This document discusses entomopathogenic viruses and their potential use for controlling insect pests. It provides background on virus structure and describes several types of viruses that infect insects, including baculoviruses. The document then discusses how genetic engineering can be used to modify viruses to optimize their speed of kill or increase virulence. Specific examples are given of genes inserted or deleted from baculoviruses to improve pest control. The conclusion emphasizes how genetic engineering combined with virus production technology may enable more effective and economical viral pesticides.
2. VIRUS
The word “virus” - Latin word venom, meaning “poison”.
Viral particle enter into a permissive (susceptible) cell -
nucleic acid takes charge of the cell’s metabolic system -
replicates into new virus particles, until the cell dies.
3. Basic structure of virus
Basic virus particle comprises a nucleic acid (DNA or
RNA), which directs the virus replication
Encapsulated within a protein case known as “capsid” -
important role in the host cell infection process.
4. Entomopathogenic viruses
Probably, there are no living forms in nature that escape
infection by at least one kind of virus.
A great variety of viruses attack and kill many insects.
These viruses are called entomopathogenic viruses
6. Insect viruses
Viral diseases found in 13 insect orders - currently placed in
12 families (Miller, 1998)
DNA Viruses: Baculoviruses (Nuclear polyhedrosis viruses-
NPV and Granuloviruses-GV), Ascoviruses, Iridoviruses,
Parvoviruses, Polydnaviruses and Poxviruses.
RNA Viruses: Reoviruses (Cytoplasmic polyhedrosis
viruses), Nodaviruses, Picorna-like viruses andTetraviruses.
7. BACULOVIRUSES
Family – Baculoviridae (Insect viruses)
Double stranded DNA viruses with rod shaped
nucleocapsid.
There are two genera of Baculoviruses:
nucleopolyhedroviruses (NPV) & granuloviruses(GV).
MODE OF ACTION
Infection occurs when susceptible host eats polyhedra or
granules which are dissolved in basic digestive gut juice
Virions are released when protein matrices dissolve
8. Infection of baculoviruses: A cross sectional representation of the anatomy of an
insect larva.
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
virions enter midgut cells after fusion with membrane epithelial
cells.The virions are uncoated and enter the nucleus where viral
genes are expressed.
9. POLYHEDROSIS VIRUS
Known to infect 500 species of insects –
best known from Lepidoptera
Virus particles enveloped singly or in groups – occluded
in protein bodies, polyhedra
Biovirus marketed in India – wettable powder
formulation for H. armigera NPV with 700 PIB/g – stable
for 2 yrs @ 40°c
It’s applied 300-500g/ha, 2-3 times @ 10-15 days interval
12. Advantages and Disadvantages of Viruses
for Controlling Pests
Advantages:
Unable to infect mammals, including humans- very
safe to handle.
Relatively specific- so the risk of non-target effects on
beneficial insects is very low.
Successful infections can perpetuate the disease
outbreak making repeat applications within a season
unnecessary.
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13. Disadvantages:
Most insect viruses take several days to kill their
host insect, during which the pest is still causing
damage.
Viruses are usually only effective against early
larval life stages.
quickly inactivated by direct sunlight or high
temperatures, some agricultural practices such as
tillage, which buries virus particles in the soil.
13
14. Suggestions for application of viruses
Scout fields before application
Apply virus when the target pests are young but actively feeding
Apply virus to maximize the longevity and effectiveness of virus
particles:
Thoroughly coat plants to maximize coverage.
Apply in the morning or evening or on cloudy days
Avoid applying on rainy days
Use formulations with ultraviolet (UV) light blockers and sticking
agents to increase longevity.
Using mixed cropping and reduce soil disturbance after application.
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15. Advent of genetic engineering
In essence, genetic engineering
provided a myriad of opportunities to enhance the
efficacy
cost effectiveness of the insect pathogens as their
control agents.
16. Steps
Isolating a gene to be inserted
Inserting the gene in a vector (agent used to
carry foreign gene)
Inserting vector into the host.
Multiplication of host cells by cloning.
Extraction of desired product.
17. Genetic Engineering of NPVs
P10 and Polyhedrin, the two most expressed proteins
required for the synthesis of occlusion bodies
The most commonly used strategy for engineering
baculoviruses has exploited the polyhedrin or p10
promoters
Construction of recombinant baculovirus achieved by
allelic replacement of the polyhedrin gene by foreign
genes - will result in over expression of chosen protein
18. Process for cloning recombinant
baculovirus
Baculovirus genomic DNA and a transfer plasmid are co-
transfected into an insect cell culture
Double homologous recombination between viral DNA
and transfer plasmid
Allelic replacement - recombinant gene in the
baculovirus genome
Viral stocks produced are amplified for recombinant
protein production
19. Baculovirus for
transduction of mammalian
cells, for production of
therapeutic proteins, or to
transduce organisms for
gene therapy or
vaccination.
Larvae are used to reduce
production costs, or when
recombinant baculovirus
are to be tested as bio
insecticides.
Insect cells used for
purification of many
proteins, including
therapeutic and vaccine
peptides.
20. Genetic engineering strategies
Genetic engineering to optimize speed of kill
Genetic engineering for increased virulence
and modify host range.
21. 1.Genetic engineering to optimize
speed of kill
A. Gene Deletion
deletion of genes that encode products prolonging
host survival
B. Gene Insertion
insertion of genes that express an insecticidal protein
during viral replication
22. A. Gene Deletion
EGT gene (Auxillary gene)
The egt gene of the insect baculovirus Autographa californica
nuclear polyhedrosis virus (AcMNPV) encodes the enzyme
ecdysteroid UDP–glucosyltransferase
Ecdysteroid UDP-glucosyl transferase (EGT), renders the
ecdysteroids inactive
Blocks molting of the host insect, thereby prolonging the actively
feeding larval stage.
Autographa californica
23. EGT
Functions to prolong the length of time insect feeds after
infection, resulting increase in the weight gain of insect
Deletion of the egt gene significantly improves the pesticide
characteristics of AcMNPV.
Larvae infected with an egt deletion mutant display considerably
reduced feeding and earlier mortality
24. Genetically modified NPVs
Deletion of ecdysteroid glucosyl transferase (EGT) gene of
Autographa californica NPV caused 40 % reduction in feeding
damage and rapid death of infected larvae of Trichoplusia ni
and Spodoptera frugiperda fall armyworm
(Reilly and Miller, 1991)
Spodoptera frugiperda
Autographa californica Trichoplusia ni
26. ... Genetically modified NPVs
Deletion of the gene encoding the polyhedral envelope
protein that surrounds the OB of AcMNPV
Resulted in 6-fold increase in infectivity against first
instar Trichoplusia ni compared to that of wild type virus.
Infected first instar T. ni
27. B. Gene Insertion
Insertion of a gene encoding a toxin, hormone or
enzyme into the baculovirus genome
Several recombinant baculoviruses - constructed for
overexpression of the host insect’s own hormones or
enzymes
28. Hormones
Several insect hormones are focused for engineering into
baculoviruses.
Eclosion hormone that initiates ecdysis, the process
leading to the shedding of old cuticle
Prothoracicotropic hormone (PTTH), which
is involved in triggering the molting process
Allatostatins and allatotropins, which regulate the
release of juvenile hormone and
Diuretic hormone (DH) that regulates water balance and
possibly blood pressure in insect.
29. Insertion of Enzymes
Interesting gene for genetic manipulation of baculovirus is
the enzyme gene, juvenile hormone esterase (JHE) that
caused the reduction in JH level.
Thus initiates metamorphosis in last instar and leads to
cessation of feeding.
30. ... Insertion of hormones
Diuretic hormone gene from Manduca sexta - introduced
into B. mori baculovirus genome
Recombinant BmNPV killed larvae of Manduca sexta about
20% faster than wild type virus .
The expression of this hormone by baculovirus - infected
larvae to rapidly lose water (Maeda, 1989).
31. ... Insertion of hormones
A recombinant isolate of AcMNPV - engineered to over-express
the gene for Manduca sexta chitinase, an enzyme that digests
chitin.
Chitinase target the chitin fibrils of the host cuticle
Weakens the cuticle so that it will rupture more easily and
release progeny occlusion bodies upon death of the host larva
32. Insertion of two or more toxin genes into
baculoviruses
Wide range of genes encoding insect-specific toxins isolated from
venomous scorpions
spiders
parasitic wasps
sea anemones
have been inserted into baculovirus genomes.
Binary mixtures of scorpion toxin, AaIT and LqhIT injected into
larvae of Helicoverpa virescens induced 5-10 fold the levels of
activity
(Hermann et al., 1995) .
33. The insect selective toxin (LqhIT2) from yellow Israeli
scorpion Leiurus quinquestriatus inserted in HzSNPV for
the control of Helicoverpa zea (DuPont, 1996)
Leiurus quinquestriatus
Helicoverpa zea
...Insertion of toxins
34. The toxin from scorpion Androctonus australis was
inserted in AcMNPV for the control of Helicoverpa zea
(Black et al., 1997)
Androctonus australis
Helicoverpa zea
...Insertion of toxins
35. ...Insertion of toxins
Inserting a toxin URF13 from maize to AcMNPV
Larvae of Trichoplusia ni were injected with this virus, all
died by 60 h post injection
(Korth and Levings, 1993)
36. 2. Genetic Engineering for Increased
Virulence
Insertion of the enhancin gene derived from Trichoplusia
ni GV enhanced AcMNPV virulence by 2 to 14-fold in
various insect species
Deletion of two enhancin genes from Lymantria dispar
MNPV reduced viral potency 12-fold compared to wild
type virus.
Lymantria dispar
Lymantria dispar MNPV
37. Conclusion
Development of baculovirus expression system and the
accomplishment of insect cell culture technology –
broadened the utility o f insect viruses as effective
insecticides
as expression vector of foreign genes in eukaryote host for
the production of useful proteins.
Production, formulation and application of technology in
conjugation with genetic engineering
for fast kill and broader host range
to enable the development of more economic and
efficacious viral products for insect control.
39. ...Entomopathogenic viruses
Insect pests are susceptible to viral infections - viruses
can be used as biological control agents.
viruses initially recovered from infected insects - later be
produced and applied in the field as bioinsecticides.
40. Successful examples of EPV
Pathogen group Pathogen Targeted
Arthropods
Crop/Host
Virus:
Baculoviridae
Alphavirus- NPV HearNPV,
HezeNPV
H. armigera, H.
zea
Corn, cotton,
Tomato,
Soyabean
SeMNPV S. exigua Vegetable, field,
flower &
ornamentals
Betavirus GV CpGV Cydia pomonella Pome fruit &
Walnut
AdorGV Adoxophyes orana Apple
41. Host range of other EPVs
Ascovirus: Ascoviridae - (Lepidoptera: Noctuidae) spp.
Iridovirus: Iridoviridae - Diptera, Lepidoptera, Coleoptera.
Polydnavirus: Polydnaviridae - Endoparasitic Hymenoptera, -
Ichneovirus infects ichneumonid wasp Campoletis sonorensis;
Bracovirus infects braconid wasp Cotesia melanocella
(Ibarra et al., 2008 )
42. ....Host range of EPV
Cypovirus: Reoviridae - ICTV recognizes 70 species, all
hosted by lepidopteran species.
Entomopoxvirus: Poxviridae - Entomopox viruses
isolated from 27 species of orthopterans, lepidopterans,
dipterans, and coleopterans
43. Bacmid technology
A major step forward in the technology of baculovirus
genetic engineering
Development of baculovirus genomes capable of
replicating in a bacterial host as bacterial artificial
chromosomes.
These recombinant baculoviruses are called bacmids -
modified to contain classical bacterial artificial
chromosomes replicons and selection markers for
selection in bacteria.
Principle advantage BACs is stability of insert
propagation over multiple generations.
44. By PCR the DNA from those colonies is purified and used to
transfect susceptible insect cells.
Naked genomic DNA from baculovirus can efficiently establish
infection when it reaches the cell nuclei.
Various commercial transfer vectors compatible with bacmid
systems to allow expression of one or two proteins (e.g.,
pFastBac1TM and pFastBacDualTM from InvitrogenTM).
45. The first bacmid developed contained the AcMNPV genome
Bacmid systems developed for
Bombyx mori NPV
Helicoverpa armigera single‐nucleocapsid nucleo polyhedrovirus
(HearSNPV) [Wang et al., 2003]
Cydia pomonella granulovirus (CpGV) [Hilton et al., 2008]
(the first report of a granulovirus bacmid).
46. Redpalmweevilcontrolbyentomopathogenicvirus
Insect viruses are obligate pathogens that can only
reproduce within a host insect.
They have to be ingested by the insect host to start the
infection process.
First case of a cytoplasmic polyhedrosis virus on RPW in
India causing deformed adults and reducing their lifespan
(Gopinadhan et al. 1990)
Viruses have little use as biological control agents (BCAs)
against RPW, except a report on their combination with
nematodes (Salama and Abd-Elgawad, 2002)
47. EPV NPVs
Entomopathogenic viruses employed as bioinsecticides - from
forest and field to food stores and greenhouses.
Baculoviruses, particularly the nucleopoly -hedroviruses (NPVs)
are the most commonly used – microbial insecticides -
lepidopterans
NPVs are formulated for application as sprays
48. MICROBIAL PESTICIDES
Market for bioinsecticides is about 4.2% of the total
insecticide market by 2010.
Although bacterial bioinsecticides represent the greatest
majority of them, viruses constitute an important
component of this type of agents, especially the
baculoviruses.
52. Limitations
Only moderate success has been achieved due to
several key limitations;
which include a relatively slow speed of kill
narrow spectrum of activity
less persistence in the field
lack of a cost-effective system for mass
production in vitro.
Fermentation technology for their mass
production on a large-scale commercial basis is
extensively investigated to reduce the
production cost.
54. .... Host range of EPV
Baculovirus: Baculoviridae : 600 isolates reported from a
variety of insect species, the ICTV only recognizes 30 species
within two genera NPVs and GVs.
The single nucleo polyhedro viruses SNPV and the multiple
nucleo polyhedro viruses MNPV type species (BmSNPV)
infects silkworms (B. mori), (AcMNPV) infects larvae of
Autographa californica (Lepidoptera: Noctuidae)
On the other hand, GV type species infects codling moth
larvae (Cydia pomonella; Lepidoptera:Tortricidae).
Editor's Notes
Most elementary biosystems that show macromolecular complexity and are able to self-replicate and evolve, but lack irritability (i.e. the ability to react to environmental factors), in contrast with the basic features that characterize all living things.
Once a viral particle gains entry into a permissive (susceptible) cell, its nucleic acid takes charge of the cell’s metabolic system and profusely replicates into new virus particles, until the cell is normally depleted of all its content and dies.
Inclusion bodies, sometimes called elementary bodies, are nuclear or cytoplasmic aggregates of stable substances, usually proteins. They typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of viral capsid proteins.
Many viruses occur naturally and may already be present in the environment. Even in cases where they are applied,
Viruses are usually not “stand alone” solutions to an insect pest problem, but are most effective in conjunction with other management strategies
The virulence and pathogenicity of pathogen - determined
microbial genome as a result of coordinated expression of a concert of genes.
The acquisition of these domains or pathogenicity islands,
may be sufficient to develop a transgenic virulent pathogen.
Polyhedrins - large structures that protect the virus particles from the outside environment for extended periods until they are ingested by other susceptible insect population.
EGT enzyme inactivates hormone ecdysone by transferring sugar molecules.
Infection with an egt defective recombinant AcMNPV resulted in 30% faster killing of larvae and significant reduction in food consumption.
Several insect hormones that play vital role in the control of insect morphogenesis and reproduction and are
focused for engineering into baculoviruses.
If JHE is inhibited, the concentration of JH remains high enough to keep the larva in the feeding stage - resulting in giant insects - so insert JHE
In BEVs, the foreign gene undergo transcription with help of a viral promoter
International Committee on Taxonomy of VIRUSES ICTV
The subfamily Entomopoxvirinae includes three genera - Entomopoxvirus A, B and C
The first one infects only coleopteran species and the type species infects Melolontha melolontha
second one infects lepidotperan and coleopteran species and the type species infects Amsacta moorei
Third one infects only dipteran species and the type species infects Chironomus luridus.
A fourth group (D) has been proposed to the ICTV which attacks hymenopterans.
BAC vectors contain a fragment of E. coli fertility factor (F‐ factor) replicon (miniF) and are maintained as circular supercoiled extrachromosomal single copy plasmid in the bacterial host [57, 58]. BACs can accept inserts up to 300 Kb in length. Once transferred into the bacterial host, the baculovirus genome can be manipulated easily through site‐specific recombination, Rec‐A mediated homologous recombination or transposition.
TRANSFECT - introduce (genetic material) by infecting a cell with free nucleic acid.
Once the recombinant bacmid is generated and the presence of transgene and the absence of the parental bacmid in the bacterial colonies are verified,
BV particles can be recovered from culture supernatant and used as inoculum to produce high titer stocks.
Autographa californica
or considered for development
as microbial insecticides mainly for the control
of lepidopteran insects on field and vegetable
crops.