Bombyx mori nucleopolyhedrovirus (BmNPV) is extremely harmful to the silk industry. The polyhedrin, which encodes the polyhedrin (Polh), can be expressed at ultra-high levels and form occlusion bodies in the nucleus, embedding the progeny virus within it. However, the detailed mechanism by which polyhedrin is transported into the host cell nucleus remains unknown. Clarifying the nuclear import mechanisms of viral proteins can help us develop better prevention and treatment measures against baculoviruses. This study employed molecular cloning, co-immunoprecipitation, and immunofluorescence to analyze in detail the expression pattern of the highly expressed polyhedrin in the very late stage of the virus, and further revealed that the host protein importin α participates in the nuclear import of polyhedrin through protein interactions. This study provides a reference for further elucidating the nuclear import mechanisms of the baculovirus proteins including polyhedrin that can enter the nucleus.
Nucleopolyhedroviruses/metabolism* ; Active Transport, Cell Nucleus ; Animals ; Bombyx/virology* ; alpha Karyopherins/metabolism* ; Cell Nucleus/metabolism* ; Viral Structural Proteins/metabolism* ; Occlusion Body Matrix Proteins

To improve the expression of heterologous genes using baculovirus expression system, we constructed a novel shuttle vector based on the Bm-Bacmid. In the Bm-Bacmid, partial sequences of Chitinase and Cystein Protease were replaced with a tandem cassette of Cm and egfp through homologous recombination. Bombyx mori bidensovirus (BmBDV) ns1 under the control of polyhedrin promoter was inserted into the modified Bm-bacmid by transposition. For comparison, BmBDV ns1 under the control of polyhedrin promoter was also cloned in the wild type Bm-bacmid. The resulting Bm-bacmids were transfected into the cultured BmN cells to prepare recombinant virus to infect silkworms for expression of BmBDV ns1. Total proteins of hemocyte from infected silkworms were subjected to Western blotting and ELISA analysis. The yield of BmBDV NS1 1 with the modified vector was three times as much as that with the unmodified vector. The method to improve the yield of BmBDV NS1 in silkworms will facilitate the function and three-dimensional structure study of BmBDV NS1.
Animals ; Baculoviridae ; Bombyx ; virology ; Cell Line ; Chitinases ; Cysteine Proteases ; Genetic Vectors ; Insect Viruses ; Promoter Regions, Genetic ; Transfection

Bombyx mori nucleopolyhedrovirus (BmNPV) bm47 gene is found in all sequenced lepidopteran nucleopolyhedroviruses (NPVs). It is one of the core genes of NPVs. However, the role of bm47 in the biological cycle of NPV remains unknown. In this study, the Red recombination system was used to knock out bm47 from BmNPV to construct bm47-ko-Bacmid in E. coli BW25113 system. Then bm47 gene was introduced back to the viral genome using the Bac-to-Bac system to create the repair virus bm47-re-Bacmid. TCID50 assay and real-time PCR (qPCR) were used to evaluate the effects of bm47 deletion on viral DNA replication, gene transcription, and protein expression. qPCR results showed that bm47 knock-out had no significant effect on viral DNA replication. However, the qPCR results showed that bm47-ko-Bacmid significantly decreased the transcription levels of early gene lef-3, late gene vp39, and very late gene p10 at 48 h and 72 h after viral transfection of BmN cells (P < 0.05). This work will provide a foundation for further studies on the biological function of BmNPV bm47 in viral replication and transcription.
Animals ; Bombyx ; virology ; Gene Deletion ; Gene Expression Regulation, Viral ; Nucleopolyhedrovirus ; genetics ; physiology ; Transcription, Genetic ; Viral Proteins ; genetics ; metabolism ; Virus Replication

Baculovirus gene expression is the most popular method to make target protein in cultured insect cells. To fast determine the generation of recombinant virus in cultured cells, donor plasmid of pFastBacI was modified by introducing egfp cassette. In the modified vector, egfp cassette was under the control of ie1 promoter, and target gene cassette was under the control of polyhedron promoter. To evaluate the convenience of the genetically modified donor plasmid used in eukaryotic expression, ns1 gene from Bombyx mori bidensovirus was ligated into the donor plasmid to generate recombinant plasmid pFastBacI-[P(ie1)-egfp-sv40]-[P(polh)-ns1-sv40]. Then the plasmid was transformed into DH10B competent cells containing Bm-Bacmid vector to produce the final recombinant Bm-Bacmid with the help of transposase. The resulting recombinant Bm-Bacmid was transfected into BmN cells to generate recombinant virus, which was easily and rapidly judged by green fluorescent signal observed in BmN cells. After infection for 96 h, the BmN cells were harvested and the total protein extracted from the infected BmN cells was subjected to Western blotting analysis. The result showed that a specific protein band about 36 kDa was detected, indicating that NS1 protein was successfully expressed in the BmN cells. In conclusion, the expression of NS1 protein with the modified expression system is useful for further research on the function of NS1 protein.
Animals ; Baculoviridae ; genetics ; Bombyx ; virology ; Cell Line ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; Plasmids ; Promoter Regions, Genetic ; Transfection ; Viral Nonstructural Proteins ; biosynthesis ; genetics

A pair of specific primers were designed according to the published 118 bp conserved sequence of polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) in GenBank and a serial dilutions of a recombinant plasmid were prepared and used to generate standard curves, to establish a real-time fluorescent quantitative PCR method for detection of BmCPV. The results showed that the linear relationship between virus concentration (X) and Ct (Y) was Y = -3. 582 lgX + 38.748 with the correlation coefficient R2 = 0999. The method was very sensitive, specific and reproducible. It can be applied in the rapid detection of BmCPV and the prevalence investigation of this disease.
Animals ; Bombyx ; virology ; Fluorescence ; Real-Time Polymerase Chain Reaction ; methods ; Reoviridae ; genetics ; isolation & purification

OBJECTIVETo construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.

METHODSHuman group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography.

RESULTSThree genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope.

CONCLUSIONWe have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.

Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; virology ; Capsid Proteins ; genetics ; Cell Line ; Gene Expression ; Genetic Vectors ; Rotavirus ; genetics ; metabolism

Three model silkworms, highly resistant strain, highly susceptible strain and their near isogenic line were established by hybridization and backcross. The resistance of silkworm (Bombyx mori L.) to BmNPV was studied at proteomic level using two-dimensional gel electrophoresis and MALDI TOF/TOF MS. Differential expression profiles of proteome in resistant strain, susceptible strain and near isogenic line were obtained, and 180, 190 and 187 of protein spots were shown, respectively. Among them, 80% were concentrated in pI 5-9. Twelve differential protein spots in total were obtained from 3 gels. Using MALDI TOF/TOF MS, five proteins, including aminoacylase, were identified from these spots. The exclusive expression of aminoacylase in highly resistant strain and near isogenic line and its absence in susceptible strain suggest that it might be a BmNPV-resistance related protein.
Amidohydrolases ; analysis ; genetics ; Animals ; Bombyx ; chemistry ; genetics ; virology ; Electrophoresis, Gel, Two-Dimensional ; Hemolymph ; chemistry ; Host-Pathogen Interactions ; genetics ; Insect Proteins ; analysis ; Nucleopolyhedrovirus ; pathogenicity ; physiology ; Proteome ; analysis ; Proteomics ; methods ; Recombination, Genetic ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

BmNPV GD isolate from China was plaque-purified and four bro genes were cloned termed as bro-a,b,c,d. The obtained sequences were aligned to the related sequences in GenBank and the BmNPV CQ1 isolate preserved in our laboratory. Compared with genome sequences of BmNPV T3 isolate, bro genes of GD isolate housed insertion and deletion, and the changes of amino acid mainly occured at the N terminal of corresponding protein. The phylogenetic analysis of bro genes indicated that GD bro-d gene belongs to subgroup A together with T3, CQ1 bro-d and SC7 bro- III; GD bro-a, c genes belong to subgroup B together with T3, CQ1 bro-a, c and SC7 bro-II; GD bro-b gene belongs to subgroup C together with T3, CQ1 bro-b, e and SC7 bro-I. The evolutionary relationship of bro genes showed vague relevance to their geographical location. The distribution character of bro genes in four BmNPV isolates is coincidence with the KANG's theory that the bro-d plays an irreplaceable functional role(s) during viral replication, while bro-a and bro-c functionally complement each other. Meanwhile, we postulate that 3 bro genes in SC7 isolate is probable the most simple form of bro genes.
Animals ; Bombyx ; virology ; Cloning, Molecular ; Genes, Viral ; Nucleopolyhedrovirus ; classification ; genetics ; Open Reading Frames ; Phylogeny ; Polymerase Chain Reaction ; Repetitive Sequences, Nucleic Acid

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3(Zhenjiang isolate), named BmDNV-3, is a kind of bidensovirus. The most obvious characteristic in the genome of BmDNV-3 is that it has 2 sets of DNA molecular (VD1, VD2),and each of them is encapsidated respectively in the form of single-stranded liner DNA ( + VD1, - VD1, + VD2, - VD2) in equal percentage. So the BmDNV-3 has 4 kinds of virions. Furthermore the sequence of BmDNV-3 is able to encode DNA polymerase itself. Some strains of silkworm revealed complete resistance to BmDNV-3, so they didn' t fall sick. To investigate the difference in the process of infection and replication between the 2 virions ( VD1, VD2) of this bidensovirus, and the difference of the increment in the resistant or susceptible host, the 5th instar larvae of the susceptible silkworm strain (HUABA 35) and the resistant silkworm strain(QIUFENG d) were inoculated determinate dose of BmDNV-3 by oral ingestion. Then the midgut were collected at 9 timepoints. The silkworm cytoplasm actin A3 was used to be normalized gene, so the number of cells in collected tissue could be determined. The result shows that whatever in the susceptible silkworm strain or in the resistant one, the copies of VD1 and VD2 in the genome of BmDNV-3 collected at the different timepoint were almost at the equal level respectively, so that the VD1 and VD2 were replicated with synchronization. The process of infection in the susceptible silkworm strain was devided into 3 partitions, latent period( 2 - 12 hours post inoculation), exponential phase (12 - 36 hours post inoculation)and stationary phase (36- 96 hours post inoculation and there are about 2 x 10(5) copies per cell) . In the resistant silkworm strain, the virus were replicated at a very low level, that was from 6 - 10 copies 2 hours post inoculation to 150 - 200 copies 96 hours post inoculation (about 20 times) . So we predict that the resistance in some of the silkworm strains from BmDNV-3 was a kind of chronic representation that the host carried virus without being caused flacherie.
Animals ; Bombyx ; genetics ; virology ; DNA, Viral ; genetics ; Densovirus ; genetics ; physiology ; Female ; Genome, Viral ; genetics ; Host-Pathogen Interactions ; Male ; Polymerase Chain Reaction ; methods ; Time Factors ; Virion ; genetics ; physiology ; Virus Replication ; genetics

BmNPV bacmid constructed recently and Red recombinant system were used to rapidly disrupted Bombyx monri nucleopolyhedrovirus (BmNPV) orf60 in Escherichia coli (E. coli) BW25113. BmNPV bacmid isolated from E. coli BmDH10Bac was electroporated into E. coli BW25113, which harbors plasmid pKD46 encoding lamda Red recombinase,to produce E. coli BW25113-Bac, which could be used for gene deletion of BmNPV. A linear fragment was amplified by PCR from plasmid pKD3 (containing a chloramphenicol acetyltransferase gene cat) using a pair of primers with length of 63bp,which had 45 bp homologous to the orf60 gene and 18bp homologous to cat sequences. The linear fragment was electroporated into E. coli BW25113-Bac and homologous recombination occurred between the linear fragment and orf60 with the help of lamda Red recombinase. Three specific primer pairs were used to confirm the replacement of orf60 by cat gene. Western blot analysis showed that orf60 was not expressed in BmN cells infected with knockout bacmid.
Animals ; Bacteriophage lambda ; enzymology ; genetics ; Bombyx ; virology ; Electroporation ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Gene Knockout Techniques ; Genes, Viral ; genetics ; Nucleopolyhedrovirus ; enzymology ; genetics ; Open Reading Frames ; genetics ; physiology ; Recombinases ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism