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.

1. VINEETHA V
2018-21-038
GENETICALLY MODIFIED
ENTOMOPATHOGENICVIRUSES

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

5. VIRUS GROUPS
ENTOMOGENOUSVIRUS
INCLUSION
VIRUSES (IV)
POLYHEDROSIS
VIRUSES (PV)
NUCLEAR
POLYHEDROSIS
VIRUSES (NPV)
CYTOPLASMIC
POLYHEDROSIS
VIRUSES (CPV)
GRANULOSIS
VIRUSES (GV)
NON-INCLUSION
VIRUSES (NIV)

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

10. GRANULOSIS VIRUS

11. INSECT VIRUSES

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.
12

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.
14

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

25. Han et al., 2015

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.

49. Production of insect viruses

50. CommerciallyavailableproductsinIndia
(Sahayaraj, 2014)
VIRUS PRODUCT NAME TARGETS
HaNPV Helicide H. armigera
Virin-H
Helocide
Biovirus- H
Helicop
Heligard
SlNPV Spodo-Cide S. litura
Spodoterin
Spodi-Cide
Biovirus-S

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).