Persistent baculovirus infection is observed frequently in insect populations. Persistent infection can be transformed to a replicative and infective state caused by stress factors and plays an important role in regulating the size of insect population and in epizoology of baculoviruses. The aim of this study is to establish a persistently baculovirus-infected cell system to explore the molecular mechanisms of baculoviral persistence. Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was serially undiluted passaged in Se301 cells to reduce virulence. Upon infection of Se301 cells with the SeMNPV up to passage 8, a few cells survived even if most of cells died due to virus infection. The surviving cells were passaged and designated as P8-Se301 cell strain. P8-Se301 cells had a population doubling time of 58-65 hours and grew slower than Se301 cells. Light microscopy and electron microscopy observation showed symptom of baculovirus infection, such as virogenic stroma, viral particles and occlusion bodies, in some of P8-Se301 cells. End-point dilution assay and infectious center assay showed that 4.14% +/- 0.99% cells continually released infectious progeny virus which replicated slower than SeMNPV in Se301 cells. The result indicated that P8-Se301 cells show a typical character trait of baculovirus persistent infection.
Animals ; Cells, Cultured ; Nucleopolyhedrovirus ; growth & development ; physiology ; Spodoptera ; virology ; Virus Cultivation ; methods

To construct a novel baculovirus expression system of Spodoptera litura multicapsid nucleopolyhedrovirus, the 5' end and 3' end-flanking fragments of ph gene were amplified from the genome DNA of SpltMNPV, Japan-C3 strain using two pairs of primers synthesized according to SpltMNPV China-G2 strain genome DNA sequence published in GenBank. To obtain the transfer vector pSplt-gfp, the fragment of gfp gene was inserted into this vector between two fragments tandem linked into pUC18. The spli cells were cotransfected with pSplt-gfp and the wild SpltMNPV genome DNA. The recombinant virus containing gfp was selected with the limited dilution method. The fluorescence can be observed in the spli cells and the 3rd instar larvae after 24 and 48 hours by infection of the recombinant virus, respectively. The result showed that the recombinant virus was obtained successfully. It will be helpful to establish Spodoptera litura multicapsid nucleopolyhedrovirus expression system and more effective pesticide for Spodoptera litura.
Animals ; Baculoviridae ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Larva ; genetics ; virology ; Nucleopolyhedrovirus ; genetics ; Spodoptera ; genetics ; virology

This study was performed to investigate the effects of different regions of the Autographa califor nica multiple nucleopolyhedrovirus envelope protein E25 on its trafficking into nucleus and nuclear localization in host cells and on virus replication. Fourteen recombinant bacmids, each containing an e25 mutant with substitution or insertion of egfp, in the absence or presence of the native e25, were constructed and used to transfect Sf9 cells. The E25-EGFP fusion proteins and native E25 expressed in the cells transfect ed with individual recombinant bacmid were traced by autofluorescence from EGFP or by immuno-fluorescence assays. Confocal microscopy revealed that the E25-EGFP fusion protein with the N-domain (2-45aa) of E25 substituted by EGFP only distributed in the cytoplasm in transfected cells; and the fusion protein with EGFP inserted at the laa/2aa site of E25 completely remained outside of the nucleus and resided along the nuclear membrane. The E25-EGFPs with 46-118aa of E25 substituted by EGFP or with EGFP inserted at the 118aa/119aa site were present outside, across from the nuclear membrane or in nuclear plasm in dot-like shapes. The fusion proteins with the C-domain substituted by EGFP or with EGFP inserted at the site of 45/46aa or at the C-terminal formed a condensed ring or spread throughout the nucleus, in a similar manner to the E25 distributed in the cells transfected by the e25-knockout repair bacmid. These results prove that the N-terminal domain is critical for nuclear transportation of E25 and possibly to its position on the cytoplasm membrane as well; and the sequence downstream of the N-terminal domain also affects trafficking and nuclear localization of the protein. In cells transfected with bacmids containing both the native e25 and individual e25-egfp mutants, the E25-EGFP fusion proteins co-localized with E25 individually, showing similar patterns of subcellular localization as E25 mutants in the absence of native E25 in most cases, suggesting that the E25 likely exists and functions as dimmers or polymers. Production of infectious BV was dramatically reduced and even completely eliminated in most cases, either in the absence or presence of the native e25.
Amino Acid Motifs ; Animals ; Cell Nucleus ; metabolism ; virology ; Mutation ; Nucleopolyhedrovirus ; chemistry ; genetics ; physiology ; Protein Transport ; Spodoptera ; virology ; Viral Proteins ; chemistry ; genetics ; metabolism ; Virus Release ; Virus Replication

OBJECTIVETo secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.

METHODSThe entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot.

RESULTSAfter transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis.

CONCLUSIONSignal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.

Animals ; Cell Line ; Dengue ; metabolism ; virology ; Dengue Virus ; genetics ; metabolism ; Gene Expression ; Humans ; Protein Structure, Tertiary ; Protein Transport ; Spodoptera ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

Present studies describe the successful establishment of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) BAC-TO-BAC expression system. The mini-F-lacZ-attTn7-kan fragment (Luckow et al, 1993) was inserted into SeMNPV US1 isolate (SeUS1) at polyhedrin gene locus by directly cloning. The recombinant virus containing low-copy-number mini-F replication, which named bacmid, can propagate in Escherichia coli. Because SeUS1 isolate is make up of several genotypes and one bacmid carries one SeMNPV genotype, the SeUS1 BAC library is established by all SeMNPV bacmids (SeBAC). REN analysis for 111 SeBAC shows that SeUS1 consists of the genotype with whole SeMNPV genetic information and several genotypes with various different deletions. Progeny virus can be produced in insect cell line after transfection with SeBAC10, which carries the whole SeMNPV genome. So SeBAC10 is a shuttle vector that can replicate in eukaryocyte as well as prokaryocyte. Considering the insert mutation of SeMNPV polyhedrin gene (Seph) in SeBAC10, Seph was reintroduced into the bacmid by site-specific transposon-mediated insertion at attTn7, the target site for the bacterial transposon Tn7. The derived recombinant SeBAC10 was named SeBAC10ph. After SeBAC10ph was transfected into Se301 cells (a susceptible insect cell line to SeMNPV), cytopathogenic effect was shown and polyhedra appeared, which indicate that the foreign gene (Seph) is expressed.
Animals ; Cell Line ; Chromosomes, Artificial, Bacterial ; genetics ; DNA Transposable Elements ; genetics ; Genetic Vectors ; genetics ; Models, Theoretical ; Nucleopolyhedrovirus ; genetics ; physiology ; Spodoptera ; cytology ; virology ; Viral Structural Proteins ; genetics

The transmembrane protein (TM) encoded by gp37 gene plays a critical role when virus fusion with cell membrane occurs. Several highly conserved regions in TM are important targets for antivirus studies. Studies on structure and function of TM will provide basic information for anti-retrovirus, especially for avian leukosis virus. In the study, gp37 gene was amplified by PCR from the Chinese strain ALV-J-WS0701. The gp37 gene was cloned into pMD18-T vector, and was sequenced. Then, pFast-BacHTb-gp37 vector was constructed and expressed by baculovirus expression vector system. The expression product of gp37 gene was analyzed by indirect immunofluorescence assay and Western blot. The results showed that positive green fluorescence was present in sf9 cells infected with recombinant virus and a protein band with a molecular weight of 21kD was present in Western blot. It is concluded that gp37 gene was expressed in sf9 cells infected with recombinant virus successfully.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; classification ; genetics ; isolation & purification ; Cell Line ; Chickens ; Cloning, Molecular ; Gene Expression ; Spodoptera ; Viral Envelope Proteins ; genetics ; metabolism

Nuclear actin which plays a key role in many nucleic processes has become a research hotspot. Baculovirus is the only reported pathogen using nuclear actin to replicate and proliferate. However, little is known about the mechanism of monomeric G-actin accumulation within nuclei of baculovirus-infected cells. It has been reported that AcMNPV ie-1, pe38, ac4, he65, ac102, and ac152 could be required for mediating nuclear localization of G-actin from transiently transfected results in TN-368 cells. In this paper, we found that IE1, AC152, PE38, AC102 localized in the whole cell and PE38, AC102 localized in the nuclear mainly, while both AC4 and HE65 localized in cytoplasm and could be mediated into the nucleus by AC102 and IE1 respectively for the first time. And ie-1 or pe38, ac4, he65 could mediate nuclear G-actin to accumulate partly, while these four genes were sufficient for recruiting G-actin accumulation within the nucleus when driven by promoter OpIE2. Determining the functions of each of these AcMNPV NLA gene products will advance our understanding of baculovirus biology and function of nuclear actin.
Actins ; genetics ; metabolism ; Animals ; Cell Nucleus ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Nucleopolyhedrovirus ; genetics ; metabolism ; Promoter Regions, Genetic ; Protein Transport ; Spodoptera ; metabolism ; virology ; Viral Proteins ; genetics ; metabolism

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program.
Animals ; Baculoviridae/genetics/*immunology ; Chickens/*virology ; DNA Primers ; Gene Amplification ; HN Protein/genetics/*therapeutic use ; Korea ; Marek Disease/immunology/prevention & control ; Newcastle Disease/immunology/*prevention & control ; Spodoptera/virology ; Vaccines, Synthetic/genetics/therapeutic use ; Viral Vaccines/genetics/therapeutic use

OBJECTIVETo clone and express VP, gene from HBoV, and the expressed VP, protein was as the antigen in order to detect serum from children in Wenling area with lower respiratory tract infections.

METHODSThe VP, gene was recombined with the genome of Baculovirus, which infected the insect cell. The fusion protein with HA tag was applied to confirm the specificity of expressed protein. Furthermore, the recombinant protein was observed using electron microscopy. The 176 serum from children in Wenling area with lower respiratory tract infections was screened using Western blot.

RESULTSThe expressed VP2 protein was more than 60% in total proteins from insect cell, and MWt about 60 x 10(3). The virus-like particle (VLP) was observed using electron microscopy, and size about 20 nm. The 176 serum from children in wenling area with lower respiratory tract infections was screened using Western blot. The HBoV positive rate was 2.28% (4/176).

CONCLUSIONThe VP2 protein from human bocavirus was expressed in insect cell successfully. Through HA tag the VP2 protein was specific, and then the assay using SDS-PAGE with Western blot could detect and screen the antibody in serum from children with lower respiratory tract infections rapidly and accurately.

Animals ; Antibodies, Viral ; blood ; Bocavirus ; genetics ; immunology ; Capsid Proteins ; genetics ; immunology ; Child, Preschool ; Female ; Gene Expression ; Humans ; Infant ; Male ; Parvoviridae Infections ; blood ; diagnosis ; immunology ; virology ; Recombinant Proteins ; genetics ; immunology ; Spodoptera

CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.
Animals ; Baculoviridae ; enzymology ; genetics ; Catalysis ; Cells, Cultured ; Chromatography, High Pressure Liquid ; methods ; Cytochrome P-450 CYP2D6 ; genetics ; metabolism ; Dextromethorphan ; metabolism ; Isoenzymes ; metabolism ; NADPH-Ferrihemoprotein Reductase ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; Spodoptera ; cytology ; virology ; Transfection