**2.1. Introduction**

The name "baculovirus" is derived from the latin "*baculum*" (stick), denoting the rod‐shaped nucleocapsids that are 230– 385 nm in length and 40–60 nm in diameter [1]. The virions are enveloped and present two phenotypes: occluded virions (OVs) and budded virions (BVs). These two types of virions differ in the origin and composition of their envelopes and their functions in the virus life cycle. In both, the genome is complexed with multiple copies of a small basic protein (p 6.9) which neutralizes the negative charge of the DNA and this structure is protected by other proteins forming the nucleocapsid. The OVs are enclosed in a paracrys‐ talline matrix forming occlusion bodies (OBs), which are orally infectious. Their morphology was initially used to define two major groups or genera of the Baculoviridae: the Nucleopo‐ lyhedrovirus (NPVs) and the Granulovirus (GVs). NPV OBs, called polyhedra, are about 0.6– 2 μm in size and their major occlusion protein is called polyhedrin. GV OBs, also known as granules or capsules, are oval‐shaped with diameters in the range of 0.2–0.4 μm (Figure 1). OBs are highly stable and can resist most normal environmental conditions thereby allowing

> Single Nucleopolyhedrovirus polyhedron

MNPV ODV SNPV ODV GV ODV

**Figure 1.** On the basis of the OB morphology, baculovirus were originally divided in two major groups: the Nucleopo‐ lyhedrovirus (NPVs) and the Granulovirus (GVs). NPVs occlusion bodies are called polyhedra and their major occlusion

Based on the fusion protein present in the BVs NPVs have been further classified into two groups: type I NPVs contain GP64, a low‐pH‐dependent membrane fusion protein required for virus entry and cell‐to‐cell transmission [7,8,9,10], BVs of group II NPVs and GVs lack a homolog of GP64, and membrane fusion during viral entry is triggered by F protein [11,12]. It was found that the entry of baculoviruses in mammalian cells is mediated by GP64. In contrast,

The natural cycle of infection by AcMNPV in insect larvae is summarized in Figure 2. Caterpillars ingest polyhedra that contaminate their food. The polyhedrin matrix is dissolved in the alkaline environment of the larvae midgut releasing ODVs (occlusion derived virions). These

protein is called polyhedrin and GV occlusion bodies and granules or capsules for GVs.

baculoviruses with F protein cannot transduce these cells [13].

Granulovirus granulum

virions to remain infectious for very long periods of time.

80 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

Multiple Nucleopolyhedrovirus polyhedron

> The high levels of expression of the very late genes has been exploited to design the first vectors for foreign gene expression based on baculoviruses. They are especially suitable regarding safety (not harmful to non‐target organisms) and easy containment in the laboratory. Baculo‐ virus vectors are used to infect insect cells or larvae where high levels of recombinant proteins are produced; the eukaryotic environment provides appropriate post-translational modifica‐ tions in comparison with prokaryotic expression systems. Insect cells to be used in the baculovirus expression system are derived from lepidopteran insects and are relatively easy to grow. No control of oxygen atmosphere is required. Moreover, insect cells can be adapted to serum‐free media and production of recombinant protein can be scaled up to pilot plant or larger bioreactors [21, 22]. The lepidopteran insect cells used are also normally free of human pathogens. Thereby, the proteins produced in the baculovirus virus expression system can be used for functional studies, vaccine preparations or diagnostics.

genome. Clonal purification requires several plaque passages. After this, viral stocks can be produced and amplified for recombinant protein production [31]. Insect cells are used for purification of many proteins, including therapeutic and vaccine peptides. Larvae are used to reduce production costs, or when recombinant baculovirus are to be tested as bioinsecticides. Finally, baculovirus can be used for transduction of mammalian cells, for production of

recombinant protein purification Larvae infection transduction of mammalian cells

Improved bioinsecticides Protein purification from larvae

**Figure 3.** General process for baculovirus cloning. Genomic DNA and a transfer plasmid are cotransfected into an in‐ sect cell culture. Recombinant virus propagates causing lysis plaques. Virus is isolated from lysis plaques and amplified. Virus can be used for recombinant protein production in insect cells or larvae. Furthermore, baculovirus can be used

Lysis plaque formation

Virus plaque isolation and amplification

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 83

Transduced organism

Protein production in mammalian cells Peptid display library Budded virus display vaccines Gene therapy and cell response mediated immunity

Transduced mammalian cells

therapeutic proteins, or to transduce organisms for gene therapy or vaccination.

Cotransfection into insect cell line

polyhedrin

Transfer plasmid containig gene of interest

baculovirus genome

Protein purification for basic studies Pharmaceutical peptides Recombinant vaccines

for transduction of mammalian cells or whole animals.

**Figure 2.** *Per os* infection of baculoviruses. A cross sectional representation of the anatomy of an insect larva is depict‐ ed. A baculovirus occlusion body (OB) is ingested in contaminated food. OBs pass through the foregut and enter the midgut where they dissolve in the alkaline midgut lumen and release occlusion derived viruses (ODVs).

A baculovirus expression vector (BEV) is a recombinant baculovirus that has been genetically modified to lead the expression of a foreign gene. BEVs are viable in insect cell culture and sometimes in larvae, depending on the baculovirus genes deleted in the process of the recombinant virus generation. In BEVs, the foreign gene coding sequence is usually placed under the transcriptional control of a viral promoter. For this reason usually viral factors are required for the transcription of the foreign gene.

The most commonly used process for cloning recombinant baculovirus is briefly summarized in Figure 3. Baculovirus genomic DNA and a transfer plasmid are cotransfected into an insect cell culture. Double homologous recombination between viral DNA and transfer plasmid causes the allelic replacement that incorporates the recombinant gene in the baculovirus genome. Clonal purification requires several plaque passages. After this, viral stocks can be produced and amplified for recombinant protein production [31]. Insect cells are used for purification of many proteins, including therapeutic and vaccine peptides. Larvae are used to reduce production costs, or when recombinant baculovirus are to be tested as bioinsecticides. Finally, baculovirus can be used for transduction of mammalian cells, for production of therapeutic proteins, or to transduce organisms for gene therapy or vaccination.

budded virions

peritrophic membrane

occlusion body ODVs

82 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

required for the transcription of the foreign gene.

**Figure 2.** *Per os* infection of baculoviruses. A cross sectional representation of the anatomy of an insect larva is depict‐ ed. A baculovirus occlusion body (OB) is ingested in contaminated food. OBs pass through the foregut and enter the

A baculovirus expression vector (BEV) is a recombinant baculovirus that has been genetically modified to lead the expression of a foreign gene. BEVs are viable in insect cell culture and sometimes in larvae, depending on the baculovirus genes deleted in the process of the recombinant virus generation. In BEVs, the foreign gene coding sequence is usually placed under the transcriptional control of a viral promoter. For this reason usually viral factors are

The most commonly used process for cloning recombinant baculovirus is briefly summarized in Figure 3. Baculovirus genomic DNA and a transfer plasmid are cotransfected into an insect cell culture. Double homologous recombination between viral DNA and transfer plasmid causes the allelic replacement that incorporates the recombinant gene in the baculovirus

midgut where they dissolve in the alkaline midgut lumen and release occlusion derived viruses (ODVs).

epithelial cell

**Figure 3.** General process for baculovirus cloning. Genomic DNA and a transfer plasmid are cotransfected into an in‐ sect cell culture. Recombinant virus propagates causing lysis plaques. Virus is isolated from lysis plaques and amplified. Virus can be used for recombinant protein production in insect cells or larvae. Furthermore, baculovirus can be used for transduction of mammalian cells or whole animals.
