon (TGA), was generated with reverse genetics. The proliferation of BmCPV was increased by the abolition of S5-sORF expression. Furthermore, the RNA transcript of S5-sORF and small peptide of S5-sORF were involved in BmCPV replication. The expression of genes related to the innate immune pathways and apoptosis in the silkworm were not significantly affected by S5-sORF overexpression. Our results suggest that a viral nucleotide sequence is utilized by the host to generate an antiviral peptide, which may be a novel strategy protecting the host from viral infection. Highlights • Small open reading frame (sORF) was found on the negative strand of BmCPV segment 5. • S5-sORF peptide was encoded by sORF and it was detected in the virion of BmCPV. • S5-sORF peptide was distributed in the cytoplasm and nuclei of BmCPV-infected cells. • The expressions of viral genes were inhibited by overexpression of S5-sORF. • RNA and small peptide of S5-sORF suppressed the replication of BmCPV. [ABSTRACT FROM AUTHOR]

Highlights • Function of tyrosine-protein kinase Src64B-like in the cell entry of BmCPV was investigated. • Silencing the expression of Src64B-like reduced the infection of BmCPV. • Overexpression of Src64B-like enhanced the infection of BmCPV. • BmCPV didn't interact with Src64B-like protein directly in the infection process. Abstract Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a non-enveloped dsRNA virus, which specifically infect the midgut epithelium of B. mori. BmCPV enters permissive cells via clathrin-dependent endocytosis employing β1 integrin mediated internalization. Until now, the cell entry mechanism of BmCPV has not been known clearly. Here, we investigated whether tyrosine-protein kinase Src64B-like is involved in the cell entry of BmCPV. The Src64B-like gene was cloned and expressed in Escherichia coli (E. coli), and the recombinant protein Src64B-like was used to immunize mouse for preparation of anti-Src64B-like polyclonal antibody (pAb). After Src64B-like gene was silenced by RNAi, the infection of BmCPV was reduced by 59.48% ± 2.18% and 92.22% ± 1.12% in vitro and in vivo autonomously. Contrary to it, BmCPV infection could be enhanced by increasing the expression of Src64B-like. In addition, immunofluorescence assay showed that Src64B-like protein did not co-localize with BmCPV in the cultured BmN cells during viral infection. These results indicate that Src64B-like protein participates and plays an important role in the cell entry of BmCPV, but not contacting directly with BmCPV. [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]

The pathogenesis of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection is unclear, although accumulating evidence indicates that circular RNAs (circRNAs), which act as competing endogenous RNAs or positive regulators, play important roles in regulating gene expression in eukaryotes and, thus, may play a role in BmCPV infections. To explore the expression and biological functions of circRNAs in the silkworm midgut following BmCPV infection, silkworm circRNA expression profiles of normal midgut tissue (control) and BmCPV-infected midgut tissue (test) were determined using high-through sequencing. A total of 9,753 and 7,475circRNAs were detected from the control and test samples, respectively. The two samples shared 6,085 circRNAs, while 646 and 737 circRNAs were expressed specifically in the control and test samples, respectively. A total of 3,638 circRNAs were shown to be differentially expressed, and 400 circRNAs were substantially differentially expressed with a fold-change ≥ 2.0 (p< 0.05 and a false discover rate < 0.05), of which 294 were up-regulated and 106 were down-regulated following infection. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to determine the principal functions of the substantially differentially regulated genes. circRNA-miRNA interaction networks were constructed based on a correlation analysis between the differentially expressed circRNAs and the nature of their microRNA (miRNA) binding sites. The network inferred that 13 miRNAs interacting with 193 circRNAs were among the 300 most abundant relationships. bmo-miR-3389-5p, bmo-miR-745-3p, and bmo-miR-3262 were related to 30, 34, and 34 circRNAs, respectively. circRNA_8115, circRNA_9444, circRNA_4553, circRNA_0827, and circRNA_6649 contained six, five, four, four, and four miRNA binding sites, respectively. We further found that alternative circularization of circRNAs is a common feature in silkworms and that the junction sites of many silkworm circRNAs are flanked by canonical GT/AG splicing signals. Our study is the first to show the circRNA response to virus infection. Thus, it provides a novel perspective on circRNA-miRNA interactions during BmCPV pathogenesis, and it lays the foundation for future research of the potential roles of circRNAs in BmCPV pathogenesis. [ABSTRACT FROM AUTHOR]

The genome of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) contains 10 double stranded RNA segments (S1–S10). The segment 7 (S7) encodes 50 kDa protein which is considered as a structural protein. The expression pattern and function of p50 in the virus life cycle are still unclear. In this study, the viral structural protein 7 (VP7) polyclonal antibody was prepared with immunized mouse to explore the presence of small VP7 gene-encoded proteins in Bombyx mori cytoplasmic polyhedrosis virus. The expression pattern of vp7 gene was investigated by its overexpression in BmN cells. In addition to VP7, supplementary band was identified with western blotting technique. The virion, BmCPV infected cells and midguts were also examined using western blotting technique. 4, 2 and 5 bands were detected in the corresponding samples, respectively. The replication of BmCPV genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using RNA interference. In immunoprecipitation experiments, using a polyclonal antiserum directed against the VP7, one additional shorter band in BmCPV infected midguts was detected, and then the band was analyzed with mass spectrum (MS), the MS results showed thatone candidate interacted protein (VP7 voltage-dependent anion-selective channel-like isoform, VDAC) was identified from silkworm. We concluded that the novel viral product was generated with a leaky scanning mechanism and the VDAC may be an interacted protein with VP7. [ABSTRACT FROM AUTHOR]

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major pathogens that pose a big challenge to the sericulture industry. Growing evidences have shown that microRNAs play key roles in the regulations of host-pathogen interactions in insects. MicroRNAs have been found in silkworms, whether and how they affect the silkworm-BmCPV interactions are still unknown. Here we investigate the effect of miR-274-3p on the BmCPV replication in the BmCPV-infected silkworm larvae. In our study, BmCPV Nonstructural protein 5 (NS5) was identified to be the target of miR-274-3p based on bioinformatics analysis and luciferase reporter assay. The abundance of NS5 was significantly increased in the presence of miR-274-3p inhibitor based on the qRT-PCR and Western blotting results. Further, qRT-PCR results revealed that the expression of polyhedrin gene of BmCPV in the larvae after applying miR-274-3p inhibitor was significantly increased comparing with that of larvae with negative control. Our results suggest that inhibition of miR-274-3p could facilitate BmCPV replication by up-regulating BmCPV NS5 gene expression and are insightful for further exploring the interactions between silkworm and BmCPV. [ABSTRACT FROM AUTHOR]

Bombyx mori cypovirus (BmCPV), a typical double-stranded RNA virus, was demonstrated to generate a viral circRNA, vcircRNA_000048, which encodes a vsp21 with 21 amino acid residues to suppress viral replication. However, the regulatory mechanism of vsp21 on virus infection remained unclear. This study discovered that vsp21 induces reactive oxygen species (ROS) generation, activates autophagy, and attenuates virus replication by inducing autophagy. Then we confirmed that the effect of vsp21-induced autophagy on viral replication was attributed to the activation of the NF-κB signaling pathway. Furthermore, we clarified that vsp21 interacted with ubiquitin carboxyl-terminal hydrolase (UCH) and that ubiquitination and degradation of phospho-IκB-α were enhanced by vsp21 via competitive binding to UCH. Finally, we validated that vsp21 activates the NF-κB/autophagy pathway to suppress viral replication by interacting with UCH. These findings provided new insights into regulating viral multiplication and reovirus-host interaction. [Display omitted] • BmCPV was demonstrated to generate a viral circRNA, vcircRNA_000048, which encodes a vsp21 to suppress viral replication. • vsp21 induces ROS generation, activates autophagy, and attenuates virus replication by inducing autophagy. • vsp21 activates the NF-κB/autophagy pathway to suppress viral replication by interacting with UCH. [ABSTRACT FROM AUTHOR]

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major viral pathogens in silkworm. Its infection often results in significant losses to sericulture. Studies have demonstrated that RNAi is one of the important anti-viral mechanisms in organisms. In this study, three dsRNAs targeting the RNA-dependent RNA polymerase (RDRP) gene of BmCPV were designed and synthesized with 2′-F modification to explore their interference effects on BmCPV replication in silkworm larvae. The results showed that injecting dsRNA in the dosage of 4–6 ng per mg body weight into the 5th instar larvae can interfere with the BmCPV-RDRP expression by 93% after virus infection and by 99.9% before virus infection. In addition, the expression of two viral structural protein genes (genome RNA segments 1 and 5) was also decreased with the decrease of RDRP expression, suggesting that RNAi interference of BmCPV-RDRP expression could affect viral replication. The study provides an effective method for investigating virus replication as well as the virus–host interactions in the silkworm larvae using dsRNA. [ABSTRACT FROM AUTHOR]

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a major pathogen of silkworms, causes serious economic losses in sericulture. The BmCPV genome contains 10 discrete dsRNA segments; among these, S1, S2, S3, S4, S6, and S7 encode virus structural proteins, whereas S5, S8, S9, and S10 encode nonstructural proteins. In an attempt to create an anti-BmCPV silkworm strain, we constructed transgenic RNAi vector pb-CNS for knockdown of S5, S8, S9, and S10, and pb-SNS targeting S1, S2, S4, S5, and S8. Transgenic silkworm line CNS and SNS were generated via microinjection of the practical diapause silkworm strain Furong. Following infection via the oral administration of a high dose of BmCPV, the mortality rates of the nontransgenic control, CNS, and SNS were 91%, 37%, and 41%, respectively. qPCR showed that the viral mRNA content in CNS and SNS was significantly lower than that in the nontransgenic line. The economic traits of CNS and SNS were not affected. These results suggest that the knockdown of multiple BmCPV genes significantly enhances the antiviral capacity of the silkworm. [ABSTRACT FROM AUTHOR]

Highlights: [•] Long term stability of dsRNA in Bm5 cells after transfection. [•] Processing of BmCPV dsRNA by RNAi machinery. [•] Presence of BmCPV dsRNA does not interfere with gene silencing. [•] Transfection of BmCPV dsRNA genome in Bm5 cells does not generate functional virus. [Copyright &y& Elsevier]

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV)that belongs to the genus Cypovirus in the family of Reoviridae is one of the problematic pathogens in sericulture. In our previous study, we have found that lipid-related constituents in the host cellular membrane are associated with the BmCPV life cycle. It is important to note that the lipids not only affect the cellular biological processes, they also impact the virus life cycle. However, the intracellular lipid homeostasis in BmN cells after BmCPV infection remains unclear. Here, the lipid metabolism in BmCPV-infected BmN cells was studied by lipidomics analysis. Our results revealed that the intracellular lipid homeostasis was disturbed in BmN cells upon BmCPV infection. Major lipids constituents in cellular membrane were found to be significantly induced upon BmCPV infection, which included triglycerides, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phospholipids, glucoside ceramide, monoetherphosphatidylcholin, ceramide, ceramide phosphoethanolamine and cardiolipin. Further analysis of the pathways related to these altered lipids (such as PE and PC) showed that glycerophospholipid metabolism was one of the most enriched pathways. These results suggested that BmCPV may manipulate the lipid metabolism of cells for their own interest. The findings may facilitate a better understanding of the roles of lipid metabolic changes during virus infection in future studies. • Lipidomics was used to uncover the lipid metabolism in BmCPV infected BmN cells. • The intracellular lipid homeostasis was disturbed in BmN cells infected by BmCPV. • Glycerophospholipid metabolism was identified as one of the most enriched pathways. [ABSTRACT FROM AUTHOR]

The biological functions of protein glycosylation have been increasingly recognized but not yet been very well understood, especially in lower organisms. Silkworm as a model lepidopteran insect and important economic insect, has been widely studied in life science, however, the current knowledge on the glycosylation status of its proteome is not satisfactory, and little is known about how pathogenic infections could affect the glycosylation status. This study performed large scale glycosite mapping for the silkworm Bombyx mori P50 strain, and quantitatively compared with that infected with the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). Some 400 glycoproteins were mapped in the silkworm, including N- and O-glycoproteins. Upon virus infection, the glycosylation levels of 41 N-glycopeptides were significantly changed, some of them belonging to transmembrane glycoproteins. The O-glycosylation profiles were also affected. In addition, 4 BmCPV-encoded viral proteins were found to be glycosylated for the first time, including polyhedrin, P101, VP3, and the NS protein. This study drafted a silkworm protein glycosylation map and underlined the potential impact of virus infection on glycosylation. This study reveals the characteristics of the glycoproteome in the silkworm strain P50, and quantitatively compared to that infected by the virus BmCPV, which underlines the impact of virus infection on the alteration of protein glycosylation in invertebrate species. Our findings add to the knowledge of the post translational modifications of this model organism, and also uncovered for the first time the glycosylation status of the viral proteins expressed by BmCPV. Unlabelled Image • Total N- and O-glycoproteins in silkworm strain P50 were revealed. • Alterations in glycosylation profiles were noted after BmCPV virus infection. • The glycosylation status of the BmCPV-encoded proteins was uncovered. [ABSTRACT FROM AUTHOR]