A high efficient method of constructing recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus based on zero‐background Tn7‐mediated transposition in Escherichia coli

A high efficient way for generation of recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus by Tn7‐mediated transposition in Escherichia coli was performed. The new system consists of a conditional replication donor vector pRCDM and an attTn7 site blocked E. coli containing BmNPV‐Bacmid. The donor vector contains a replication origin derived from R6Kγ, which propagated only in host cells with pir gene expression decreased in the transposition background greatly. Compared with original vector derived from pUC, the transposition efficiency increased from 5.7 to 66% (≈10 fold) when using conditional replication vector pRCDM transposition into original BmDH10Bac. A further effort to decrease the transposition background was made by blocking the attTn7 site in host E. coli genome. The resulting attTn7 occupied BmDH10BacΔTn7 resulted in a significant increase from 5.7 to 23% (≈4 fold) in the efficacy of generate recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10BacΔTn7 with pRCDM resulted typically in 100% white colonies, and it indicated that a zero transposition background was accomplished. This high efficient and zero background transposition system provides a new simple and rapid method for construction of recombinant BmNPV used to express target genes or produce gene‐delivery virus particles in silkworm. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Inflorescences vs leaves: a distinct modulation of carbon metabolism process during Botrytis infection

Plant growth and survival depends critically on photo assimilates. Pathogen infection leads to changes in carbohydrate metabolism of plants. In this study, we monitored changes in the carbohydrate metabolism in the grapevine inflorescence and leaves using Botrytis cinerea and Botrytis pseudo cinerea. Fluctuations in gas exchange were correlated with variations in chlorophyll a fluorescence. During infection, the inflorescences showed an increase in net photosynthesis (Pn) with a stomatal limitation. In leaves, photosynthesis decreased, with a non‐stomatal limitation. A decrease in the effective photosystem II (PSII) quantum yield (ΦPSII) was accompanied by an increase in photochemical quenching (qP) and non‐photochemical quenching (qN). The enhancement of qP and ΦPSII could explain the observed increase in Pn. In leaves, the significant decline in ΦPSII and qP, and increase in qN suggest that energy was mostly oriented toward heat dissipation instead of CO₂ fixation. The accumulation of glucose and sucrose in inflorescences and glucose and fructose in the leaves during infection indicate that the plant's carbon metabolism is differently regulated in these two organs. While a strong accumulation of starch was observed at 24 and 48 hours post‐inoculation (hpi) with both species of Botrytis in the inflorescences, a significant decrease with B. cinerea at 24 hpi and a significant increase with B. pseudo cinerea at 48 hpi were observed in the leaves. On the basis of these results, it can be said that during pathogen attack, the metabolism of grapevine inflorescence and leaf is modified suggesting distinct mechanisms modifying gas exchange, PSII activity and sugar contents in these two organs.     

Identification of BV/ODV-C42, an Autographa californica Nucleopolyhedrovirus orf101-Encoded Structural Protein Detected in Infected-Cell Complexes with ODV-EC27 and p78/83

orf101 is a late gene of Autographa californica nucleopolyhedrovirus (AcMNPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (1629K) and ODV-EC27 (orf144). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.     

Eclipse period of baculovirus infection in larvae of the armyworm, Pseudaletia unipuncta

Two strains of a nuclear polyhedrosis virus (baculovirus) infect larvae of the armyworm, Pseudaletia unipuncta. The hypertrophy strain (HNPV) produces a gradient of infected epithelial cells along the tracheae indicating the movement of infectious material to adjacent cells. Cytopathology of the eclipse period up to the appearance of the virogenic stroma has been separated into three phases during which the chromatin disappears and is replaced by dense interconnected strands of fibrils and dense punctate bodies. Cellular hypertrophy occurs in phase 1 and the virogenic stroma appears in phase 3. The typical strain (TNPV) does not produce structures comparable to those of HNPV infection.     

Cytopathological process by multiple nucleopolyhedrovirus in the testis of Bombyx mori L., 1758 (Lepidoptera: Bombycidae)

A cytopathological methodology was used to analyze infection by Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), a geographic isolate of the family Baculoviridae, in the caterpillar testes of the B. mori. Japanese B. mori strain caterpillar, fifth instar, were inoculated with BmMNPV and their testes were collected and processed for light and transmission electronic microscopy. Epithelial coating cells and interfollicular septa in testes were susceptible to BmMNPV. The first evidence of infection was detected on the 6th day post-inoculation (p.i.) in the external epithelium, and on the 7th day p.i. in the internal epithelium and interfollicular septa. Cytopathological characteristics consisted of hypertrophied nuclei, the formation of virogenic stroma, and the occlusion of virions in polyhedron protein crystals in several stages of development. At the end of the infectious process, cell lysis and release of polyhedra into the extracellular medium occurred. Histopathology revealed early infection foci in the surrounding regions of tracheal insertions, thus underlining the role of the trachea as an infection-spreading organ in insects. This spreading occurs through penetration of the basal lamina, which facilitates entry of the budded virus into the testis. Additionally, an alignment of a partial sequence of the ORF 14 of the BmMNPV geographic isolate with other NPV certified the virus genera.     

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

The yeast‐like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP‐28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP‐28 sterilized Prototheca cultures within 30–60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3–6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70–90% killing, suggesting they act via non‐lytic mechanisms. In circular dichroism studies, the conformation of BMAP‐28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP‐28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non‐lytic mechanisms may be exploited for the development of target‐selective drugs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

cDNA cloning and induction of tyrosine hydroxylase gene from the diamondback moth,Plutella xylostella

We cloned a full‐length tyrosine hydroxylase cDNA from the integument of the diamondback moth, Plutella xylostella. In the phylogenetic tree, tyrosine hydroxylase (PxTH) clustered with the other lepidopteran THs. Serine residues in the PxTH sequence, namely Ser²⁴, Ser³¹, Ser³⁵, Ser⁵³, and Ser⁶⁵, were predicted to be the target sites for phosphorylation based on PROSITE analysis. In particular, Ser³⁵ of PxTH is highly conserved across a broad phylogenetic range of animal taxa including rat and human. Western blot analysis using both PxTH‐Ab1 and PxTH‐Ab2 polyclonal antibodies verified the expression of PxTH in all life cycle stages of P. xylostella, namely the larval, pupal, and adult stages. To examine the possible immune function of PxTH in P. xylostella, PxTH gene expression was investigated by RT‐PCR and western blotting analysis after challenging P. xylostella with bacteria. PxTH expression was elevated 1 h post‐infection and was continued till 12 h of post‐infection relative to control larvae injected with sterile water. © 2010 Wiley Periodicals, Inc.     

荨麻蛱蝶核型多角体病毒在病虫脂肪体中的形态发生

本文叙述了一种在高山草原地区较低温度下感染荨麻蛱蝶(Vanessa urticae)的核型多角体病毒的形态发生过程。荨麻蛱蝶幼虫经其病毒多角体感染5日后出现明显变化:细胞核膨大,核仁消失,核内出现清晰区及病毒发生基质。在病毒发生基质的周围,核衣壳大量产生。核衣壳是从这些病毒发生基质四周的模样结构碎片上获得套膜,装配成病毒粒子。随后病毒粒子逐渐进入多角体蛋白中,形成了成熟的单粒包埋型的多角体。观察结果表明,在较低温度下生长的荨麻蛱蝶NPV与在常温下生长的其它NPV有着类似的形态发生过程。     

Inhibition of Orientia tsutsugamushi infection by a truncated recombinant 56‐kDa outer membrane protein

Aims: The objective of this study was to evaluate recombinant 56‐kDa outer membrane protein as a potential inhibitor to infection from Orientia tsutsugamushi. Methods and Results: The 56‐kDa protein was cloned and expressed in an Escherichia coli system, and the degree of target cell attachment to immobilized 56‐kDa protein was measured in a cell adhesion assay. The results showed that the 56‐kDa protein has an ability to attach HeLa cells. Furthermore, treatment of target cells with a truncated 56‐kDa 1–418 (amino acid residues) protein inhibited target cell infection by O. tsutsugamushi, demonstrated with an indirect immunofluorescence antibody assay. Conclusions: The truncated 56‐kDa protein (a.a. 1–418) plays an important role in O. tsutsugamushi infection, and the 56‐kDa protein could be useful and effective in the inhibition of O. tsutsugamushi attachment and infection. Significance and Impact of the Study: The attachment of the 56‐kDa protein to target cells was directly determined by in vitro adherence test, and the invasion of target cells by O. tsutsugamushi was inhibited by treating the target cells with a truncated 56‐kDa protein.     

Proteomic Analysis of Differentially Expressed Proteins in Resistant Soybean Leaves after Phakopsora pachyrhizi Infection

Soybean rust caused by Phakopsora pachyrhizi is a destructive foliar disease in nearly all soybean‐producing countries. Understanding the host responses at the molecular level is certainly essential for effective control of the disease. To identify proteins involved in the resistance to soybean rust, differential proteomic analysis was conducted in soybean leaves of a resistant genotype after P. pachyrhizi infection. A total of 41 protein spots exhibiting a fold change >1.5 between the non‐inoculated and P. pachyrhizi‐inoculated soybean leaves at 12 and 24 h postinoculation (hpi) were unambiguously identified and functionally grouped into seven categories. Twenty proteins were up‐regulated and four proteins were down‐regulated at 12 hpi, whereas 18 proteins were up‐regulated and eight proteins were down‐regulated at 24 hpi. Generally, proteins involved in photosynthesis were down‐regulated, whereas proteins associated with disease and defense response, protein folding and assembly, carbohydrate metabolism and energy production were up‐regulated. Results are discussed in terms of the functional implications of the proteins identified, with special emphasis on their putative roles in defense. Abundance changes of these proteins, together with their putative functions reveal a comprehensive picture of the host response in rust‐resistant soybean leaves and provide a useful platform for better understanding of the molecular basis of soybean rust resistance.     

Morphogenesis of nuclear polyhedrosis virus from Autographa californiea in a cell line from Mamestra brassicae (cabbage moth)

Summary Cultures of cell line IZD-MB 0503 from Mamestra brassicae were inoculated with nonoccluded nuclear polyhedrosis virus of Autographa californica (AcNOV). At 1 h postinoculation (p.i.) nucleocapsids were found in the cytoplasm near nuclear pores and within the nucleoplasm. Formation of virogenic stroma was observed at 7 h p.i. The first short empty capsids were seen at 10 h postinoculation (p.i.), followed by partially and completely filled nucleocapsids (11–12h p.i.) with most capsids filled at 12h and later. This suggests that nucleocapsid components, such as capsid and DNA-core, assemble in successive stages rather than simultaneously. Viral progeny, being released from the cells by budding, were observed at 13h p.i.Supported by Bundesministerium für Forschung und Technologie, Bonn, Kennzeichen PTB 8141