Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix

Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion binding to midgut cells. We compared the potency of wild-type Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) with that of single and double enhancin deletion viruses in L. dispar in the presence and absence of an intact PM. Compared to wild-type virus, the double enhancin deletion virus was 6-fold and 14-fold less potent, respectively, indicating that within this homologous system the LdMNPV enhancin genes have a function beyond PM degradation.     

A baculovirus enhancin alters the permeability of a mucosal midgut peritrophic matrix from lepidopteran larvae

The peritrophic matrix (PM) in lepidopterous larvae may function as a defensive barrier against ingested viral pathogens. PMs isolated from Trichoplusia ni and Pseudaletia unipuncta larvae, were treated with a baculovirus-encoded metalloprotease (enhancin) from Trichoplusia ni granulosis virus (TnGV) and their in vitro permeability to blue dextran and fluorescent-labelled Autographa californica nuclear polyhedrosis virus (AcMNPV) was determined using a dual chamber permeability apparatus. Incubation of T. ni PMs with 0.0, 0.5, 1.0, and 2.0mg/ml enhancin resulted in a blue dextran 2000 flux of 4.4, 6.3, 9.9, and 15.6&mgr;g/mm(2)/h, respectively. In addition, T. ni PMs treated with enhancin were found to be significantly more permeable to fluorescent-labelled AcMNPV than non-treated control PMs. The permeability of T. ni PMs treated with 3.0mg/ml enhancin was 0.017 cumulative percent crossing/mm(2)/h, whereas the permeability of the control PM was below the detectable limit. Similarly, enhancin treatment greatly increased the permeability of P. unipuncta PMs to AcMNPV. These results provide evidence that the PM from two lepidopteran species can block the passage of baculovirions across this matrix thus reducing the probability of larval infection. Furthermore, these results support the hypothesis that enhancin facilitates NPV infection of larvae by altering the permeability of the PM.     

Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.     

Vertical transmission of TnSNPV,TnCPV, AcMNPV,and possibly recombinant NPV in Trichoplusia ni

Four viruses were tested for vertical transmission in Trichoplusia ni: T. ni nucleopolyhedrovirus (TnSNPV), T. ni cypovirus (TnCPV), Autographa californica nucleopolyhedrovirus (AcMNPV), and AcMNPV engineered to express a scorpion toxin (AcMNPV.AaIT). Fifth instars were exposed to each virus, the survivors were reared and mated, and second-generation (F(1)) insects were examined for infection. TnSNPV was transmitted to offspring at a prevalence rate of 15.4%, TnCPV at 10.2%, and AcMNPV at 10.1%. Only one of 2484 F(1) insects was infected with AcMNPV.AaIT; this experiment was repeated, and none of 4774 insects was infected. Thus, vertical transmission is unlikely to contribute to AcMNPV.AaIT contacting non-target organisms after its field release. There was evidence that TnCPV and possibly TnSNPV were activated to overt infections by ingestion of a different virus. TnCPV, but not the NPVs, routinely infected 0.3-1.7% of non-treated insects, probably indicating that it is vertically transmitted at enzootic levels.     

粉纹夜蛾颗粒体病毒增强蛋白锌离子结合域定点突变

粉纹夜蛾颗粒体病毒（Trichoplusia ni granulovirus, TnGV）增强蛋白（enhancin）具有增强病毒感染力的作用。该蛋白包含一个多种杆状病毒增强蛋白都具有的保守结构域HELGH,是典型的金属蛋白酶锌离子结合域,但该结构域对增强蛋白生物活性的重要性尚未得到研究。本研究通过定点突变构建了该结构域的5个氨基酸分别突变为2种不同氨基酸的共10种增强蛋白突变体基因,并用杆状病毒载体进行了重组表达。活性测定发现,10种突变型增强蛋白大部分都丧失了野生型增强蛋白所具有的降解粉纹夜蛾幼虫围食膜粘蛋白的生物学功能,只有1种（第4位G突变为A）保留该生物学活性。这一结果表明锌离子结合域对增强蛋白生物活性具有重要作用,也提示增强蛋白确是一种金属蛋白酶。     

增强蛋白与多角体蛋白共包埋的研究

颗粒体病毒的增强蛋白（enhancin)是一种能显著提高核型多角体病毒（NPV）对昆虫感染力的病毒蛋白。构建了一种不形成多角体但表达粉纹夜蛾颗粒体病毒增强蛋白的重组病毒AcBBH-TnEn,将它与野生型AcMNPV共同感染SF21细胞,经SDS－PAGE、免疫印迹分析、荧光免疫等方法检测证实,增强蛋白与多角体可在同一细胞中同时表达,而且发现所形成的病毒多角体带有增强蛋白。这表明,可以通过混合感染的方式生产带有增强蛋白的病毒多角体。     

Benzylideneacetone, an eicosanoid biosynthesis inhibitor enhances baculovirus pathogenicity in the diamondback moth, Plutella xylostella

Benzylideneacetone (BZA) is a monoterpenoid compound produced by an entomopathogenic bacterium, Xenorhabdus nematophila. BZA inhibits phospholipase A₂ to suppress biosynthesis of eicosanoids that mediate immune responses in insects. In response to per os infection of Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), the diamondback moth, Plutella xylostella, developed red spots on the midgut epithelium. The midgut exhibiting red spot formation suffered abnormal cell integrity, such as genomic DNA fragmentation and condensed spots in the nucleoplasm. The number of red spots increased with viral dose and incubation time after the viral treatment. BZA inhibited the formation of the midgut red spots in a dose-dependent manner. However, the inhibitory effect of BZA on the red spot formation was reversed by addition of arachidonic acid, suggesting that the red spot response may be mediated by eicosanoids. BZA treatment resulted in significant enhancement of AcMNPV occlusion body (OB) pathogenicity to P. xylostella.     

The Lymantria dispar nucleopolyhedrovirus enhancins are components of occlusion-derived virus

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of approximately 84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.     

Comparative activity of baculoviruses against the codling moth Cydia pomonella and three other tortricid pests of tree fruit

The granulovirus of Cydia pomonella (L.) (CpGV) offers potential for selective control of codling moth. Two major limitations of CpGV are its narrow host range and lack of persistence in the orchard agroecosystem. The nucleopolyhedroviruses of the alfalfa looper Autographa californica (Speyer) (AcMNPV) and those of the celery looper Anagrapha falcifera (Kirby) (AfMNPV) have broad host ranges. Comparative assays of CpGV, AcMNPV, and AfMNPV against codling moth neonate larvae revealed a 54-93-fold greater susceptibility of codling moth to the granulovirus than to the two nucleopolyhedroviruses based on the LC(50) values for each virus. The LC(50)s for CpGV, AfMNPV, and AcMNPV were 32.7 capsules/mm(2), 1.77 x 10(3) occlusion bodies (OBs)/mm(2), and 3.05 x 10(3)OBs/mm(2), respectively. The LT(50) determined for AfMNPV using an approximate LC(95) of the virus against neonate larvae was 3.6 days. Histological examination of tissues in moribund codling moth larvae that had been treated with AfMNPV revealed the presence of nonoccluded and unenveloped virus rods in midgut tissue. Neither OBs nor signs of infection were detected in other tissues. The activity of AfMNPV was also evaluated in three other tortricid apple pests (obliquebanded leafroller, Choristoneura rosaceana (Harris); Pandemis leafroller, Pandemis pyrusana Kearfott; and the oriental fruit moth, Grapholitha molesta (Busck)). Codling and Oriental fruit moths were significantly more susceptible to AfMNPV than were the two leafroller species.     

Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera: Noctuidae)

A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixed infected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of ThorMNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band profiles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from different virion sample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 values of 17 and 242OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera frugiperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as the fat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA transfection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21 was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame (ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phylogenetic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and is related but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster.     

Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

Bacillus thuringiensis Bel Protein Enhances the Toxicity of Cry1Ac Protein to Helicoverpa armigera Larvae by Degrading Insect Intestinal Mucin

Bacillus thuringiensis has been used as a bioinsecticide to control agricultural insects. Bacillus cereus group genomes were found to have a Bacillus enhancin-like (bel) gene, encoding a peptide with 20 to 30% identity to viral enhancin protein, which can enhance viral infection by degradation of the peritrophic matrix (PM) of the insect midgut. In this study, the bel gene was found to have an activity similar to that of the viral enhancin gene. A bel knockout mutant was constructed by using a plasmid-free B. thuringiensis derivative, BMB171. The 50% lethal concentrations of this mutant plus the cry1Ac insecticidal protein gene were about 5.8-fold higher than those of the BMB171 strain. When purified Bel was mixed with the Cry1Ac protein and fed to Helicoverpa armigera larvae, 3 μg/ml Cry1Ac alone induced 34.2% mortality. Meanwhile, the mortality rate rose to 74.4% when the same amount of Cry1Ac was mixed with 0.8 μg/ml of Bel. Microscopic observation showed a significant disruption detected on the midgut PM of H. armigera larvae after they were fed Bel. In vitro degradation assays showed that Bel digested the intestinal mucin (IIM) of Trichoplusia ni and H. armigera larvae to various degrading products, similar to findings for viral enhancin. These results imply Bel toxicity enhancement depends on the destruction of midgut PM and IIM, similar to the case with viral enhancin. This discovery showed that Bel has the potential to enhance insecticidal activity of B. thuringiensis-based biopesticides and transgenic crops.Bacillus thuringiensis is a ubiquitous gram-positive, spore-forming soil bacterium and produces insecticidal crystal proteins during the sporulation phase of its growth cycle. Because these insecticidal crystal proteins have activity against certain insect species, B. thuringiensis has been extensively used as a biopesticide to control crop pests in commercial agriculture and forest management. It is also a key source of genes for transgenic expression and provides pest resistance in plants (2, 20, 30).The viral enhancin protein was originally described for granuloviruses (GVs) as a 126-kDa protein that showed an ability to enhance the infectivity of nucleopolyhedroviruses (NPVs) (36, 37, 39). It has also been found in several other GVs (13) and NPVs (19, 27). Considered a pathogenicity factor, it is not essential for growth of viruses in cell culture or infected insects but has the function of facilitating GV and NPV infection and decreasing larval survival time (14, 17, 19, 27).The widely accepted action mode of the viral enhancin protein, which has been identified as a metalloprotease (17), is that it can disrupt the protective peritrophic matrix (PM), allowing virion access to the underlying epithelial cells of the insect gut (17). The PM has a lattice structure formed by chitin and insect intestinal mucin (IIM), and the viral enhancin protein targets the IIM for degradation (33).Enhancin-like genes with 24 to 25% nucleotide identity to viral enhancin genes have been found in Yersinia pestis, Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus genome sequences (16, 25, 28). When B. cereus enhancin-like protein was expressed in recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded viruses and polyhedral inclusion bodies, it was found to be cytotoxic compared to viral enhancin protein. However, larval bioassays indicated that this enhancin-like protein did not enhance infection (8). Hajaij-Ellouze et al. (12) isolated a B. thuringiensis enhancin-like gene from a 407 crystal-minus strain and found that this enhancin-like protein has a typical metalloprotease zinc-binding domain (HEIAH) and belongs to the PlcR regulon. When the enhancin-like mutant was fed to Galleria mellonella larvae, no significant reduction in virulence was observed.In the present study, we report a B. thuringiensis enhancin-like gene (bel) encoding a protein (Bel) that has 20 to 30% identity to the viral enhancin protein and 95% identity to bacterial enhancin-like proteins. Therefore, Bel function may have a synergistic action similar to that of the virus enhancin protein. To understand the biochemical activity of this novel bacterial gene, bel was knocked out in the plasmid-free strain BMB171. We expected that this bel mutant would have no significant reduction in toxicity according to the reports of Galloway et al. (8) and Hajaij-Ellouze et al. (12). However, the bel mutant surprisingly resulted in dramatically reduced Cry1Ac toxicity to Helicoverpa armigera larvae. To further confirm this result, purified Bel was fed together with the Cry1Ac protein to H. armigera larvae. We found that Bel can function as a synergist of Cry1Ac toxicity against H. armigera. In vivo and in vitro observations showed that Bel can disrupt the insect midgut PM and degrade IIM of insect midgut PM. The target of Bel is the IIM of PM, similar to the results found with viral enhancin.     

Infection Status of Hospitalized Diarrheal Patients with Gastrointestinal Protozoa, Bacteria, and Viruses in the Republic of Korea

To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged ≤ 5 years was significantly higher, while C. perfringens among bacterial infection was higher for ≥ 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea.     

Eicosanoids mediate nodulation reactions to a mollicute bacterium in larvae of the blowfly, Chrysomya megacephala

Nodulation is the temporally and quantitatively most important cellular defense response to bacterial, fungal and some viral infections in insects. We tested the hypothesis that prostaglandins and other eicosanoids are responsible for mediating nodulation reactions to bacterial infection in larvae of the blowfly Chrysomya megacephala. Third-instar larvae treated with Ureaplasma urealyticum formed nodules in a challenge dose-dependent manner. Nodulation was evoked shortly after injection and reached a maximum of approximately 25 nodules/larva within 8 h. Larvae treated with the glucocorticoid, dexamethasone and the cyclooxygenase inhibitors, indomethacin and piroxicam were impaired in their ability to form nodules following U. urealyticum infection. The number of nodules decreased with increasing doses of piroxicam. Contrarily, treating larvae with the lipooxygenase inhibitor, esculetin, and the dual cyclooxygenase/lipooxygenase inhibitor, phenidone did not influence nodulation reactions to infection. Supplying dexamethasone-treated larvae with the eicosanoid precursor, arachidonic acid, reversed the inhibitory effect of dexamethasone on nodulation. We infer from these results that eicosanoids mediate nodulation reactions to infection of a bacterial species that lacks cell walls in larvae of the blowfly, C. megacephala.     

Improving baculovirus resistance to UV inactivation: increased virulence resulting from expression of a DNA repair enzyme

The use of baculoviruses as biological control agents is hampered by their susceptibility to inactivation by ultraviolet (UV) light. In an attempt to reduce UV inactivation, an algal virus pyrimidine dimer-specific glycosylase, cv-PDG, was expressed in the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV), and the infectivity of recombinant viruses expressing cv-PDG was measured after exposure to UV light. Expression of cv-PDG resulted in a 3-fold decrease in inactivation of budded virus by UV as measured by plaque assay in Spodoptera frugiperda Sf21 cells. However, occluded viruses expressing cv-PDG were not more resistant to UV inactivation than wild type AcMNPV when fed to either S. frugiperda or Trichoplusia ni neonate larvae. Surprisingly, however, viruses expressing cv-PDG showed a significant decrease in both the dose of occluded virus required for oral lethality and the time required for lethality compared to control virus, but these effects were only seen in S. frugiperda and not in T. ni larvae.     

Functional study of Capsicum annuum fatty acid desaturase 1 cDNA clone induced by Tobacco mosaic virus via microarray and virus-induced gene silencing

A series of microarray analyses employing the expressed sequence tags (ESTs) of hot pepper was conducted in an effort to elucidate the molecular mechanisms inherent to hypersensitive response (HR) by viral or bacterial pathogens. There were 2535 ESTs exhibiting differential expression (over 2-fold changes) among about 5000 ESTs during viral or bacterial response. Further, via virus-induced gene silencing (VIGS) and TMV-infection studies, we were able to isolate several ESTs, which may be relevant to defense response against TMV. Of these ESTs, Capsicum annuum fatty acid desaturase 1 (CaFAD1) showed the distinct phenotype against TMV infection and thus was subjected to further study. CaFAD1-silenced plants showed weaker resistance against TMV-P0 infection compared to TRV2 control plants. Also the suppression of FAD1 expression caused blocking of cell death induced by Bcl2-associated X (Bax) protein in tobacco plants. Therefore, this report presents that both microarray and VIGS approaches are feasible in hot pepper plants and the TMV-induced CaFAD1 plays a role in HR response.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.