3H-117, a structural protein of Heliothis virescens ascovirus 3h (HvAV-3h).

The open reading frame 117 (3h-117) of Heliothis virescens ascovirus 3h (HvAV-3h), which is a conserved coding region present in all completely sequenced ascovirus members, was characterized in this study. By RT-PCR detection, 3h-117 transcription began at 6-h post-infection (hpi) and remained stable until 168 hpi in HvAV-3h-infected Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. In addition, 3h-117 putatively encodes a 21.5-kDa protein (3H-117) predicted to be a CTD-like phosphatase. Western blot analysis using a prepared rabbit polyclonal antibody specific to 3H-117 showed that the product could be detected at 24 hpi, which remained stably detectable until 168 hpi. The same analysis also demonstrated that the 3H-117 protein localized in the virions of HvAV-3h. Immunofluorescence analysis showed that at 24 hpi, 3H-117 was mainly located in the nuclei of H. armigera larval fat body cells and later spread into the cytoplasm. In summary, our results indicate that 3H-117 is a structural protein of HvAV-3h.

Functional characterization of a plant-produced infectious bursal disease virus antigen fused to the constant region of avian IgY immunoglobulins.

Infectious bursal disease virus (IBDV) is the cause of an economically important highly contagious disease of poultry, and vaccines are regarded as the most beneficial interventions for its prevention. In this study, plants were used to produce a recombinant chimeric IBDV antigen for the formulation of an innovative subunit vaccine. The fusion protein (PD-FcY) was designed to combine the immunodominant projection domain (PD) of the viral structural protein VP2 with the constant region of avian IgY (FcY), which was selected to enhance antigen uptake by avian immune cells. The gene construct encoding the fusion protein was transiently expressed in Nicotiana benthamiana plants and an extraction/purification protocol was set up, allowing to reduce the contamination by undesired plant compounds/proteins. Mass spectrometry analysis of the purified protein revealed that the glycosylation pattern of the FcY portion was similar to that observed in native IgY, while in vitro assays demonstrated the ability of PD-FcY to bind to the avian immunoglobulin receptor CHIR-AB1. Preliminary immunization studies proved that PD-FcY was able to induce the production of protective anti-IBDV-VP2 antibodies in chickens. In conclusion, the proposed fusion strategy holds promises for the development of innovative low-cost subunit vaccines for the prevention of avian viral diseases.

The Epstein-Barr Virus Immunoevasins BCRF1 and BPLF1 Are Expressed by a Mechanism Independent of the Canonical Late Pre-initiation Complex.

Subversion of host immune surveillance is a crucial step in viral pathogenesis. Epstein-Barr virus (EBV) encodes two immune evasion gene products, BCRF1 (viral IL-10) and BPLF1 (deubiquitinase/deneddylase); both proteins suppress antiviral immune responses during primary infection. The BCRF1 and BPLF1 genes are expressed during the late phase of the lytic cycle, an essential but poorly understood phase of viral gene expression. Several late gene regulators recently identified in beta and gamma herpesviruses form a viral pre-initiation complex for transcription. Whether each of these late gene regulators is necessary for transcription of all late genes is not known. Here, studying viral gene expression in the absence and presence of siRNAs to individual components of the viral pre-initiation complex, we identified two distinct groups of late genes. One group includes late genes encoding the two immunoevasins, BCRF1 and BPLF1, and is transcribed independently of the viral pre-initiation complex. The second group primarily encodes viral structural proteins and is dependent on the viral pre-initiation complex. The protein kinase BGLF4 is the only known late gene regulator necessary for expression of both groups of late genes. ChIP-seq analysis showed that the transcription activator Rta associates with the promoters of eight late genes including genes encoding the viral immunoevasins. Our results demonstrate that late genes encoding immunomodulatory proteins are transcribed by a mechanism distinct from late genes encoding viral structural proteins. Understanding the mechanisms that specifically regulate expression of the late immunomodulatory proteins could aid the development of antiviral drugs that impair immune evasion by the oncogenic EB virus.

Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt-mTORC1 pathway depending on activation of p70S6K and Cdc2.

We previously demonstrated that coxsackievirus B3 (CVB3) infection upregulated heat shock protein 70 (Hsp70) and promoted CVB3 multiplication. Here, we report the underlying mechanism by which Hsp70 enhances viral RNA translation. By using an Hsp70-overexpressing cell line infected with CVB3, we found that Hsp70 enhanced CVB3 VP1 translation at two stages. First, Hsp70 induced upregulation of VP1 translation at the initiation stage via upregulation of internal ribosome entry site trans-acting factor lupus autoantigen protein and activation of eIF4E binding protein 1, a cap-dependent translation suppressor. Second, we found that Hsp70 increased CVB3 VP1 translation by enhancing translation elongation. This was mediated by the Akt-mammalian target of rapamycin complex 1 signal cascade, which led to the activation of eukaryotic elongation factor 2 via p70S6K- and cell division cycle protein 2 homolog (Cdc2)-mediated phosphorylation and inactivation of eukaryotic elongation factor 2 kinase. We also determined the position of Cdc2 in this signal pathway, indicating that Cdc2 is regulated by mammalian target of rapamycin complex 1. This signal transduction pathway was validated using a number of specific pharmacological inhibitors, short interfering RNAs (siRNAs) and a dominant negative Akt plasmid. Because Hsp70 is a central component of the cellular network of molecular chaperones enhancing viral replication, these data may provide new strategies to limit this viral infection.

Yeast as an expression system for producing virus-like particles: what factors do we need to consider?

The development of various types of virus-like particles (VLPs) has accelerated over the past two decades as the importance of VLPs for generating next-generation vaccines has been appreciated. Yeast has advantages such as scalable fermentation, low risk of contamination by adventitious agents, low production costs and the ability to produce VLPs with reliable qualities. It is generally recognized that yeast is suitable for producing VLPs that have simple structures and are produced intracellularly. However, recently there has been much effort to extend its applicability, and there is now evidence that it can be used as an expression platform for the productions of VLPs not only of nonenveloped viruses but also of enveloped viruses. Moreover, evidences indicated that yeast allows secretory VLP productions. Meanwhile, it has become evident that the quality and quantity of yeast-derived VLPs are influenced by the choice of plasmid and promoter, the ratio of the structural proteins produced. Here, we review the characteristics of the yeast expression system in terms of the production of VLP and compare it with other expression systems. We also consider strategies for VLP production in yeast and factors that need to be taken into account.

A locus encompassing the Epstein-Barr virus bglf4 kinase regulates expression of genes encoding viral structural proteins.

The mechanism regulating expression of late genes, encoding viral structural components, is an unresolved problem in the biology of DNA tumor viruses. Here we show that BGLF4, the only protein kinase encoded by Epstein-Barr virus (EBV), controls expression of late genes independent of its effect on viral DNA replication. Ectopic expression of BGLF4 in cells lacking the kinase gene stimulated the transcript levels of six late genes by 8- to 10-fold. Introduction of a BGLF4 mutant that eliminated its kinase activity did not stimulate late gene expression. In cells infected with wild-type EBV, siRNA to BGLF4 (siG4) markedly reduced late gene expression without compromising viral DNA replication. Synthesis of late products was restored upon expression of a form of BGLF4 resistant to the siRNA. Studying the EBV transcriptome using mRNA-seq during the late phase of the lytic cycle in the absence and presence of siG4 showed that BGLF4 controlled expression of 31 late genes. Analysis of the EBV transcriptome identified BGLF3 as a gene whose expression was reduced as a result of silencing BGLF4. Knockdown of BGLF3 markedly reduced late gene expression but had no effect on viral DNA replication or expression of BGLF4. Our findings reveal the presence of a late control locus encompassing BGLF3 and BGLF4 in the EBV genome, and provide evidence for the importance of both proteins in post-replication events that are necessary for expression of late genes.

Melatonin modulates the autophagic response in acute liver failure induced by the rabbit hemorrhagic disease virus.

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin-1, ubiquitin-like autophagy-related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3-I to autophagosome-associated LC3-II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome-associated membrane protein (LAMP)-1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 mRNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase-3 activity, and immunoglobulin-heavy-chain-binding protein (BiP) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF.

Bacteriophage lambda display systems: developments and applications.

Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of λ and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique formation of the λ capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future applications of λ display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage λ and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control.

Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 cells, infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants.

During cell infection, the fp25k gene of baculoviruses frequently mutates, producing the few polyhedra (FP) per cell phenotype with reduced polyhedrin (polh) expression levels compared with wild-type baculoviruses. Here we report that the fp25k gene of the model baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), contains two hypermutable seven-adenine (A7) mononucleotide repeats (MNRs) that were mutated to A8 MNRs and a TTAA site that had host DNA insertions, producing fp25k mutants during Sf21 cell infection. The FP phenotype in Sf9 and Hi5 cells was more pronounced than in Sf21 cells. AcMNPV fp25k mutants produced similar levels of polyhedra or enhanced GFP, which were both under the control of the AcMNPV polh promoter for expression, in Sf21 cells but lower levels in Sf9 and Hi5 cells compared with AcMNPV with an intact fp25k gene. This correlated with the polh mRNA levels detected in each cell line. The majority of Sf21 cells infected with fp25 mutants showed high polh promoter-mediated GFP expression levels. Two cell lines subcloned from Sf21 cells that were infected with fp25k mutants showed different GFP expression levels. Furthermore, a small proportion of Hi5 cells infected with fp25k mutants showed higher production of polyhedra and GFP expression than the rest, and the latter was not correlated with increased m.o.i. Therefore, these data suggest that AcMNPV polh promoter-mediated gene expression activities differ in the three cell lines and are influenced by different cells within the cell line.

Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice.

Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni-NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

Characterization of Magnaporthe oryzae chrysovirus 1 structural proteins and their expression in Saccharomyces cerevisiae.

Magnaporthe oryzae chrysovirus 1 (MoCV1), which is associated with an impaired growth phenotype of its host fungus, harbors four major proteins: P130 (130 kDa), P70 (70 kDa), P65 (65 kDa), and P58 (58 kDa). N-terminal sequence analysis of each protein revealed that P130 was encoded by double-stranded RNA1 (dsRNA1) (open reading frame 1 [ORF1] 1,127 amino acids [aa]), P70 by dsRNA4 (ORF4; 812 aa), and P58 by dsRNA3 (ORF3; 799 aa), although the molecular masses of P58 and P70 were significantly smaller than those deduced for ORF3 and ORF4, respectively. P65 was a degraded form of P70. Full-size proteins of ORF3 (84 kDa) and ORF4 (85 kDa) were produced in Escherichia coli. Antisera against these recombinant proteins detected full-size proteins encoded by ORF3 and ORF4 in mycelia cultured for 9, 15, and 28 days, and the antisera also detected smaller degraded proteins, namely, P58, P70, and P65, in mycelia cultured for 28 days. These full-size proteins and P58 and P70 were also components of viral particles, indicating that MoCV1 particles might have at least two forms during vegetative growth of the host fungus. Expression of the ORF4 protein in Saccharomyces cerevisiae resulted in cytological changes, with a large central vacuole associated with these growth defects. MoCV1 has five dsRNA segments, as do two Fusarium graminearum viruses (FgV-ch9 and FgV2), and forms a separate clade with FgV-ch9, FgV2, Aspergillus mycovirus 1816 (AsV1816), and Agaricus bisporus virus 1 (AbV1) in the Chrysoviridae family on the basis of their RdRp protein sequences.

The transcription analysis of duck enteritis virus UL49.5 gene using real-time quantitative reverse transcription PCR.

Duck enteritis virus (DEV) UL49.5 encoding glycoprotein N was a conserved gene. The transcription dynamic process of UL49.5 homologous genes in herpesviruses was reported. However, the transcription dynamic process of DEV UL49.5 gene has not yet been established. In this study, a real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay was established to test the transcription dynamic process of DEV UL49.5 gene, and the recombinant plasmid pUCm-T/UL49.5 was constructed as the standard DNA. The samples prepared from DEV-infected (at different time points) and uninfected cell were detected and calculated. The results demonstrated that the real-time qRT-PCR assay was successfully established. The transcription product of DEV UL49.5 gene was first detected at 0.5 h post infection (p.i.), increased at 8 h p.i. and reached a peak at 60 h p.i. Our results illustrated that DEV UL49.5 gene could be regarded as a late gene. The transcription dynamic process of DEV UL49.5 gene may provide a significant clue for further studies of DEV UL49.5 gene.

Cell-based analysis of Chikungunya virus E1 protein in membrane fusion.

BACKGROUND: Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored. METHODS: A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity. RESULTS: Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion. CONCLUSIONS: Cells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.

The screening and functional study of proteins binding with the BmNPV polyhedrin promoter.

BACKGROUND: The polyhedrin gene promoter has an essential role in regulating foreign gene expression in baculovirus expression vector systems (BEVS); however, the high-level transcription mechanism is still unknown. One-hybrid screening in yeast is a powerful way of identifying rapidly heterologous transcription factors that can interact with the polyhedrin promoter DNA sequence. In the current study, total RNA was extracted from the fat bodies of fifth-instar silkworm larvae that had been infected with Bombyx mori nuclear polyhedrosis virus (BmNPV) for 5 days; complementary DNA (cDNA) was then generated using reverse-transcription (RT)-PCR to construct a silkworm gene expression library. Key polyhedrin promoter bait sequences were synthesized to generate a bait yeast strain, which was used to screen the one-hybrid cDNA library. RESULTS: In total, 12 positive yeast colonies were obtained from the SD/-Leu/AbA plates; sequencing analysis showed that they belong to two different protein cDNA colonies. Positive colonies underwent bioinformatics analysis, which revealed one colony to be ribosomal proteins [B. mori ribosomal protein SA (BmRPSA)] and the other to be NPV DNA-binding proteins (DBP). To further verify the regulatory function of these two protein groups, transient expression vectors (pSK-IE-dbp and pSK-IE-BmRPSA) were constructed. The recombinant plasmids were then transfected into cultured B. mori N (BmN) cells, which had been infected with a recombinant bacmid containing the gene encoding luciferase (luc). The results showed that overexpression of either dbp or BmRPSA upregulated the polh promoter-driven transcription of luc in BmN cells. In addition, dbp or BmRPSA RNA interference (RNAi) resulted in the downregulation of luciferase reporter expression in BmN cells, demonstrating that DBP and BmRPSA are important for luc transcription. EMSA results further confirmed that DBP could directly bind to the conserved single-stranded polh promoter region in intro. However, EMSA assay also showed that BmRPSA did not bind to this region, precluding a direct DNA association. CONCLUSIONS: Both DBP and BmRPSA are important for polh transcription. DBP can regulate polh promoter activity by direct binding to the conserved single-stranded polh promoter region, BmRPSA may regulate polh promoter activity by indirect binding to this region.

Pathological changes in the pancreas of fulminant type 1 diabetes and slowly progressive insulin-dependent diabetes mellitus (SPIDDM): innate immunity in fulminant type 1 diabetes and SPIDDM.

OBJECTIVE: The contribution of innate immunity responsible for beta-cell destruction in fulminant type 1 diabetes (FT1D) and slowly progressive insulin-dependent diabetes mellitus (SPIDDM) is unclear. RESEARCH DESIGN AND METHODS: Islet-cell expression of Toll-like receptors (TLRs) including TLR3 and TLR4, the cytoplasmic retinoic acid-inducible protein I (RIG-I)-like helicases, RIG-I, melanoma differentiation-associated gene-5 and laboratory of genetics and physiology 2 in the affected islets were studied immuno-histochemically on three pancreases obtained 2-5 days after the onset of FT1D and a pancreas from a patient with SPIDDM. RESULTS: Laboratory of genetics and physiology 2 and RIG-I strongly expressed in beta cells in all three FT1D pancreases infected with enterovirus (VP1 antigen). Melanoma differentiation-associated gene-5 was hyper-expressed in all subsets of islet cells including beta cells and alpha cells. TLR3 and TLR4 were expressed in mononuclear cells that infiltrated to islets. IFN-alpha/beta was strongly expressed in islet cells. In contrast, pancreas of a patient with SPIDDM, enterovirus and expression of innate immune receptors including RIG-I, melanoma differentiation-associated gene-5, hyperexpression of laboratory of genetics and physiology 2 and mononuclear cells, which were positive for TLR3 and TLR4, and infiltration to the islets were not detected. CONCLUSIONS: These findings demonstrate that retinoic acid-inducible protein I (RIG-I)-like helicases and TLRs play a crucial role on beta-cell destruction in enterovirus-induced FT1D. The presence of distinct mechanism(s) of slowly progressive beta-cell failure in SPIDDM was suggested.

Characterization of a late gene, ORF134, from Bombyx mori nucleopolyhedrovirus.

OBJECTIVE: ORF134 of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homolog of Autographa californica multiple NPV ORF5, but its function is unknown so far. The aim of this study is to characterize BmNPV ORF134 (Bm134). METHODS: Transcription, protein expression and subcellular localization of Bm134 in BmN cells and silkworm midguts were analyzed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence, respectively. RESULTS: Both the transcription and protein expression of Bm134 were not detected until 24 h post-infection (p.i.) by RT-PCR and Western blot, indicating that Bm134 is a late gene. Western blot revealed that Bm134 encodes an expected 12.4-kDa structural protein that is associated with occlusion-derived virus (ODV), not with budded virus. Immunofluorescence analysis showed that the Bm134 was first detected in the cytoplasm 24 h p.i. and then transported to the nucleus during later infection. Transcripts of Bm134 and the corresponding protein were only detected 48-72 h p.i. in BmNPV-infected larvae of 306, a highly BmNPV-susceptible silkworm strain, but not in the NB strain that is resistant to BmNPV infection. CONCLUSION: Taken together, our data suggest that Bm134 is a late gene of BmNPV and may function as an ODV structural protein.

Expression of a recombinant chimeric protein of hepatitis A virus VP1-Fc using a replicating vector based on Beet curly top virus in tobacco leaves and its immunogenicity in mice.

We describe the expression and immunogenicity of a recombinant chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus VP1 and an Fc antibody fragment using a replicating vector based on Beet curly top virus (BCTV) in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Recombinant HAV VP1-Fc was expressed with a molecular mass of approximately 68 kDa. Recombinant HAV VP1-Fc, purified using Protein A Sepharose affinity chromatography, elicited production of specific IgG antibodies in the serum after intraperitoneal immunization. Following vaccination with recombinant HAV VP1-Fc protein, expressions of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Recombinant VP1-Fc from infiltrated tobacco plants can be used as an effective experimental immunogen for research into vaccine development.

[Construction and toxicity the recombinant SpltMNPV expressing the scorpion toxin gene].

OBJECTIVE: To develop a high toxic recombinant Spodoptera litura multicapsid nucleopolyhedroviruse (SpltMNPV) insecticide. METHODS: We constructed a recombinant transfer vector that was characterized by disrupting of ecdysterioid UDP-glucosyltransferase (egt) gene and expressing the mature peptide of the Chinese scorpion, B. martensi Karsch (BmK ITal) gene at the control of ie-1 promoter. The transfer vector and the SpltMNPV II DNA cotransfected the SpLi cells. Recombinant viruses were purified by the end point dilution and fluorescent spot purification. RESULTS: We successfully screened the recombinant SpltMNPV-deltaegt-Pph-egfp-ie-1-BmK ITal of which the egt gene was knocked out and expressed the mature peptide of the BmK ITal gene at the control of ie-1 promoter. Bioassays showed that, compared to the wide-type SpltMNPV, the speed of the recombinant virus killing the S. litura (LT50) increased by 0.7-0.8 days. CONCLUSION: The insecticidal effect of SpltNPV could be increased by inserting the foreign gene, which provided a further opportunity to develop the SpltNPV into commercially viable products to control the S. litura.

[Coronaviruses].

Coronaviruses contain positive-stranded RNA with ca. 30 kb as a genome, which is wrapped by the envelope, and constitute Nidovirales together with Arteriviridae. The feature of viruses in Nidovirales is the unique structure of the mRNA set, called 3' co-terminal nested set. Coronaviruses have several to more than 10 different species of subgenomic mRNA and generally only the OFR located in the 5' end of each mRNA is translated. The 5' 20 kb of the coronavirus genome or mRNA-1 consists of two ORFs, 1a and 1b, between that there is a unique RNA structure called pseudoknot. From mRNA-1, 1a as well as 1a+1b are translated; the latter 1a+1b results from the translation due to ribosomal frame-shifting facilitated by the pseudoknot structure. From those two proteins, totally 16 proteins are produced as a result of auto-cleavage by the proteases included in la protein. Those proteins exhibit different functions, such as RNA-dependent RNA polymerase, helicase, proteases and proteins that regulate cellular functions, mRNAs smaller than mRNA-2 translate in general the structural proteins, nucleocapsid (N) protein, spike (S) protein, integrated membrane (M) protein and envelope (E) proteins. Those proteins assemble to the vesicles located from ER to Golgi (ER Golgi intermediate compartment) and virions bud into the vesicles. Those virions are released from infected cells via exocytosis.

Nipah virus edits its P gene at high frequency to express the V and W proteins.

Nipah virus (NiV) is predicted to encode four proteins from its P gene (P, V, W, and C) via mRNA editing and an alternate open reading frame. By use of specific antibodies, the expression of the V, W, and C proteins in NiV-infected cells has now been confirmed. Analysis of the P-gene transcripts shows a ratio of P:V:W mRNA of 1:1:1, but this differs over time, with greater proportions of V and W transcripts observed as the infection progresses. Eighty-two percent of transcripts are edited, with up to 11 G insertions observed. This exceptionally high editing frequency ensures expression of the V and W proteins.