Search

Bombyx mori Nucleopolyhedrovirus (BmNPV), which is a member of the Baculoviridae family, is a significant pathogen of the silkworm. The infection of BmNPV is often lethal and causes about 20% loss of cocoon in the silk industry annually. To explore the effects of different gene inhibition strategies on the replication cycle of baculovirus, we constructed the mutant virus to infect BmN cells directly and further identified ie0, ie1, and gp64 as the essential viral genes of BmNPV. To elucidate the significance of the inhibition effect of different interference strategies, we characterized and constructed the recombinant BmNPV that carried a single or multigene-interfering cassette. The results showed that the inhibition effect of dsie1 on target gene expression, virus titer, and silkworm mortality was significantly better than that of dsie0 and dsgp64. It also showed that the dsie1 interference produced fewer progeny virions and was less lethal, which indicates that ie1 played a more critical role in the BmNPV replication cycle. Furthermore, the inhibitory effect of the virus titer and mortality indicated that the multigene co-interference constructed by the baculovirus expression system was significantly better than the interference of any single-gene (p < 0.05). In summary, the strategy of multigene synergy can achieve the function of continuous interference and provide a new platform for the breeding of silkworm disease resistant. In addition, this strategy improves the various traits of the silkworm. [ABSTRACT FROM AUTHOR]

Nuclear polyhedrosis virus (NPV) is the most harmful virus responsible for the manifestation of grasserie disease in the larvae of silkworm,Bombyx mori. It causes a huge economic loss in the sericulture industry. An attempt was made in the present investigation for the screening of antiviral activity using medicinal plants such asLantana camara, Phyllanthus amarusand marine seaweeds such asSargassum wightii, Turbinaria ornataagainstBmNPV. Crude extracts were prepared using different solvents, such as hexane, ethyl acetate, methanol and water. The silkworm feeding bioassay study was carried out with the crude extracts to investigate the presence of anti-BmNPV activity after inoculating fifth instar larvae of silkworm with occlusion bodies (OBs) ofBmNPV. Each extract was tested for their anti-BmNPV activity using various concentrations of crude extracts ranging from 200 μg to 1000 μg. Among the crude extracts tested, methanol and aqueous extracts ofP. amarusshowed significant anti-BmNPV activity. [ABSTRACT FROM PUBLISHER]

Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Since microRNAs (miRNAs) have been shown to play important roles in host-pathogen interactions, in this study we investigated the effects of BmNPV infection on silkworm microRNAs expression profile. To achieve this, we constructed and deep-sequenced two small RNA libraries generated from BmNPV infected and un-infected larvae. The results revealed that 38 silkworm miRNAs were differentially expressed after BmNPV infection. Based on the GO analysis, their predicted target genes were found to be involved in diverse functions such as binding, catalytic, virion and immune response to stimulus suggesting their potential roles in host-virus interactions. Using the dual-luciferase reporter assay, we confirmed that Bmo-miR-277-5p, up-regulated in BmNPV-infected larvae, targeted the B. mori DNA cytosine-5 methyltransferase (Dnmt2) gene which may play potential role in silkworm-BmNPV interaction. These results provide new insights into exploring the interaction mechanism between silkworm and BmNPV. [ABSTRACT FROM AUTHOR]

Viruses and high temperature (HT) are the primary threats to silkworms. Changes in the expression of stress-response genes can be measured using quantitative polymerase chain reaction (qPCR) after exposure to viruses or HT. However, appropriate reference genes (RGs) for qPCR data normalization have not been established in this organism. In this study, we summarized the RGs used in the previous silkworm studies after infection with Bombyx mori nucleopolyhedrovirus (BmNPV), B. mori cytoplasmic polyhedrosis virus (BmCPV), or B. mori densovirus (BmDNV) or after HT treatment. The expression levels of these RGs were extracted from silkworm transcriptome data to screen for candidate RGs that were unaffected by the experimental conditions. Actin-1 ( A1), actin-3 ( A3), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), and translation initiation factor 4a ( TIF- 4A) were selected for further qPCR verification. The results of RNA-seq and qPCR showed that GAPDH and TIF- 4A were suitable RGs after BmNPV challenge or HT stress, whereas TIF- 4A was an appropriate RG for BmCPV or BmDNV-Z challenge in silkworms. These results suggested that TIF-4A may be the most appropriate RG for gene expression analysis after challenge with viruses or HT in silkworms. [ABSTRACT FROM AUTHOR]

We previously established two silkworm cell lines, BmN-SWU1 and BmN-SWU2, from Bombyx mori ovaries. BmN-SWU1 cells are susceptible while BmN-SWU2 cells are highly resistant to BmNPV infection. Interestingly, we found that the entry of BmNPV into BmN-SWU2 cells was largely inhibited. To explore the mechanism of this inhibition, in this study we used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative protein expression profiling and identified 629 differentially expressed proteins between the two cell lines. Among them, we identified a new membrane protein termed BmREEPa. The gene encoding BmREEPa transcribes two splice variants; a 573 bp long BmREEPa-L encoding a protein with 190 amino acids and a 501 bp long BmREEPa-S encoding a protein with 166 amino acids. BmREEPa contains a conserved TB2/DP, HVA22 domain and three transmembrane domains. It is localized in the plasma membrane with a cytoplasmic C-terminus and an extracellular N-terminus. We found that limiting the expression of BmREEPa in BmN-SWU1 cells inhibited BmNPV entry, whereas over-expression of BmREEPa in BmN-SWU2 cells promoted BmNPV entry. Our results also indicated that BmREEPa can interact with GP64, which is the key envelope fusion protein for BmNPV entry. Taken together, the findings of our study revealed that BmREEPa is required for BmNPV to gain entry into silkworm cells, and may provide insights for the identification of BmNPV receptors. [ABSTRACT FROM AUTHOR]

The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication. [ABSTRACT FROM AUTHOR]

Grasserie, a polyorganotrophic disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV), accounts for lethal infection to fifth instar silkworm larvae. It was found that nanoparticle (NP)-induced morphological transformation of BmNPV polyhedra could reduce the infectivity of BmNPV both in cell line and in silkworm larvae. Initially, 11 NPs were screened for evaluation of their nature of interaction with polyhedra surface through scanning electron microscopy. Amongst these NPs, lipophilically coated silica nanoparticle (SNPL), alumina nanoparticles in the hexagonal close-packed α structure and aspartate capped gold nanoparticle transformed polyhedra were tested for their infectivity in B. mori cell line using cytopathic effect and plaque reduction assay. SNPL was evaluated for its bio-efficacy in fifth instar silkworm larvae. The study of polyhedra morphology as a function of NP concentration showed severe 'roughening' of the polyhedra with replacement of the regular facets by a large number of irregular ones by SNPL, and this caused transition of highly infectious polyhedra into a nearly spherical, non-infectious structure. A moderate polyhedra roughening was observed for alumina NPs, and no roughening was noticed for gold NPs. The morphological changes could be correlated with reduction of virus-induced cytopathic effect and plaque formation, and increased survival rate of SNPL transformed polyhedra infected silkworm larvae to 70.09 ± 6.61 % after 96 h. In this group, 61.04 ± 8.03 % larvae formed normal cocoons from which moths eclosed, laid eggs and larvae emerged. This study could lead to open up newer pathways for designing nano pharmaceuticals to combat other viral diseases. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 ( Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle. [ABSTRACT FROM AUTHOR]

Nuclear polyhedrosis is the most-common viral disease of the silkworm Bombyx mori. Tolerance to nuclear polyhedrosis virus (NPV) was evaluated in 10 breeds of silkworms. After IIIrd moult the worms were orally inoculated with B. mori NPV two dilutions (10-1 & 10-3) for fourth and fifth instar worms and reared up to the spinning. The biological and economic characteristics were measured in different groups. The results obtained, on fifth instar larval duration is found to be maximum in pure breed CSR16 10.67 and 10.33 days in both the instar when inoculated with two viral dilutions and same has been seen in the control lots 9.67 & 9.67 days. The pupal duration is decreased from 11.33 to 11.67 days & 14.67 to 15.00 days and 11.67 to 12.00 days & 14.33 to 15.33 days in PM and CSR2 at both the dilutions of same instars. However among hybrids, CSR2xCSR4 recorded significantly longer pupal duration of 16.67 (10-3) days in fourth instar instars and 16.67 & 15.67 days in fifth instar respectively. The same trend was noticed in control lots 16.33 and 16.33 days. The silk productivity of experimented breeds was CSR2 and CSR4xCSR16 found highest value of 2.53 & 3.03cg/day and 2.50 & 2.50cg/day (10-1 & 10-3) and 2.83 & 3.14 and 2.12 & 2.20cg/day for both the instars inoculated. Whereas, CSR2xCSR4 (10-1) in fourth instar and CSR2xCSR16 in both the instars at (10-1 & 10-3) both the dilutions did not recorded any of the parameters due to their high sensitivity to BmNPV. [ABSTRACT FROM AUTHOR]

Infection of the silkworm cell line, Bm-N, with the Bombyx mori nuclear polyhedrosis virus (BmNPV) was enhanced by adding lecithin (phosphatidyl choline) to culture media. The enhancement index increased by 2.5 times when added 100 μg/ml. Cationic detergents, i.e., cetyltrimethyl-ammonium bromide and dodecylamine hydrochloride, were effective in enhancing BmNPV infection in vitro, their optimal concentrations ranged from 10−2 to 10−1 μg/ml. Both nonionic (Triton X-100) and anionic (sodium dodecyl sulfate) detergents were suppressive to BmNPV infection in Bm-N cells. Zusammenfassung Faktoren zur Förderung der Kernpolyedervirusinfektion in Bombyx mori-Zell-Linien Die Kernpolyedrose-Virus(BmNPV-)Infektion der Seidenspinner-Zell-Linie, Bm-N, konnte durch Zugabe von Lezithin (Phosphatidylcholin) zum Kulturmedium gefördert werden. Der Förderungs-index wurde um das 2,5fache erhöht, wenn 100 μg/ml zugegeben wurden. Kationen-Detergentien wie Cetyltrimethylammoniumbromid und Dodecylaminhydrochlorid waren in vitro als Förderungssubstanzen für das BmNPV wirksam. Ihre optimalen Konzentrationen lagen zwischen 10−2 und 10−1 μg/ml. Sowohl das nonionische Triton X-100 als auch das anionische Natriumdodecylsulfat unterdrückten als Detergentien die BmNPV-Infektion in den Bm-N-Zellen. [ABSTRACT FROM AUTHOR]

Bombyx mori Nucleopolyhedrovirus (BmNPV), which is a member of the Baculoviridae family, is a significant pathogen of the silkworm. The infection of BmNPV is often lethal and causes about 20% loss of cocoon in the silk industry annually. To explore the effects of different gene inhibition strategies on the replication cycle of baculovirus, we constructed the mutant virus to infect BmN cells directly and further identified ie0, ie1, and gp64 as the essential viral genes of BmNPV. To elucidate the significance of the inhibition effect of different interference strategies, we characterized and constructed the recombinant BmNPV that carried a single or multigene-interfering cassette. The results showed that the inhibition effect of dsie1 on target gene expression, virus titer, and silkworm mortality was significantly better than that of dsie0 and dsgp64. It also showed that the dsie1 interference produced fewer progeny virions and was less lethal, which indicates that ie1 played a more critical role in the BmNPV replication cycle. Furthermore, the inhibitory effect of the virus titer and mortality indicated that the multigene co-interference constructed by the baculovirus expression system was significantly better than the interference of any single-gene (p < 0.05). In summary, the strategy of multigene synergy can achieve the function of continuous interference and provide a new platform for the breeding of silkworm disease resistant. In addition, this strategy improves the various traits of the silkworm. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important pathogen of Bombyx mori , silkworm and causes severe losses in the silk industry. During the virus infectious cycle, budded virus (BVs) and occlusion-derived virus (ODVs) particles, which have identical genetic content but different phenotypes, are produced. The envelope glycoprotein GP64, specific in BVs, is involved in host cell receptor binding and is sufficient to mediate membrane fusion during the viral entry. However, the host cell factors, interacting with GP64 to mediate BVs infection, are still unknown. In this study, a cDNA library of Bombyx mori cells (BmN) was constructed and yeast two-hybrid screening was used to identify the host cell factors interacting with GP64. One of the eight candidate proteins encoded the E3 ubiquitin-protein ligase SINA-like 10 (SINAL10), was further confirmed through coimmunoprecipitation assays as novel GP64 binding protein. Moreover, overexpression of SINAL10 significantly enhances viral reproduction, and conversely, silencing its expression by small interfering RNAs showed significant inhibitory effects. Collectively, we demonstrated that SINAL10 is a novel GP64-binding protein that stimulates BmNPV proliferation. [ABSTRACT FROM AUTHOR]

Nuclear polyhedrosis virus (NPV) is the most harmful virus responsible for the manifestation of grasserie disease in the larvae of silkworm,Bombyx mori. It causes a huge economic loss in the sericulture industry. An attempt was made in the present investigation for the screening of antiviral activity using medicinal plants such asLantana camara, Phyllanthus amarusand marine seaweeds such asSargassum wightii, Turbinaria ornataagainstBmNPV. Crude extracts were prepared using different solvents, such as hexane, ethyl acetate, methanol and water. The silkworm feeding bioassay study was carried out with the crude extracts to investigate the presence of anti-BmNPV activity after inoculating fifth instar larvae of silkworm with occlusion bodies (OBs) ofBmNPV. Each extract was tested for their anti-BmNPV activity using various concentrations of crude extracts ranging from 200 μg to 1000 μg. Among the crude extracts tested, methanol and aqueous extracts ofP. amarusshowed significant anti-BmNPV activity. [ABSTRACT FROM PUBLISHER]

Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Since microRNAs (miRNAs) have been shown to play important roles in host-pathogen interactions, in this study we investigated the effects of BmNPV infection on silkworm microRNAs expression profile. To achieve this, we constructed and deep-sequenced two small RNA libraries generated from BmNPV infected and un-infected larvae. The results revealed that 38 silkworm miRNAs were differentially expressed after BmNPV infection. Based on the GO analysis, their predicted target genes were found to be involved in diverse functions such as binding, catalytic, virion and immune response to stimulus suggesting their potential roles in host-virus interactions. Using the dual-luciferase reporter assay, we confirmed that Bmo-miR-277-5p, up-regulated in BmNPV-infected larvae, targeted the B. mori DNA cytosine-5 methyltransferase (Dnmt2) gene which may play potential role in silkworm-BmNPV interaction. These results provide new insights into exploring the interaction mechanism between silkworm and BmNPV. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 ( Bm5 ) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. [ABSTRACT FROM AUTHOR]

Viruses and high temperature (HT) are the primary threats to silkworms. Changes in the expression of stress-response genes can be measured using quantitative polymerase chain reaction (qPCR) after exposure to viruses or HT. However, appropriate reference genes (RGs) for qPCR data normalization have not been established in this organism. In this study, we summarized the RGs used in the previous silkworm studies after infection with Bombyx mori nucleopolyhedrovirus (BmNPV), B. mori cytoplasmic polyhedrosis virus (BmCPV), or B. mori densovirus (BmDNV) or after HT treatment. The expression levels of these RGs were extracted from silkworm transcriptome data to screen for candidate RGs that were unaffected by the experimental conditions. Actin-1 ( A1), actin-3 ( A3), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), and translation initiation factor 4a ( TIF- 4A) were selected for further qPCR verification. The results of RNA-seq and qPCR showed that GAPDH and TIF- 4A were suitable RGs after BmNPV challenge or HT stress, whereas TIF- 4A was an appropriate RG for BmCPV or BmDNV-Z challenge in silkworms. These results suggested that TIF-4A may be the most appropriate RG for gene expression analysis after challenge with viruses or HT in silkworms. [ABSTRACT FROM AUTHOR]

We previously established two silkworm cell lines, BmN-SWU1 and BmN-SWU2, from Bombyx mori ovaries. BmN-SWU1 cells are susceptible while BmN-SWU2 cells are highly resistant to BmNPV infection. Interestingly, we found that the entry of BmNPV into BmN-SWU2 cells was largely inhibited. To explore the mechanism of this inhibition, in this study we used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative protein expression profiling and identified 629 differentially expressed proteins between the two cell lines. Among them, we identified a new membrane protein termed BmREEPa. The gene encoding BmREEPa transcribes two splice variants; a 573 bp long BmREEPa-L encoding a protein with 190 amino acids and a 501 bp long BmREEPa-S encoding a protein with 166 amino acids. BmREEPa contains a conserved TB2/DP, HVA22 domain and three transmembrane domains. It is localized in the plasma membrane with a cytoplasmic C-terminus and an extracellular N-terminus. We found that limiting the expression of BmREEPa in BmN-SWU1 cells inhibited BmNPV entry, whereas over-expression of BmREEPa in BmN-SWU2 cells promoted BmNPV entry. Our results also indicated that BmREEPa can interact with GP64, which is the key envelope fusion protein for BmNPV entry. Taken together, the findings of our study revealed that BmREEPa is required for BmNPV to gain entry into silkworm cells, and may provide insights for the identification of BmNPV receptors. [ABSTRACT FROM AUTHOR]

Baculoviruses have been known to induce hyperactive behavior in their lepidopteran hosts for over a century. As a typical lepidopteran insect, the silkworm Bombyx mori displays enhanced locomotor activity (ELA) following infection with B . mori nucleopolyhedrovirus (BmNPV). Some investigations have focused on the molecular mechanisms underlying this abnormal hyperactive wandering behavior due to the virus; however, there are currently no reports about B . mori . Based on previous studies that have revealed that behavior is controlled by the central nervous system, the transcriptome profiles of the brains of BmNPV-infected and non-infected silkworm larvae were analyzed with the RNA-Seq technique to reveal the changes in the BmNPV-infected brain on the transcriptional level and to provide new clues regarding the molecular mechanisms that underlies BmNPV-induced ELA. Compared with the controls, a total of 742 differentially expressed genes (DEGs), including 218 up-regulated and 524 down-regulated candidates, were identified, of which 499, 117 and 144 DEGs could be classified into GO categories, KEGG pathways and COG annotations by GO, KEGG and COG analyses, respectively. We focused our attention on the DEGs that are involved in circadian rhythms, synaptic transmission and the serotonin receptor signaling pathway of B . mori . Our analyses suggested that these genes were related to the locomotor activity of B . mori via their essential roles in the regulations of a variety of behaviors and the down-regulation of their expressions following BmNPV infection. These results provide new insight into the molecular mechanisms of BmNPV-induced ELA. [ABSTRACT FROM AUTHOR]

The mulberry silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is affected by numerous diseases like Grassery, caused by the nuclear polyhedrosis virus (BmNPV). The management and control of these diseases mainly consist of prevention and early diagnosis. The objective of this work was aimed at the diagnosis of the virus BmNPV associated with the symptoms of Grassery observed in the production areas of the Mulberry silkworm in Cuba. Molecular diagnostic methods for BmNPV based on radioactive and non-radioactive hybridization of nucleic acids (HNA), from larvae's polyhedra extractions in the 3rd and 4th instars, were developed. In both hybridization methods, BmNPV presence was detected. Positive results of 5.5% of samples analyzed in the 3rd larval stage were obtained using radioactive hybridization, which agreed with non- radioactive hybridization results. Nevertheless, in the 4th stage, 28.8% of positive samples were detected using radioactive hybridization and 22.2% of total analyzed samples for non-radioactive hybridization. Although radioactive hybridization showed higher sensibility, both methods allowed to confirm that it was possible to detect BmNPV from the 3rd larval stage, which allows to establish the enhancement of preventive treatment and management measures of mulberry litters with the objective of minimizing production loses. [ABSTRACT FROM AUTHOR]

The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication. [ABSTRACT FROM AUTHOR]

Grasserie, a polyorganotrophic disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV), accounts for lethal infection to fifth instar silkworm larvae. It was found that nanoparticle (NP)-induced morphological transformation of BmNPV polyhedra could reduce the infectivity of BmNPV both in cell line and in silkworm larvae. Initially, 11 NPs were screened for evaluation of their nature of interaction with polyhedra surface through scanning electron microscopy. Amongst these NPs, lipophilically coated silica nanoparticle (SNPL), alumina nanoparticles in the hexagonal close-packed α structure and aspartate capped gold nanoparticle transformed polyhedra were tested for their infectivity in B. mori cell line using cytopathic effect and plaque reduction assay. SNPL was evaluated for its bio-efficacy in fifth instar silkworm larvae. The study of polyhedra morphology as a function of NP concentration showed severe 'roughening' of the polyhedra with replacement of the regular facets by a large number of irregular ones by SNPL, and this caused transition of highly infectious polyhedra into a nearly spherical, non-infectious structure. A moderate polyhedra roughening was observed for alumina NPs, and no roughening was noticed for gold NPs. The morphological changes could be correlated with reduction of virus-induced cytopathic effect and plaque formation, and increased survival rate of SNPL transformed polyhedra infected silkworm larvae to 70.09 ± 6.61 % after 96 h. In this group, 61.04 ± 8.03 % larvae formed normal cocoons from which moths eclosed, laid eggs and larvae emerged. This study could lead to open up newer pathways for designing nano pharmaceuticals to combat other viral diseases. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 ( Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs (BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794bp DNA fragment from Nosema bombycis, 471bp fragment from B. mori NPV (BmNPV) and 391bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers. [Copyright &y& Elsevier]

Abstract: The global transcriptional profile of host genes in the silkworm cell line during the early phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection was analyzed by oligonucleotide microarray. Our analysis showed 35 genes were significantly up-regulated and 17 genes were significantly down-regulated. This is the first report of changes in the expression of these genes in response to NPV infection. We further quantified the levels of mRNA expression by quantitative reverse transcriptase-polymerase chain reaction and confirmed that the expression of 13 (such as BmEts and BmToll10-3) genes significantly increased and 7 genes (such as Hsp20-1) significantly decreased after BmNPV infection. However, the expression levels of most genes were not dramatically changed except BmEts expression increased approximately 8.0-fold 12h after BmNPV infection. [Copyright &y& Elsevier]

Abstract: It has been previously reported that the fp25K product of Bombyx mori nucleopolyhedrovirus (BmNPV) is required for post-mortem host degradation, but the mechanism by which it regulates host degradation is still unknown. This study shows that disruption of BmNPV fp25K attenuates the expression of viral cathepsin gene (v-cath) at a late stage of infection, and thus reduces the secretion of its product V-CATH. Western blot analysis showed that secretion of V-CATH was severely reduced in BmN cells and B. mori larvae infected with Bm25KD, a BmNPV mutant lacking functional fp25K, compared to that of wild-type BmNPV. Also, reduced accumulation of pro-V-CATH in Bm25KD-infected cells was observed from 4 days postinfection (dpi), during which V-CATH was first detected in the medium of BmNPV-infected cells. qRT-PCR experiments showed that the expression levels of v-cath mRNA in wild-type- and Bm25KD-infected BmN cells were comparable at 3 dpi, but showed a marked decrease in Bm25KD-infected BmN cells at 4 dpi. Collectively, these results suggest that BmNPV FP25K is essential for the proper transcriptional regulation of v-cath and efficient secretion of V-CATH, and a steady-state level of v-cath expression during the period of V-CATH secretion (after 4 dpi) is required for post-mortem host degradation. [Copyright &y& Elsevier]

Although microarray and expressed sequence tag (EST)-based approaches have been used to profile gene expression during baculovirus infection, the response of host genes to baculovirus infection and the interaction between baculovirus and its host remain largely unknown. To determine the host response to Bombyx mori nucleopolyhedrovirus infection and the dynamic interaction between the virus and its host, eight digital gene expression libraries were examined in a Bm5 cell line before infection and at 1.5, 3, 6, 12, 24, 48, and 96 h postinfection. Gene set enrichment analysis of differentially expressed genes at each time point following infection showed that gene sets including cytoskeleton, transcription, translation, energy metabolism, iron ion metabolism, and the ubiquitin-proteasome pathway were altered after viral infection. In addition, a time course depicting protein-protein interaction networks between the baculovirus and the host were constructed and revealed that viral proteins interact with a multitude of cellular machineries, such as the proteasome, cytoskeleton, and spliceosome. Several viral proteins, including IE2, CG30, PE38, and PK-1/2, were predicted to play key roles in mediating virus-host interactions. Based on these results, we tested the role of the ubiquitin-proteasome pathway and iron ion metabolism in the viral infection cycle. Treatment with a proteasome inhibitor and deferoxamine mesylate in vitro and in vivo confirmed that these pathways regulate viral infection. Taken together, these findings provide new insights into the interaction between the baculovirus and its host and identify molecular mechanisms that can be used to block viral infection and improve baculovirus expression systems. [ABSTRACT FROM AUTHOR]