Detection of prawn white spot baculovirusby polymerase chain reaction

INTRODUCTIONAprawnbaculovirushasbeenresponsibleformostoftheseriousshrimpdiseaseinChinasince1992.Studiesonthepathology,Pathogenesisandmorphologyofthevirusshowedthatitwasanon-occlUSiontheybaculoviruswhichcouldinfectPenaeusjaponicus,P.nzonham,P.chinests...

Baculovirus vector-mediated transfer of NIS gene into colon tumor cells for radionuclide therapy

AIM:To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter(NIS) gene.METHODS:A recombinant baculovirus plasmid carrying the NIS gene was constructed,and the viruses(BacNIS) were prepared using the Bac-to-Bac system.The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein(GFP) expressing baculovirus(Bac-GFP) at different multiplicities of infection(MOI) with various concentrations of sodium butyrate was determined by flow cytometry.An in vitro cytotoxicity assay was also conducted after infection of SW1116 cells with Bac-NIS.Iodine uptake of Bac-NIS infected SW1116 cells and inhibition of this uptake by sodium perchlorate was examined,and the effect of Bac-NISmediated 131 I in killing tumor cells was evaluated by cell colony formation tests.RESULTS:Infection and transgene expression in SW1116with Bac-GFP were significantly enhanced by sodium butyrate,as up to 72% of SW1116 cells were infected with the virus at MOI of 400 and sodium butyrate at 0.5 mmol/L.No obvious cytotoxicity was observed under these conditions.Infection of SW1116 with Bac-NIS allowed uptake of 131 I in these tumor cells,which could be inhibited by sodium perchlorate.The viability of SW1116 cells infected with Bac-NIS was significantly lower than with Bac-GFP,suggesting that NIS gene-mediated 131 I uptake could specifically kill tumor cells.CONCLUSION:Baculovirus vector-mediated NIS gene therapy is a potential approach for treatment of colon cancer.

Study on purification and ultrastructure of a baculovirus in Penaeus chinensis

A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathin section of the gills, stomach and mid-gut tissues also revealed the presence of rod-shaped baculoviral particles with the same size in the affected cell nuclei, where most of the virions arranging in cluster assembled and caused a series of cytopathic changes. The virion covered with bilaminal envelope was 320 ~ 400 nm × 100 ~ 130 nm in size, whereas the nucleocapsid ranged in size of 250~ 300 nm in length and 70 ~ 100 nm in breadth respectively. No nuclear polyhedron or granulin occlusion theies have been found in cells. According to the principle of viral classification, this newly found virus could probably belong to the non-occluded subgroup of insect baculoviridae, i. e., C subgroup baculovirus.

Baculovirus-expressed FAdV-4 penton base protein protects chicken against hepatitis-hydropericardium syndrome

Hepatitis-hydropericardium syndrome(HHS)is an infectious disease caused by fowl adenovirus serotype 4(FAdV-4).Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculovirus expression system to develop candidate subunit vaccines.However,the protective efficiency of baculovirus-expressed penton base protein has not been assessed.In this study,two recombinant capsid proteins,penton base and fiber-2,were constructed.And then,penton base and fiber-2 were administrated alone or together to specific pathogen-free(SPF)chickens at 14 days of life and boosted at 28 days of life.At 42 days of life,the immunized groups and the control group were challenged with FAdV-4 virulent strain.Results show that inoculating penton base or penton base+fiber-2 provided 100%protection to the chickens.All groups vaccinated with the recombinant protein produced detectable antibodies and showed no apparent lesions.Thus,baculovirus-expressed penton base protein is a promising candidate subunit vaccine.

Interstitial tissue-specific gene expression in mouse testis by intra-tunica albuguineal injection of recombinant baculovirus

The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein （GFP）-expressing recombinant baculovirus （GFP-baculovirus）, in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus （CMV）-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineal injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction （RT-PCR）, and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. lntra-tunica albuguineal injection of GFP- baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein （IGFBP）-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intratunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.

Transgene expression and differentiation of baculovirus-transduced adipose-derived stem cells from dystrophin-utrophin double knock-out mouse

In this study, recombinant baculovirus carrying the microdystrophin and β-catenin genes was used to infect adipose-derived stem cells from a dystrophin-utrophin double knock-out mouse. Results showed that, after baculovirus transgene infection, microdystrophin and β-catenin genes were effectively expressed in adipose-derived stem cells from the dystrophin-utrophin double knock-out mouse. Furthermore, this transgenic expression promoted adipose-derived stem cell differentiation into muscle cells, but inhibited adipogenic differentiation. In addition, protein expression related to the microdystrophin and Wnt/β-catenin signaling pathway was upregulated. Our experimental findings indicate that baculovirus can successfully deliver the microdystrophin and β-catenin genes into adipose-derived stem cells, and the microdystrophin and Wnt/β-catenin signaling pathway plays an important role in myogenesis of adipose-derived stem cells in the dystrophin-utrophin double knock-out mouse.

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by Western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.

High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in Insect Cells with a Baculovirus Expression System

HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain ac- tive polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombi- nant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Re- combinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were in- fected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy in Lung Adenocarcinoma

Objective: To investigate the feasibility of temporally and spatially restricted Kringle5 expression induced by radiation, as well as the dual effect of radiotherapy and antiangiogenic therapy in lung adenocarcinoma in vitro. Methods: We first constructed recombinant baculovirus vectors containing Egr1 promoter and human plasminogen Kringle5 gene (rhK5), then transfected them into lung adenocarcinoma cells (A549). Transfect efficiency of the baculovirus for gene transfer in A549 cells and the activity of Egr1 promoter induced by X-radiation were detected by fluorescence microscopy. The rhK5 mRNA transcription and rhK5 protein expression were detected by Real-time PCR and Western blot assay, respectively. The apoptosis asssay of human umbilical veins endothelial cells (HUVEC) was analyzed by flow cytometry. Results: The recombinant baculovirus were successfully transfected into A549 and HUVEC cells. As for the temporal regulation, the rhK5 mRNA transcription and rhK5 protein expression were elevated with the irradiation time significantly. And the HUVEC apoptotic percentage increased in relation to the irradiation time as well. As for the spatial regulation, rhK5 mRNA transcription level of A549 cell lines transfected with recombinant baculovirus Egr1-K5 was significantly higher than that of control groups after the same dose of X-radiation. When we analyzed the dose and frequency of X-radiation, no difference was observed among each dose after continuously three-times of irradiation. Conclusion: Baculovirus-mediated Egr1-K5 can be used in gene radiotherapy for its temporary and spatial controllable rhK5 expression by X-radiation and the consequent HUVEC apoptosis in vitro study. And low dose and more times of irradiation might be more effective. It would provide a promising way for the tumor treatment.

Expression of Green Fluorescent Protein Gene with Baculovirus Vectorin Insect Cells

The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.

Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system

Objective：CYP1A2 and NADPH-CYP450 oxidoreductase（POR）were expressed in the baculovirus/ Spodoptera frugiperda（sf9）system.The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR.Methods：The heme precursors[δ-Aminolaevulinic Acid（5-ALA）,Fe^3＋and hemin]were introduced into the system to evaluate their effects on the expression of CYP1A2,POR and their co-expression.All the proteins were identified using immunoblotting,CO-difference spectroscopy,or cytochrome c assay.Results：In the present study,functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system,and both of them showed high activities.Co-addition of 5-ALA and Fe^3＋significantly improved expression of CYP1A2 by about 50%compared with the addition of 5-ALA,Fe^3＋or hemin alone.Either co-addition of 5-ALA and Fe^3＋or addition of 5-ALA or Fe^3＋alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control（no addition）.However,unlike CYP1A2,there was no difference between the co-addition and addition of these heme precursors alone.Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR,with a 3：1 ratio of BvCYP1A2/BvPOR significantly increasing their co-expression.Surprisingly,the addition of 0.1 mM 5-ALA or Fe^3＋alone,but not their co- addition,could significantly improve the CYP1A2 and POR co-expression（P〈0.05）.Conclusion：5-ALA and Fe^3＋increased the expression of CYP1A2 and POR in a baculovirus/sf9 system,but the pattern of their expression was different between their expression alone and co-expression.

Inhibiting Mechanism of Baculovirus p35 Gene to Apoptosis

We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35 gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNA electrophoresis, Sf9-35 cells were found to resist apoptosis induced by infection of vAcΔp35 deleting p35 gene and actinomycin D treatment; And it was also found that apoptosis induced by viral infection and actinomycin D treatment can only be delayed, but can not be stopped in Sf9-35.

Expression of Recombinant Baculovirus Carrying Schistosoma japonicum 26 ku GST in Mammalian Cells

In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene （Sj26）, and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into T vector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells, The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro, By using Western blot, the expression of 26 ku glutathione S-transferase （GST） was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24×10^8. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells, It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

Expression of p30, the major surface antigen of Toxoplasma gondii, in baculovirus-insect cell system and the evaluation of immune response induced by p30

Objective: To produce the major surface antigen (p30) of Toxoplasma gondii from the Baculovirus Expression System. Methods: The p30 coding sequence was cloned into a transfer vector, then the recombinant baculovirus containing p30 gene was cloned and purified by the co-transfection and plaque assay. The expression and immunoactivity of the recombinant p30 were analyzed by SDS-PAGE and Western blot. The immune responses in mice for being immunized with recombinant p30 were tested. Results: About 750μg of purified (95% purity) p30 was obtained from a culture of 108 in- sect Sf21 cells. Mice in injected with the recombinant protein produced specific humoral and cellular immune responses. Immunization with p30 also prolonged the period of mice survival infected by Toxoplasma gondii. Conclusion: It is indicated that the recombinant p30 from baculovirus expression system can stimulate mice to produce effective protection from Toxo- plasma gondii infection.

Localization of the VP2 Protein of Canine Parvovirus Type 2 on the Baculovirus Envelop and Its Immunogenicity in a Mouse Model

In this study, the full-length VP2 gene of canine parvovirus type 2 (CPV-2) was cloned into the pBacSC vector which possesses baculovirus transmembrane domain (gp64 TM) gene, baculovirus cytoplasmic domain (gp64 CTD) gene, and green florescence protein (GFP) gene. Baculovirus gp64 TM and gp64 CTD in the pBacSC vector were designed to display heterologous proteins on the baculovirus envelope. After cloning the VP2 gene of CPV-2 into pBacSC vector, the recombinant plasmid pBacSC-VP2 was transformed into E. coli DH10Bac competent cells to form recombinant bacmid DNA. One recombinant baculovirus BacSC-VP2 that expresses the VP2 protein of CPV-2 was obtained. Confocal microscopy and immunogold electron microscopy were used to verify whether VP2 expressing on baculovirus envelope or cell membrane. Immunization of BALB/c mice with recombinant baculovirus BacSC-VP2, demonstrated that serum from the BacSC-VP2 treated models had higher levels of virus neutralization titers than the control groups. The results show that the recombinant baculovirus BacSC-VP2 can induce a strong immune response in a mouse model, suggesting that the pseudotyped baculovirus BacSC-VP2 can serve as a potential vaccine against CPV infections.

Phylogenetic Analysis of Baculovirus Isolates from Diseased Insects in Southern Vietnam

The aim of this study was to investigate the molecular identification and assess the genetic relationship of baculovirus isolated from Southern Vietnam. The diseased insect samples were collected from the different fields. The partial sequence of 450 base pairs of lef-8 gene was amplified and sequenced to assess the genetic variations of baculovirus isolates specific for Spodoptera litura, Helicoverpa zea, and Helicoverpa armigera. The sequences alignment demonstrated that Helicoverpa zea specific isolates exhibited six single nucleotide polymorphic sites. Whereas, twenty five single polymorphic sites were found in Spodoptera litura specific isolates. Thus, Spodoptera litura specific isolates were higher polymorphic than Helicoverpa zea specific isolates. The genetic distance analyses showed that the distance between Vietnamese baculovirus isolates and Group II Alphabaculovirus isolates was lower than other Baculovirus groups. The phylogeny of Vietnamese isolates in relation to other baculovirus isolates was also determined using partial sequences of lef-8 gene. The phylogenetic tree placed all Vietnamese isolates in Group II Alphabaculovirus, where seven Vietnamese Helicoverpa zea specific isolates were most closely related to Helicoverpa zea SNPV, fourteen Vietnamese Spodoptera litura specific isolates were located with Spodoptera litura NPV-G2 in one clade and a Vietnamese Helicoverpa armigera isolate was appeared to be closely related to Helicoverpa armigera SNPV-NNg1, Helicoverpa armigera NPV-C1, Helicoverpa armigera NPV-G4.

Nucleotide Sequence of Polyhedrin Gene of LsNPV and Analysis of Baculovirus Polyhedron Proteins

The intact 741 hp polyhedrin gene of LsNPV was sequenced by Silver Sequencing System, and shares 90.6% and 97.0% nucleotide identity, 97.2% and 97. 6% amino acid identity with PfNPV and MdNPV polh genes respectively. The 14 hp conservative sequence with the core element GTAAG,is located in the 5'untranslated region of the gene. The polh gene was predicted to encodes a 246 amino sold residures with molecular weight of 29.0 kd, in which the number of acidic amino acids and alkaline amino acids was roughly equal resulting in almost no charges in polyhedrin protein molecule and hence occlusion body. It gives a valuable implication that ionic bonds as well as hydrophobic bonds and hydrogen bond may Play an important role in the crystallization or polyhedrin, by comparing amino acid variation of twenty-one polyhedrin. The comparison of promoter regions of polyhedrin gene and class Ⅲ gene shown that they are very similar, but also have differences in GC content.This could explain that both categories of gene are highly expressed, and polyhedrin genes are expressed more higher than class Ⅲ gene.

Roadblock to Mammalian Cell Entry of Baculovirus

Baculoviruses infect insects in nature by targeting their larvae.They can also pass on genes into mammalian cells(including human cells)without producing progeny viruses via a process termed transduction.For that,they are promising vectors for human gene therapy.There is,however,an unsolved obstacle that the mammalian transduction is very inefficient.To address that,scientists from the CAS Wuhan Institute of Virology(WHIOV)sought to figure out the major roadblock to mammalian cell entry of baculovirus and find out a solution to overcome it.

Baculoviruses as Vectors in Mammalian Cells

The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy.

Processing of Baculovirus Late and Very Late mRNAs

Baculoviruses encode a DNA-directed RNA polymerase that is evolutionarily divergent from cellular polymerases. This RNA polymerase is a multifunctional complex that has the ability to recognize late promoters, transcribe linked genes, and process transcripts at both the 5’ and 3’ ends. The LEF-4 subunit of the viral RNA polymerase is the mRNA capping enzyme, with both RNA triphosphatase and guanylyltransferase activities. Conversion to cap 1 structures is mediated by the viral enzyme MTase1 and another as yet unidentified methyltransferase. Termination is an intrinsic property of the viral RNA polymerase and occurs at oligoU rich sequences. Polyadenylation of the released transcripts is also a function of the viral RNA polymerase. Thus, although viral mRNAs resemble host messages with respect to their 5’ and 3’ end structures, the processing is mediated by viral enzymes and, in the case of the 3’ ends, by mechanisms that differ from the host cell.