Simulated Sunlight-UV Sensitivity of Engineered Juvenile Hormone Esterase and Scorpion Toxin Recombinants of the Nuclear Polyhedrosis Virus of Autographa californica

There has been an effort over the last decade to enhance the effectiveness of wild-type baculoviruses using genetic engineering. Wild-type viruses are extremely sensitive to sunlight-ultraviolet, but, what about engineered, recombinant baculoviruses? We found that insertion of a foreign gene did not result in recombinant baculoviruses being more or less sensitive to simulated sunlight-UV than a parental wild-type baculovirus. The half-life of activity for all recombinants and the wild-type parental isolate we tested was within that previously reported for other baculoviruses

Toxins for Transgenic Resistance to Hemipteran Pests

The sap sucking insects (Hemiptera), which include aphids, whiteflies, plant bugs and stink bugs, have emerged as major agricultural pests. The Hemiptera cause direct damage by feeding on crops, and in some cases indirect damage by transmission of plant viruses. Current management relies almost exclusively on application of classical chemical insecticides. While the development of transgenic crops expressing toxins derived from the bacterium Bacillus thuringiensis (Bt) has provided effective plant protection against some insect pests, Bt toxins exhibit little toxicity against sap sucking insects. Indeed, the pest status of some Hemiptera on Bt-transgenic plants has increased in the absence of pesticide application. The increased pest status of numerous hemipteran species, combined with increased prevalence of resistance to chemical insecticides, provides impetus for the development of biologically based, alternative management strategies. Here, we provide an overview of approaches toward transgenic resistance to hemipteran pests

Next Generation Sequencing Technologies for Insect Virus Discovery

Insects are commonly infected with multiple viruses including those that cause sublethal, asymptomatic, and latent infections. Traditional methods for virus isolation typically lack the sensitivity required for detection of such viruses that are present at low abundance. In this respect, next generation sequencing technologies have revolutionized methods for the discovery and identification of new viruses from insects. Here we review both traditional and modern methods for virus discovery, and outline analysis of transcriptome and small RNA data for identification of viral sequences. We will introduce methods for de novo assembly of viral sequences, identification of potential viral sequences from BLAST data, and bioinformatics for generating full-length or near full-length viral genome sequences. We will also discuss implications of the ubiquity of viruses in insects and in insect cell lines. All of the methods described in this article can also apply to the discovery of viruses in other organisms

Autographa californica multiple nucleopolyhedrovirus ODV-E56 is a per os infectivity factor, but is not essential for binding and fusion of occlusion-derived virus to the host midgut

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) envelope protein ODV-E56 is essential for oral infection of larvae of Heliothis virescens. Bioassays with recombinant clones of AcMNPV lacking a functional odv-e56 gene showed that ODV-E56 was required for infectivity of both polyhedra and to a lesser extent, purified ODV. However, binding and fusion assays showed that ODV lacking ODV-E56 bound and fused to midgut cells at levels similar to ODV of wild-type virus. Fluorescence microscopy of midguts from larvae inoculated with ODV-E56-positive and -negative viruses that express GFP indicated that ODV-E56 was required for infection of the midgut epithelium. Purified ODV-E56 bound to several proteins in midgut-derived brush border membrane vesicles, but failed to rescue infectivity of ODV-E56-negative viruses in trans. These results indicate that ODV-E56 is a per osinfectivity factor (pif-5) required for primary midgut infection at a point before or after virion binding and fusion

Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification