The document discusses various types of viruses that can be used as biopesticides, including baculoviruses like nucleopolyhedrosis viruses and granulosis viruses which infect insect hosts and form crystalline protein structures allowing them to survive outside the host, as well as less classified group C baculoviruses and entomopox viruses. Baculoviruses like NPVs and GVs produce occlusion bodies within infected cells that protect the virus and allow it to be transmitted between hosts, making them useful for biocontrol.
5. Introduction
• Increased resistance to chemical pesticides and
concern over their use has resulted in rewed interet
in the application of biological means to control
pests of commercial importance
• Insect-specific viruses can be highly effective
natural controls of several caterpillar pests. Different
strains of naturally occurring nuclear polyhedrosis
virus (NPV) and granulosis virus are present at low
levels in many insect populations.
5
10. What are Biopesticides?
• Biopesticides are certain types of pesticides derived from such
natural materials as animals, plants, bacteria, and certain minerals.
For example, canola oil and baking soda have pesticidal applications
and are considered biopesticides
Recent efforts to reduce broad spectrum toxins added to the
environment have brought biological insecticides back into vogue. An
example is the development and increase in use of Bacillus
thuringiensis, a bacterial disease of Lepidopterans and some other
insects. It is used as a larvicide against a wide variety of caterpillars.
10
12. Biopesticides fall into three major classes:
• Microbial pesticides consist of a microorganism (e.g., a bacterium, fungus, virus or protozoan) as the
active ingredient. Microbial pesticides can control many different kinds of pests, although each separate
active ingredient is relatively specific for its target pest[s]. For example, there are fungi that control
certain weeds, and other fungi that kill specific insects.
• The most widely used microbial pesticides are subspecies and strains of Bacillus thuringiensis, or Bt.
Each strain of this bacterium produces a different mix of proteins, and specifically kills one or a few
related species of insect larvae. While some Bt's control moth larvae found on plants, other Bt's are
specific for larvae of flies and mosquitoes. The target insect species are determined by whether the
particular Bt produces a protein that can bind to a larval gut receptor, thereby causing the insect larvae
to starve.
• Plant-Incorporated-Protectants (PIPs) are pesticidal substances that plants produce from genetic
material that has been added to the plant. For example, scientists can take the gene for the Bt pesticidal
protein, and introduce the gene into the plant's own genetic material. Then the plant, instead of the Bt
bacterium, manufactures the substance that destroys the pest. The protein and its genetic material, but
not the plant itself, are regulated by EPA.
• Biochemical pesticides are naturally occurring substances that control pests by non-toxic mechanisms.
Conventional pesticides, by contrast, are generally synthetic materials that directly kill or inactivate the
pest. Biochemical pesticides include substances,
12
14. Baculoviruses
• Description: Baculoviridae are the safest insect viruses to use as pathogens, since
no similar viruses are known to infect vertebrates or plants. They have double-
stranded DNA and are protected by a protein coat which improves their
persistence.
Infection: Infection occurs after susceptible insect larvae eat food contaminated
with virus. The virus then attacks the haemolymph, fatty tissue and mid gut. The
insect becomes paralysed.
Virulence: Highly virulent; the presence of very few particles can initiate infections
and hosts die within 3-10 days.
Susceptibility: The gut of the host insect must be alkaline so that the occlusion
body can dissolve.
14
15. Baculoviruses
• The baculovirus genome is non-segmented and contains a molecule of circular, double-
stranded DNA. The complete genome sequence is 80000-180000 nucleotides long.
Interspersed throughout the genome are sections of repetitive sequences of DNA known as
homologous regions, or hrs. The complex structure of these hrs are formed by 60bp repeats,
with each repeat containing a 28bp-long imperfect palindrome. These homologous regions
enhance early transcription as well as act as origins for DNA replication. Many of the genes
in the baculovirus genome overlap at the ends, which allows a large number of genes to be
encoded in a small amount of DNA. (sources: ICTVdB and Viral Bioinformatics Resource
Center)
• Recent analysis of the genome sequence of baculoviruses suggests that the taxonomy of
baculoviruses needs to be altered. More specifically, it has been discovered that the
phylogeny of baculoviruses is more closely related to the classification of the host organism
than the morphological traits of the virus, which had been used previously to classify
baculoviruses. (source: Jehle et al. and ICTVdB)
15
16. Baculoviruses
Infection: Infection occurs
after susceptible insect
larvae eat food
contaminated with virus.
The virus then attacks the
haemolymph, fatty tissue
and mid gut. The insect
becomes paralysed.
16
17. Viron Structure of a Baculovirus
• Baculovirus virions have a complex structure which consists of an envelope and a rod-shaped
nucleocapsid. The capsid is 200-450nm in length, and 30-100nm in diameter. The capsid has helical
symmetry.
• When the baculoviruses are extracellular, they can be found in two forms: budded virus (BV) and
occluded virus (OV). OVs are polyhedral or oval-shaped crystalline protein matrices in which one or
several mature virions are embedded. The OVs are large, measuring 0.15-15μm in length. OV particles
are formed inside infected cells and are released when the cell lyses. The crystalline protein matrix of the
OVprotects the virus while in the extracellular environment; because of this, OVs are used for transfer of
the virus between hosts.
• The two genera in the family Baculoviridae are definied by their different OV structure. Granulovirus OVs
contain only one virion, and do not have a polyhedral envelope (known as a calyx). These OV are small,
giving a "granular" appearance when many OVs are seen together. The protein that forms the crystalline
matrix of Granulovirus OVs is known as granulin. Nucleopolyhedrovirus OVs are much larger than
Granulovirus OVs, holding 20 or more virions in each OV particle. The virions are either seperate or
bundled together inside a calyx. The protein that forms the crystalline matrix of Nucleopolyhedrovirus
OVs is called polyhedrin.
• BVs are used for cell-to-cell transmission within an infected host. BV particles consist of a single capsid
enclosed in an envelope which the capsid obtains when it "buds" out through the cell wall and into the
host's system. Unlike OVs, BVs cannot survive outside the host organism. (sources: Herniou et al.,
ICTVdB, Viral Bioinformatics Resource Center)
17
18. • The most studied baculovirus is Autographa californica multicapsid
nucleopolyhedrovirus (AcMNPV). The virus was originally isolated
from the alfalfa looper (a lepidopteran) and contains a 134-kbp
genome with 154 open reading frames (ORF). The major capsid
protein VP39 together with some minor proteins forms the
nucleocapsid (21 nm x 260 nm) that encloses the DNA with p6.9
protein.
• BV acquires its envelope from the cell membrane and requires a
glycoprotein, gp64, to be able to spread systemic infection. This
protein forms structures called peplomers on one end of the budded
virus particle but is not found on ODV (although several other
proteins are only associated with the ODV form). Some differences
also exist in the lipid composition of the viral envelope of the two
forms. While BV envelope consists of phosphatidylserine, ODV
contains phosphatidylcholine and phosphatidylethanolamine
18
19. 4/28/2012 Free Template from www.brainybetty.com 19
20. Genome
• Baculoviruses have a circular, double stranded DNA genome. The genome size of
these viruses range in size from 80 - 180 kbp. Of the fully sequenced baculovirus
genomes the number of open reading frames (orfs) ranges from approximately 120
to 160. In addition to the genes encoded in the genome the are also a number of
small repeated sequences known as homologous regions (hrs) interspersed in the
genome. These regions have been shown to enhance early gene transcription and
also to act as origins of replication. Many of the genes in a baculovirus genome
have overlapping ends allowing a large number of genes to be encoded in a
smaller amount of DNA. A diagram of the Eppo MNPV genome map is shown
below.
20
22. Molecular biology of baculoviruses
• Baculoviruses are the major group of arthropod viruses well known due to their potential as agents of
biological control of pests in agriculture and forestry. They are also widely used as expression vectors
inbiotechnology. The family Baculoviridae comprises two genera: the Nucleopolyhedrovirus (NPVs) and
the Granulovirus (GVs) NPVs can be phylogenetically subdivided into group Iand group II. These viruses
produce a large number of occlusion bodies in infected cells which allow virus to survive in the
environment and to transmit the
• disease from one insect to another. Baculoviruses infect arthropods and they do not replicate in
vertebrates, plants and microorganisms. However, though they do not replicate, they may, under special
conditions, enter animal cells. This unexpected property made them a valuable tool in the last few years
for studies of transient expression of foreign genes undervertebrate promoters introduced into
baculovirus genomeBaculoviruses are a large group of double-stranded DNAviruses (over 600 species
have been described); the majority have been isolated from a few insect orders: Lepidoptera, Diptera,
Hymenoptera and Coleoptera. Individual baculoviruses usually have a narrow host range
• limited to a few closely related species. Virions consist of one (SNPV) or more (MNPV) nucleocapsids
embedded in a membranous envelope. Viral genome ranges in size from 80 to 200 kb in length. The
most widely studied baculovirus is the Autographa californica nuclepolyhedrovirus (AcMNPV). Early
work on AcMNPV was directed towards the development of viral pesticides and construction of
baculovirus-based expression vectors
22
23. Baculovirus expression system
• Recombinant baculovirus have become widely used as vectors to
express heterologous genes in cultured insect cells and insects
larvae
• Heterologous genes placed under the transcriptional control of the
strong polyhedrin promoter of the Autographa californica
polyhedrosis virus (AcNPV)
• Based on site specific transposition of an expression cassette (pfast
Bac with gene of interest) into a baculovirus shuttle vector (bacmid)
• Starting from a DNA sequence, cDNA or recombinant virus, Paragon
scientists will clone your gene (± affinity tags) into a commercially
available expression vector or a custom vector that you provide
23
25. Steps in recombinant baculovirus production
• Clone the gene of interest in pfast Bac donor plasmid
• Expression cassette in pfast Bac is flanked by left and right arms of
Tn7 and also an SV40 polyadenylation signal to form a miniTn7
• Cloned pfast Bac is transformed in E.coli host strain (DH10Bac)
which contains a baculovirus shuttle vector bacmid having a mini-
attTn7 target site
• Helper plasmid which allows to transpose the gene of interest from
pfast to bacmid (shuttle vector)
• Transposition occurs between the mini-att Tn7 target site to generate
a recombinant bacmid
• This recombinant bacmid can now be used to transfect insect cell
lines.
25
26. 4/28/2012 Free Template from www.brainybetty.com 26
31. Nuclear polyhedrosis viruses (NPV)
• Description: About 280 species known. Rounded cubic or hexagonal polyhedra.
0.5-1.5 microns (µm) in size. Singly or multiply envelopped.
Infection: Infection occurs in the adipose tissue of the hypodermis, in the tracheae
and the middle intestine.
Host: Approximately 120 species of Lepidoptera and Hymenoptera (particularly
saw-flies). Each virus is highly specific to its host.
Survival: Nuclear polyhedrosis viruses form particles inside a crystalline protein
structure (occlusion body). This allows the virus to survive outside the host for
years out of sunlight.
Biocontrol agents: The following NPVs have all been produced on a commercial
or semi-commercial scale: Autographa californica NPV, Lymantria dispar NPV,
Malacasoma disstria NPV, Mamestra brassicae NPV, Neodiprion sertifer NPV,
Spodoptera NPV and Heliothis NPV
31
36. Granulosis viruses (GV)
• Description: There are about 65 species of granulosis virus, with
oval or ovoid granules.
Infection: Granulosis viruses attack the adipose tissue.
Host: Lepidoptera larvae.
Survival: Granulosis viruses form particles inside a crystalline
protein structure (occlusion body). This allows the virus to survive
outside the host. Can survive for years out of sunlight.
Biocontrol agents: Include Cydia pomonella GV (codling moth),
Phthorimaea operculella GV (potato tuber moth).
36
39. Group C Baculoviruses
• NB This group of viruses is currently unclassified
• Description: Double stranded DNA. Viruses with non-included virions. Only visible
using an electron microscope. 22-30 nm in size. These viruses are unusual since
they have no protective protein coat to help them to survive.
Infection: These viruses attack the haemolymph, fat body, mid-gut. Insects
become paralysed.
Host: These viruses are restricted to Arthropoda. Larvae and adults of Coleoptera,
Hymenoptera and mites.
Biocontrol agents: Baculovirus oryctes: - Used for the control of rhinoceros
beetles, Oryctes spp. This virus is excreted from the living diseased insect as
virions. These are passed on to other adults during mating. Some spread occurs
from contamination of adult breeding and larval feeding sites, but the virus does not
survive long in the environment.
39
40. Entomopox viruses
• Description: Entomopox viruses have inclusion bodies (i.e. they are
occluded viruses) which are important for identification. Spherical or
ovoid particles. 5-20 µm in size.
Infection: These viruses must be ingested by the host. They then
attack the fat body.
Virulence: They kill hosts more slowly than baculoviruses.
Host: Lepidoptera larvae, Diptera, Coleoptera, Orthoptera.
Survival: Little is known.
Locusts: Entomopox viruses have been recorded from locusts and
grasshoppers
40
41. Baculovirus life cycle
• The baculovirus life cycle involves two distinct forms of virus. Occlusion derived virus (ODV) is present in
a protein matrix (polyhedrin or granulin) and is responsible for the primary infection of the host while the
budded virus (BV) is released from the infected host cells later during the secondary infection.
• Typically, the initial infection occurs when a susceptible host insect feeds on plants that are
contaminated with the occluded form of the virus. The protein matrix dissolves in the alkaline
environment of the host midgut (stomach), releasing ODV that then fuse to the columnar epithelial cell
membrane of the host intestine and are taken into the cell in endosomes. Nucleocapsids escape from
the endosomes and are transported to nucleus. This step is possibly mediated by actin filaments. Viral
transcription and replication occur in the cell nucleus and new BV particles are budded out from the
basolateral side to spread the infection systemically. During budding, BV acquires loosely fitting host cell
membrane with expressed and displayed viral glycoproteins.
• Baculovirus infection can be divided to three distinct phases, early (0-6 h post-infection), late (6-24 h p.i.)
and very late phase (18-24 to 72 h p.i.). While BV is produced in the late phase, 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. These occlusion bodies are released when cells lyse to further spread
baculovirus infection to next host. The extensive lysis of cells frequently causes the host insect to literally
melt, thus the reason for the historic name "wilting disease." To adapt survival in the wild, ODV-
polyhedrin particles are resistant to heat and light inactivation, whereas BV is more sensitive to
environment.
41
44. What are the advantages of using
biopesticides?
1-Biopesticides are usually inherently less toxic than conventional pesticides.
2-Biopesticides generally affect only the target pest and closely related organisms, in
contrast to broad spectrum, conventional pesticides that may affect organisms as
different as birds, insects, and mammals.
3-Biopesticides often are effective in very small quantities and often decompose
quickly, thereby resulting in lower exposures and largely avoiding the pollution
problems caused by conventional pesticides.
4-Greater public acceptance
5-Production is relatively inexpensive
6-A renewable resource
7-High specific activity
8-Usually target specific
44
46. Genetic engineering in virus
• Genetic engineering offers a potential solution to this shortcoming. A foreign
pesticidal gene can be inserted into the viral genome and expression of the
pesticidal gene product during replication will allow the virus to kill insects faster or
cause quick cessation of feeding. Foreign genes which have been inserted into
baculoviruses for this purpose include the Buthus eupeus insect toxin-1,the Bacillus
thuringiensis ssp. kurstaki HD-73 delta-endotoxin, the Pyemotes tritici TxP-I toxin,
Androctonus australis neurotoxin.The most efficacious gene inserts have been the
neurotoxins and the T-urf 13 gene which is responsible for cytoplasmic male
sterility of maize. Several major pesticide companies are currently involved in the
commercial development of these and other genetically enhanced viral pesticides.
46
47. Modification of baculovirus genome
• Modification of baculovirus genome by introduction of a
specific toxin gene was much widely exploited.Historically,
introduction of cry toxin gene of B. thuringiensis was one of the
first attempts.
• The most promising insect-specific toxin gene used for
construction of baculovirus recombinants is probably the gene
coding for AaIT toxin originating from scorpion A. australis.
47
48. major problems in using baculoviruses
One of the major problems in using
baculoviruses as pesticides is their
slow action and lack of morphological
• changes in larvae in first stages of
baculovirus development.
• Their major disadvantage is that for
most baculoviruses it typically takes
from 4 to 14 days to kill the insect
host.(During this time, the
• insects can still cause serious
damage to the crop.)
48
51. REFERENCES
1-Bartelt, R.J., McGuire, M.R., Black, D.A., 1990. Feeding stimulantsfor the European corn borer (Lepidoptera: Pyralidae):
additives toa starch-based formulation for Bacillus thuringiensis. Environ.Entomol. 19, 182–189.
2-Ca~nas, L.A., O’Neil, R.J., 1998. Applications of sugar solutions tomaize, and the impact of natural enemies on fall
armyworm. Int. J.Pest Manag. 44, 59–64
3-Cisneros, J., Perez, J.A., Penagos, D., Ruiz, V.J., Goulson, D.,Caballero, P., Cave, R.D., Williams, T., 2002b.
Formulation of a
baculovirus with boric acid for control of Spodoptera frugiperda(Lepidoptera: Noctuidae) in maize. Biol. Contr. 23, 87–95.
4-Crawley, M.J., 1993. GLIM for Ecologists.Methods in Ecology Series.Blackwell, Oxford, UK.
5-Dunkle, R.L., Shasha, B.S., 1988. Starch-encapsulated Bacillus thuringiensis:a potential new method for increasing
environmental
stability of entomopathogens. Environ. Entomol. 17, 120–126.
6-Escribano, A., Williams, T., Goulson, D., Cave, R.D., Chapman,J.W., Caballero, P., 1999. Selection of a
nucleopolyhedrovirus for
control of Spodoptera frugiperda (Lepidoptera: Noctuidae): structural,genetic and biological comparison of four isolates
from the
Americas. J. Econ. Entomol. 92, 1079–1085.
7-Harris, J., Dent, D., 2000. Priorities in Biopesticide Research andDevelopment in Developing Countries. CABI
Publishing, Wallingford,UK.
51
52. REFERENCES
8-Hruska, A.J., Gould, F., 1997. Fall armyworm (Lepidoptera: Noctuidae)and Diatraea lineolata (Lepidoptera:
Pyralidae): impact of
larval population level and temporal occurrence on maize yield inNicaragua. J. Econ. Entomol. 90, 611–622.
9-Hunt, L.M., Ojanguren, R., Schwartz, N., Halperin, D., 1999. Balancingrisks and resources: applying
pesticides without safety equipment
in Southern Mexico. In: Hahn, R. (Ed.), Anthropology inPublic Health. Oxford University Press, Oxford, UK,
pp. 265–289.
10-Hunter-Fujita, F.R., Entwistle, P.F., Evans, H.F., Crook, N.E., 1998.Insect Viruses and Pest Management.
Wiley, Chichester, UK.
11-Jones, K.A., Cherry, A.J., Grzywacz, D., 1997. Formulation: is it anexcuse for poor application? In: Evans,
H.F. (Ed.), Microbial
Insecticides: Novelty or Necessity?. Proc. Brit. Crop Prot. CouncilNo. 68, Farnham, UK, pp. 11–19.
12-Jones, K.A., Burges, H.D., 1998. Formulation of bacterial, viruses andprotozoa to control insects. In:
Burges, H.D. (Ed.), Formulation of
Microbial Biopesticides: Beneficial Micro-organisms, Nematodesand Seed Treatments. Kluwer Academic
Publishers, Dordrecht,Netherlands, pp. 32–127
52
