Ph.D. Scholar, IGKV, Raipur (C.G.)

1. INSECT TOXIN: GENE STRUCTURE
AND USE FOR INSECT CONTROL
USING BACULOVIRUS VECTOR
Seminar Incharge :
Dr. S. S. Shaw
Professor
Presented By:
Aishwarya Ray
Ph.D. Scholar
Department Of Entomology, IGKV,Raipur (C.G)

2. INTRODUCTION
• Baculovirus belong to the
family Baculoviridae.
• Baculo has been derived from
the Latin word ″baculus″
meaning ″rod or stick shaped″.
• Baculovirus are lytic viruses,
primarily pathogenic for insects
and non pathogenic to
vertebrates and plants.

3. Historical Influence
• The earliest records of baculovirus can be found in the literature from
as early as the sixteenth century in reports of "wilting disease"
infecting silk-producing larva.
• Paillot (1926) described the first granulosis disease from the larvae of
Pieris brassicae
• Bergold (1947) provided definitive evidence of the viral nature of
disease.
• In 1970 Elcar was the first baculovirus to be developed for commercial
use.
• Since the 1990s Baculoviruses have been utilized for producing
complex eukaryotic proteins in insect cell cultures .

4. Structure Of Baculovirus
• Baculovirus nucleocapsids are rod-shaped and surrounded by an
envelope, and they contain circular genomes of double-stranded DNA
that range in size from about 80–180 kbp in length.
• It is associated with a highly basic (arginine-rich) protein of 6.5KDa.
• Baculoviruses are the only known nuclear-replicating DNA viruses that
encode a DNA-directed RNA polymerase.

5. Contd…
• They produce two distinct types of enveloped virions: occlusion-
derived virions (ODV), which are embedded in large (5–10 micron)
protein crystals called occlusion bodies and are responsible for
horizontal transmission between insects, and budded virions (BV) are
non-occluded which spread infection from cell to cell.
- Hawtin,1993

6. Figure showing Occluded derived virions and Budded
virions of Baculovirus

7. Classification
Baculoviridae
Nucleopolyhedrovirus (NPV)
SNPV
(single packaged
nucleocapsids)
MNPV
(multiple packaged
nucleocapsids)
Granulovirus (GV)
- Murphy et al.,1995

8. Contd…..
• The genus NPV is characterized by the presence of polyhedral shaped
viral occlusions (polyhedra) containing randomly occluded viral
particles.
• The genus granulose virus (GV) has one nucleocapsid per envelope
and has granule shaped viral occlusions (capsules) containing one or
rarely 2 or more virions.
• GVs are more specific than NPVs as they have been reported only
from Lepidoptera.

9. Taxonomic Status of Baculoviridae
The family has been divided into four genera:
1) Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses)
2) Betabaculovirus (lepidopteran-specific Granulovirus
3) Gammabaculovirus (hymenopteran-specific nucleopolyhedroviruses)
4) Deltabaculovirus (dipteran-specific nucleopolyhedroviruses)
(Source- www.ictvonline.org)

10. Mode of Action of Baculovirus

11. Cont…
• Baculovirus infection can be divided to three distinct phases:
Early (0–6 h post-infection),
Late (6–24 h p.i.)
Very late phase (18–24 to 72 h p.i)
* BV is produced in the late phase whereas the ODV form is produced in
the very late phase acquiring the envelope from host cell nucleus and
embedded in the matrix of occlusion body protein.

12. SYMPTOMS
• They have a characteristic
shiny oily appearance and
stop feeding.
• Infected larvae may initially
turn white and granular or very
dark.
• They climb to the top of the
crop canopy, becomes limp, and
hang from the upper leaves or stems,
hence the common name “caterpillar
wilt” or “tree top” disease.

13. Structure of the Virion
• The most studied baculovirus is Autographacalifornica multicapsid
nucleopolyhedrovirus (AcMNPV).
The virus was originally isolated from the alfalfa looper.
• A nucleocapsid assembly-essential element (NAE) was identified in
the AcMNPV genome. The NAE is an internal cis-element within
the ac83 gene.
• A major envelope of glycoprotein Gp64 is also present in the virion.

14. Baculoviruses: Sophisticated
Pathogen of Insects
Baculovirus-infected larvae exhibit several interesting examples of
pathogen-modified physiology and behavior.
1) Inhibition of larval development :-baculovirus-infected larvae do not
molt, due to the action of a viral enzyme called ecdysteroid UDP-
glucosyltransferase (EGT).
2) Increased larval locomotory activity:- induced by expression of a
viral protein called protein tyrosine phosphatase (PTP).
Phylogenetic analyses indicate that both the egt and ptp genes appear to
have a lepidopteran origin, indicating that baculoviruses have acquired
many advantageous genes during coevolution with their hosts.

15. Figure showing Baculovirus-induced climbing behavior, historically known as
“Wipfelkrankheit” (tree top disease), attributing to the viral egt gene.

16. Baculovirus Expression Vector System
• The Baculovirus Expression Vector System (BEVS) is one of the most
powerful and versatile eukaryotic expression systems available.
• The BEVS is a helper- independent viral system which has been used to
express heterologous gene from many different cells.
• AcNPV is the widely used Baculovirus DNA in BEVS.
• Co-transfection of the transfer vector and AcNPV DNA into Spodoptera
frugiperda cells allows recombination between homologous sites.
• Major difference between the naturally occuring invivo infection and the
recombinant invitro infection is that the naturally occuring polyhedrin gene
within the wildtype baculovirus genome is replaced with a recombinant
gene.

17. Figure Showing Invivo Baculovirus Infection And Replication

18. Methods of Generating Recombinant
Virus
HOMOLOGOUS
RECOMBINATION
BAC-to-BAC
SYSTEM

19. HOMOLOGOUS COMBINATION

20. BAC-to-BAC SYSTEM

21. Figure showing Bac-to-Bac Expression System

22. Advantages of using BEVS
1) Functional activity of the recombinant protein
2) Post translations modifications
3) High expression levels
4) Capacity for large inserts
5) Capacity to express unspliced genes
6) Simplicity of technology
7) Simultaneous expression of multiple genes
8) Localization of recombinant proteins
9) Ease of purification
10)Direct cloning

23. Neurobactrus
• Neurobactrus, a novel, highly effective and environmentally friendly
recombinant Baculovirus Insecticide was constructed to develop an
improved baculovirus insecticide with additional beneficial properties
such as higher insecticidal activity and improved recovery, compared to
wild-type baculovirus.
• It was constructed by introducing Bt cry1-5 gene into the AcMNPV
genome and on the opposite direction, an insect –specific neurotoxin
gene, AaIT, from Androctonus australis was introduced.
• It was activated by treatment with trypsin.

24. Cont…
• Neurobactrus showed a high level of insecticidal activity
against Plutella xylostella larvae and a significant
reduction in the median lethal time against Spodoptera
exigua larvae compared to those of wild-type AcNPV.
(Source-http://aem.asm.org)

25. Commercialization of Baculoviruses
*The first baculovirus to be developed for commercial use was Elcar, the
NPV of Helicoverpa zea (Boddie), primarily developed for use on cotton
and registered by Environmental Protection Agency in USA in 1975.
* It was infective against all the major Helicoverpa species and provided
efficient control in soybean, sorghum, maize, tomato and chickpea.

26. List of commercially available viral pesticides registered
for pest control
Viral Species Commercial
Names
Target Pests Crops
Autographa
californica
Speyer MNPV
VPN-80 Lepidopteran
larvae
Cabbage, cotton
Phthorimaea
opercullea
Boyd GV
PTM
Baculovirus
Potato tuber
moth
Potatoes
Heliothis
virescens Fab.
NPV
Elcar Heliothines Cotton,soybean.chi
ckpea,tomato
Plodia
interpunctella
Hubner GV
Nutguard-V Indian meal
moth
Stored almonds,
raisins
Spodoptera
exigua MNPV
SPOD-X Beet armyworm Ornamentals,Veget
ables

27. Resistance to Baculoviruses
Cydia pomonella Linnaeus GV in apple orchards has led to a
high degree of resistance in some populations in Europe.
-Koul, 2011

28. Advantages of Baculovirus
• Baculoviruses are incapable of replicating within the cells of mammals
and plants. Because baculoviruses are not harmful to humans, they are
considered a safe option for use in research applications.
• They are also used as biological agents as in the case of the Indian
mealmoth, a grain-feeding pest.
• Baculovirus expression in insect cells represents a robust method for
producing recombinant glycoproteins or membrane proteins.
• Baculovirus-produced proteins are currently under study as therapeutic
cancer vaccines with several immunologic advantages over proteins
derived from mammalian sources.[

29. Conclusions
• Baculoviruses are the most important tool for the study of basic
virology and biology.
• Baculoviruses are highly beneficial viruses since they do not infect
man or plants but they do provide effective natural biological control
of many insect species.
• Baculoviruses are also versatile vectors for the expression of proteins
for basic research and medical applications.