DNA-Experimental Design

[Name of the Institute]

DNA-Experimental Design

Introduction and Purpose

The present situation with regard to the use of baculoviruses in insect control is outlined. By virtue of their high degree of host specificity, they offer considerable advantages over chemical insecticides, but their practical use is limited by a number of factors, particularly their slow speed of action. Various approaches to the genetic modification of baculoviruses to overcome these problems are described. These have resulted in improvements in insecticidal activity in laboratory trials which are now being confirmed in the field. Thus, genetically modified baculoviruses have a promising future in pest-control programmes. Our increasing knowledge of the genetic factors which regulate their behavior is showing how other aspects of their performance may be controlled and exploited.

Baculoviruses are invertebrate pathogens which offer an alternative to the use of chemicals for the control of insect pests. They have a large, double-stranded, covalently closed, circular DNA genome (c. 90È160 kilobase pairs). The virus genome is packaged within a rod shaped nucleocapsid which is further surrounded by a lipoprotein envelope to form the virus particle. This structure is then occluded by a crystalline matrix largely comprising a single occlusion protein (28 kDa), which serves to protect it in the environment. The Baculoviridae are sub-divided into two genera: nucleopolyhedroviruses (NPVs) and granuloviruses (GVs).1 The NPVs are distinguished by the inclusion of several virus particles within each occlusion body or polyhedron.

Baculovirus Infection In VIVO

The most common mode of entry of a baculovirus into its insect host is by ingestion during larval feeding on foliage contaminated with virus. Although the pupal and adult stages of some species may be found to be infected, these insects are considered to have acquired the virus as larvae.13 Other modes of entry and infection include transovarial (passage within the egg) and transovum (contamination of the egg surface) passage, through spiracles into the tracheal system, or by parasitism.

However, these are not considered important mechanisms of virus transmission because of their relatively low frequency of occurrence.

The occluded form of the virus is involved in the process of horizontal infection. Following ingestion of virus, the occlusion bodies are dissolved in the high pH conditions (pH 8É5 to 11É0) of the insect midgut, releasing virus particles into the gut lumen.15h17 The foregut and hindgut of the insect are of ectodermal origin and are lined with cuticle, presenting a barrier to infection.

Therefore, sites in the midgut epithelium, which are not lined with cuticle, are involved in primary virus attachment and entry. Released virions bind to the columnar epithelial cells and enter the tips of the microvilli on the apical brush border of cells 0É25È2 h postingestion.

Following fusion with the cell membrane, the nucleocapsids are released into the cytoplasm and are transported to the nucleus, where viral DNA replication occurs.15,20 Virus-infected-cell morphology is evident at 8 hours postinfection (h.p.i.), when the nucleus of the cell becomes enlarged. Nucleocapsids pass into the cytoplasm at approximately 12 h.p.i.15 From the cytoplasm, nucleocapsids are transported to the basal membrane of the midgut cells, and ...