Comparison of the bacterial Enhancin-like proteins from Yersinia and Bacillus spp. with a baculovirus Enhancin

Enhancins are a class of metalloproteases found in some baculoviruses that enhance viral infection by degrading the peritrophic, membrane (PM) of the insect midgut. However, sequencing has revealed enhancin-like genes with 24-25% homology to viral enhancins, in the genomes of Yersinia pestis and Bacillus anthracis. AcMNPV does not encode enhancin therefore recombinant AcMNPV budded viruses (BVs) and polyhedra inclusion bodies (PIBs) were generated expressing the bacterial Enhancins. Bacterial Enhancins were found to be cytotoxic when compared to viral enhancin, however, larval bioassays suggested that the bacterial Enhancins did not enhance infection in the same way as viral Enhancin. This suggests that the bacterial Enhancins may have evolved a distinct biochemical function. (c) 2005 Elsevier Inc. All rights reserved.

The glycosylation pattern of baculovirus expressed envelope protein E2 affects its ability to prevent infection with bovine viral diarrhoea virus

We have investigated the role of glycosylation of the envelope glycoprotein E2 of bovine viral diarrhoea virus (BVDV), produced in insect cells, in BVDV infection. When amino acids predicated to code for the C-terminal N-linked glycosylation site were mutated the resulting protein was less efficient than wild type protein at preventing infection of susceptible cells with BVDV. In addition, mutational analysis showed that a further two predicted N-terminal N-linked glycosylation sites of E2 are required for efficient production of recombinant protein. (c) 2005 Elsevier B.V. All rights reserved.

Improved display of synthetic IgG-binding domains on the baculovirus surface

Improved display of foreign protein moieties in combination with beneficial alteration of the viral surface properties should be of value for targeted and enhanced gene delivery. Here, we describe a vector based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) displaying synthetic IgG-bincling domains (ZZ) of protein A fused to the transmembrane anchor of vesicular stomatitis virus (VSV) G protein. This display vector was equipped with a GFP/EGFP expression cassette enabling fluorescent detection in both insect and mammalian cells. The virus construct displayed the biologically active fusion protein efficiently and showed increased binding capacity to IgG. As the display is carried out using a membrane anchor of foreign origin, gp64 is left intact for virus entry, which may increase gene expression in the transduced mammalian cells. In addition, the viral vector can be targeted to any desired cell type via binding of ZZ domains when an appropriate IgG antibody is available.

Improving baculovirus recombination

Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector. Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation.

Probing the receptor interactions of an H5 avian influenza virus using a baculovirus expression system and functionalised poly(acrylic acid) ligands

Influenza viruses attach to host cells by binding to terminal sialic acid (Neu5Ac) on glycoproteins or glycolipids. Both the linkage of Neu5Ac and the identity of other carbohydrates within the oligosaccharide are thought to play roles in restricting the host range of the virus. In this study, the receptor specificity of an H5 avian influenza virus haemagglutinin protein that has recently infected man (influenza strain A/Vietnam/1194/04) has been probed using carbohydrate functionalised poly(acrylic acid) polymers. A baculovirus expression system that allows facile and safe analysis of the Neu5Ac binding specificity of mutants of H5 HA engineered at sites that are predicted to effect a switch in host range has also been developed. (C) 2007 Elsevier Ltd. All rights reserved.

Improving promiscuous mammalian cell entry by the baculovirus AcMNPV

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) enters many mammalian cell lines, prompting its application as a general eukaryotic gene delivery agent, but the basis of entry is poorly understood. For adherent mammalian cells we show that entry is favoured by low pH and increasing the available cell surface area through transient release from the substratum. Low pH also stimulated baculovirus entry into mammalian cells grown in suspension which, optimally, could reach 90% of the transduced population. The basic loop, residues 268-281, of the viral surface glycoprotein gp64 was required for entry and a tetra mutant with increasing basicity increased entry into a range of mammalian cells. The same mutant failed to plaque in Sf9 cells, instead showing individual cell entry and minimal cell to cell spread, consistent with an altered fusion phenotype. Viruses grown in different insect cells showed different mammalian cell entry efficiencies suggesting additional factors also govern entry.

Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening

In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells coexpressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the reinfection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.

Expression and purification of a recombinant adenovirus fiber knob in a baculovirus system

Objectives: To construct a recombinant baculovirus expressing the fiber knob domain of human adenovirus type 2 modified by the insertion of a foreign peptide, purify this protein after its production in insect cells, and to test its properties. Methods: Recombinant baculoviruses expressing the fiber knob were produced in Sf9 cells. The recombinant fiber knob was recovered from culture supernatants of infected cells and purified by a combination of Ni-NTA and ion-exchange chromatography. Results: Fiber knob was recovered from the culture media as a soluble protein. In the system used, the fiber knob is expressed fused with the V5 epitope and a histidine tag, which allowed purification by Ni-NTA chromatography. The protein was further purified by ion-exchange chromatography. We show that the recombinant fiber knob produced, with 31 extra amino acids in the C-terminus, can oligomerize and bind to the adenovirus receptor CAR, as it can block the infection of a recombinant type 5 adenovirus. Conclusions: The modified form of the fiber knob, produced in insect cells and purified by Ni-NTA and ion-exchange chromatography, retains the properties of oligomerization and binding to the fiber natural receptor, CAR. This construct has the potential to be a new adjuvant. Copyright (C) 2008 S. Karger AG, Basel.

Biological activity of astilbin from Dimorphandra mollis against Anticarsia gemmatalis and Spodoptera frugiperda

Astilbin was isolated in high yield from Dimorphandra mollis, and its insecticidal and growth inhibiting activity by stomach ingestion were evaluated against Anticarsia gemmatalis and Spodoptera frugiperda. The insecticidal activity of astilbin, the weight reduction of the larval phase and the prolongation of the larval and pupal phases were verified for both species. Astilbin was identified on the base of its NMR, MS and physical data. (C) 2002 Society of Chemical Industry.

Velocidade do fluxo de ar em barra de pulverização no controle químico de Anticarsia gemmatalis, Hübner e percevejos na cultura da soja

A lagarta Anticarsia gemmatalis (Hübner) e os percevejos fitófagos são pragas importantes na cultura da soja no Brasil. Este trabalho objetivou avaliar o controle desses insetos após o tratamento com inseticidas, sob diferentes velocidades do fluxo de ar junto à barra de pulverização nessa cultura. O experimento foi desenvolvido em Botucatu, na cultura da soja [Glycine max (L.) Merrill], var. Conquista (safra 2007/08), no delineamento experimental de blocos ao acaso (quatro velocidades do fluxo de ar: 0, 9, 11 e 29 km h-1), mais testemunha, totalizando cinco tratamentos e quatro repetições. No estádio de desenvolvimento vegetativo (V10) realizou uma aplicação do inseticida deltametrina na dosagem de 6,5 g do i.a. ha-1 para o controle de lagartas e no estádio de desenvolvimento reprodutivo (R6) aplicou o inseticida tiametoxam associado com lambda-cialotrina na dosagem de 25,38 + 19,08 g do i.a. ha-1 para o controle de percevejos. A aplicação foi feita com um pulverizador Advanced Vortex 2000, com pontas de pulverização jato cônico JA2, conferindo um volume de calda de 200 L ha-1. As avaliações antes e após a aplicação foram realizadas pelo método de batidas no pano. Avaliaram-se os danos causados por percevejos, considerando-se a porcentagem de danos às sementes, ao poder germinativo e à produtividade. No geral, o número médio de lagartas e percevejos foram significativamente menores nas parcelas tratadas em relação ao obtido na testemunha. Não houve diferença de produtividade entre os tratamentos. A porcentagem de emergência e sementes picadas por percevejos foram significativamente menores nos tratamentos que receberam o controle em comparação à testemunha.

Morphological study of the hindgut in larvae of Anticarsia gemmatalis Hübner(Lepidoptera: Noctuidae)

A lagarta da soja (Anticarsia gemmatalis Hübner) tem grande interesse econômico, pois afeta significativamente a cultura da soja em todo o mundo. Este trabalho descreve a morfologia do intestino posterior de larvas de A. gemmatalis, com ênfase nos seus aspectos histológicos. O intestino posterior é constituído por regiões morfologicamente distintas, identificadas como piloro, íleo, cólon e reto. Independente da região, a parede do intestino posterior é constituída por fina cutícula, epitélio simples e camada muscular. A íntima cuticular apresenta espículas no anel intersticial posterior, entre o intestino médio e o posterior, e na região posterior do piloro. A musculatura do reto é formada por camada única de largas fibras circulares, diferindo das demais regiões do intestino posterior que apresentam duas camadas de fibras musculares. As extremidades distais dos túbulos de Malpighi atravessam as paredes do reto, constituindo o sistema criptonefridial característico de Lepidoptera.

Ultramorphology of digestive tract of Anticarsia gemmatalis (Hubner, 1818) (Lepidoptera : Noctuidae) at final larval development

The digestive tract of insects is an important natural, physical, and chemical defense barrier against pathogen invasion. Certain. lepidopteran caterpillars are serious pests of agricultural crops and their biology has received much attention, but little is known about the larval noctuid gut. The morphological analysis of the digestive tract in Anticarsia gemmatalis under scanning electron microscopy (SEM) is a good model for studies about its defense mechanism. The material was fixed (2,5% glutaraldehyde solution; 0.1 M-phosphate buffer, pH 7.3), post-fixed (1% osmium tetroxide in the same buffer), dried at critical point, gold coated and analyzed in a SEM 515-Philips. A. gemmatalis digestive tract consists of a straight duct of varying length and diameter, subdivided in three main regions: the foregut formed by the oral cavity, pharynx, esophagus, and crop; the midgut that is the largest portion of the digestive tract without noticeable morphological differentiation along its length; and the hindgut that is morphologically differentiated in pylorus, ileum, colon, and rectum. Although the general morphology of the A. gemmatalis digestive tract is quite similar to the other Lepidoptera species, the anatomical array of the crop muscular layers is quite different comparing with the description for other larval insect.

A morphometric study of the midgut in resistant and non-resistant Anticarsia gemmatalis (Hubner) (Lepidoptera: Noctuidae) larvae to its nucleopolyhedrovirus (AgMNPV)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)