Insect Viruses

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Insect Viruses
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Viruses found in insects

• those being vectored to plants or animals
• Mechanical
• biological
• those infecting and damaging insect

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Mechanical transmission

• stylet-borne
• Viruses cannot survive long in vectors
• Very infectious right after infected

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vectored Biological transmission

• must have minimal effect on insect
• so it will move and bite
• especially in biological transmission
• must have time to go through cycle in insect

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process

• insect becomes infectious only after an
  incubation period of multiplication and the
  migration
• insect retains its infectious power for a very
  long time.
• enters in sap or blood meal

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Process for mosquito vectors

• blood meal goes to midgut
• through epithelial cells and basal lamina
• line mid gut mid gut barrier

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Process for mosquito vectors

• to haemoceol where replicates
• cross salivary gland barrier
• grow to high titers in salivary gland
• passes on when feeds next time
• leaves most of insect intact

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Mosquito anatomy

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in cell culture

• vertebrate
• inhibit cell function
• destroy cell
• invert
• retain activity and morphology
• after initial high virus out put become
  persistent infection

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INSECT PATHOGENIC

• FOCUS

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History phase 1 as pathogens of valuable insects
viruses kill the good guys

• sixteenth and seventeenth centuries
• Literature and scientific reports
• detailed descriptions of the wilting disease of
  silkworms
• Now know caused by virus
• early nineteenth century,
• crystalline polyhedral bodies found associated
  with wilting disease

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History phase 1 as pathogens of valuable insects
viruses kill the good guys

• late nineteenth century,
• Polyhedra established as causal agents of the
  disease
• 1920, wilting disease was attributed to a
  filterable virus
• 1940s, Bergold discovered rod-shaped virions,
  embedded in polyhedral

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History Phase 2 - viruses as biological control -
viruses kill the bad guys

• 1949, idea of insect pathogens as biological
  pesticides
• 1960s
• discovery of many new types of insect viruses,
• irido
• nodaviruses,
• polydnavirus-like particles,
• entomopox
• establishing insect cell lines

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History Phase 2 - viruses as biological control -
viruses kill the bad guys

• 1975, the first insect virus was registered by
  EPA as a pesticide
• 1977, a catalog of viral diseases of insect and
  mite viruses
• 640 insect or mite species belonging to 10
  different orders,\
• 21 different types of viral disease had been
  reported.

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History Phase III object of study and tool for
molecular virology

• mid- I 970s
• molecular biological research on insect viruses.
  
• insect viruses such as their use as gene
  expression vectors

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Viruses Pathogenic to Insects

• dsDNA Viruses
• Baculoviridae
• Iridoviridae
• Poxviridae
• Ascoviridae
• Polydnaviridae
• ssDNA Viruses
• Parvoviridae

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Viruses Pathogenic to Insects

• dsRNA Viruses
• Reoviridae
• Birnavirida
• Positive ssRNA Viruses
• Picornaviridae
• Tetraviridae
• Nodaviridae
• Negative ssRNA Viruses
• Rhabdoviridae

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insect pathogenic

• multiply and get passed on to other insects
• multiply in intestinal tract
• released in excrement
• infect food source of others

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insect pathogenic

• multiply through out body
• when insect dies virus "spills out"
• contaminates environment of others

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insect pathogenic

• Insect viruses eaten by an insect
• spread from insect to insect during mating or egg
  laying.
• stay out in environment longer than plant and
  animal viruses

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why interested

• disease in economically important insects
• silkworms
• Insects comprise over 80 of existing animal
  species
• critically important to the ecosystem
• Parasitoids
• Both
• bees

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why interested use as biological control in pests
and disease vectors

• 40 percent of the world's crops and livestock are
  lost annually due to insects
• United States loses 30 percent to 40 percent
  every year,
• most of it before harvest,
• gypsy moths, corn earworms. and mosquitoes
  caterpillar pests. alfalfa looper, cabbageworm,
  cabbage looper, cotton bollworm, cotton leafworm,
  tobacco budworm, armyworms, European corn borer,
  almond moth, spruce budworm, Douglas fir tussock
  moth, pine sawfly and gypsy moth. fall webworm,

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why interested use as biological control in pests
and disease vectors

• number of chemical pesticides available for
  control is decreasing,
• undesirable effect on the environment
• insect resistance."
• naturally occurring epizootics for insect pest
  control is not economically feasible
• By time have population levels high enough to
  initiate epizootics, Crop damage exceeds
  economically and/or aesthetically acceptable
  thresholds.

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why interested use as biological control in pests
and disease vectors

• Brazil, over a million hectares of land treated
  with a baculovirus for soybean looper
• South Pacific, a baculovirus is used on many
  islands to control coconut beetles
• Europe, a baculovirus is used to control the
  apple maggot.
• Several baculoviruses are now registered
• Used primarily by government agencies
• U.S. Forest Service baculoviruses
• tussock moth larvae
• Gypsy moth

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why interested use as biological control in pests
and disease vectors

• Narrow spectrum - infect one species
• Strength - do not hurt other insects
• Weakness relatively small market.
• Cost effectiveness to make
• Mode of Action
• invade via the gut.
• replicate in many tissues
• disrupt insect's physiology, interfering with
  feeding, egg laying, and movement.

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insect pathogenic

• many have occlusion bodies polyhedra
• protect outside of insect

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NPV
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insect pathogenic

• crystals of protein coded by virus
• resistant to proteolytic enzymes
• may also have lipid trace metals silicon
• virus embedded
• Crystallize in nuclear sap where replicate
• excreted

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insect pathogenic

• released in mild alkaline environment
• Insect midgut pH10
• So released at site of primary infection in midgut

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Outcomes

• little or no overt effects on insect population
  size
• influence the "health" o f the population
• stress factor on insect populations.
• infections by some tetraviruses and cypoviruses,
• cause a gut disease similar to diarrhea.

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Outcomes

• striking effects on population levels,
• widespread epizootics
• morbidity in dense insect populations.
• high population densities
• usually observed under unnatural situations
• accidental introduction of a species
• intensive chemical pesticide application.

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challenge - metamorphosis

• extreme changes in host
• some only infect specific stages
• most larvae

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NPV nuclear polyhedrosis virus caterpillar wilt

• Symptoms
• NPV-infected larvae
• turn white and granular or very dark.

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NPV caterpillar wilt

• climb to the top of the crop canopy,
• stop feeding,
• become limp,
• hang from the upper leaves or stems,
• "caterpillar wilt"
• .

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• granulosis virus
• turn milky white
• stop feeding.

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NPV and GSV

• body contents of the dead larvae are liquefied
• cuticle ruptures to release infectious viral
  particles.
• Death within three to eight days

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gene expression vectors

• Started to genetically engineer viruses to be
  more effective as pesticides
• Moved into using as expression vectors
• Baculoviruses
• BEVS, or Baculovirus Expression Vector System
• can stably accommodate large amounts of foreign
  DNA
• produce very high levels of biologically active
  foreign eukaryotic proteins
• posttranslational modification and tertiary
  protein folding similar mammals

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gene expression vectors

• proteins of research, vaccine, diagnostic, or
  pharmaceutical utility.
• over 500 different eukaryotic genes expressed
  using baculovirus vectors.
• nodaviruses
• RNA polymerase to amplify RNA
• insect viruses to produce vaccines. BVES
• AIDS vaccines trials
• influenza vaccine
• breast and colon cancer vaccine

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• The general process of a baculoviral infection
  begins with ingestion of the occlusion bodies
  (OB) on the diet. Once the OB reaches the midgut
  of the insect the alkaline pH causes the
  dissolution of the occlusion body releasing the
  virions (ODV). The released virions then pass
  through the peritrophic membrane of the the
  midgut. It has been suggested that there may be
  some baculoviral proteins incorporated into the
  ODV which may enhance the ability of the virions
  to pass through the peritrophic membrane. These
  proteins include forms of chitinases and
  metalloproteases. The basic virus infection
  process is shown in the following diagram

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• Once the virions have entered the midgut
  epithelia the nucleocapsids migrate to the
  nucleus of the cell. This migration may be in
  association with the cellular actin. Once the
  nucleocapsids reach the nucleus the DNA is
  uncoated into the nucleus. Phosphorylation of the
  DNA binding protein p6.9 causes the DNA to unwind
  allowing expression and replication of the viral
  genome. This process is slightly different for
  the granuloviruses. These viruses release their
  DNA into the nucleus through the nuclear pore.
  For MNPVs it is postulated that some of the
  nucleocapsids may bypass the nucleus and bud out
  of the cell, allowing the virus to infect other
  cells faster than if they have to replicate the
  genome. The following table out line the various
  phases of baculovirus replication. For more
  information on each stage click here or on the
  name of the phase.

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Phase Description
Immediate early Expression of viral transregulators and genes which do not require transregulators for efficient transcription. Many of the genes expressed in this phase are involve in establishing the infection.
Delayed early Expression of genes involved in the replication of the virus and manipulation of the host. Delayed early genes often require the presence of viral transregulators (e.g. IE-0, IE-1, PE38) for efficient transcription.
Late Transition from early to late is characterised by shutdown of the host cell DNA replication and protein synthesis. Nucleocapsids are produced. Budded virus is produced and disseminates the virus throughout the host.
Very late (or occlusion) Advanced stage of virus infection. Virions become occluded in the protein polyhedrin. Viral proteases liquefy the host and degrade the chitinous exoskeleton. Occluded progeny virus is disseminated onto surrounding material for horizontal spread.
An general overview
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• Members of the Baculoviridae family have various
  tissue tropisms. The NPVs which affect
  lepidopteran insects (caterpillars) tend to
  infect all of the major tissue types. They
  initially infect the midgut cells before budding
  out and infecting other tissues such as
  haemocytes, fat bodies, the epidermis and the
  tracheal matrix. Very few if any occlusion bodies
  are formed in the midgut cells. Most of the ODV
  is produced in the other tissues.
• Almost all of the other baculoviruses only infect
  midgut cells (and produce occluded virus in these
  cells). A small number of the GVs (including the
  type species, the Cydia pomenella GV) which have
  a cellular tropism similar to the lepidopteran
  NPVs, i.e. initially infect the midgut epithelium
  and then spread to almost all other tissues and
  produce occluded virus in those tissues.

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Protein Function
EGT Inactivates the host moulting hormones by conjugating a UDP-sugar group to them. Reduces stress on the insect and prevents sloughing/apoptosis of midgut cells.
P35 IAP-1, -2, -3, -4 Apoptosis inhibitors. Prevent the process of "programmed cell death" which is a response to viral infection (among other things)
Chitinase/Cathepsin Involved in the liquefaction of the host and hence the dissemination of the occluded form of the virus for horizontal spread.
 
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Baculoviridae Properties of Virion

• Morphology.
• Virions of two different phenotypes found
• occluded by protein bodies or crystals in thin
  sections,
• not occluded bacilliform.
• Virions enveloped
• 1 nucleocapsid(s) per envelope,
• Nucleocapsids rod-shaped 200-450 nm long 30-100
  nm in diameter.

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Baculoviridae Properties of Virion

• Genome.
• Circular (supercoiled) double stranded DNA.
• monopartite.
• ORFs on both strands
• some overlap
• genome 90000-230000 nucleotides long.

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Family Baculoviridae

• Members
• Genus Nucleopolyhedrovirus
• Many virions per occlusion body
• Some have each virion in occlusion body in own
  membrane
• Some have 2 /membrane
• replicate in nucleus
• Genus Granulovirus
• One virion per occlusion body
• found in cytoplasm

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Family Baculoviridae

• Special tricks
• egt gene
• enzyme blocks hormones that trigger ecdysis
• insect doesnt molt
• prolongs virus multiplication
• Alter nuclear and cytosketetal structure
  dramatically
• if cell can undergo apoptosis during replication
  infection is aborted
• virus makes proteins to block

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Family Iridoviridae

• five genera
• iridovirus (insect)
• chloriridovirus (insect)
• ranavirus (frog)
• lymphocystrivirus (fish)
• unnamed genus (fish)

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Genus Vernacular name Host species Type species
Iridovirus Small iridescent insect virus Invertebrates (mainly insects) Chilo iridescent virus (IV6)
Chloriridovirus Large iridescent insect viruses Mosquitos Mosquito iridescent virus (IV3)
Lymphocystivirus Lymphocystis disease virus Fish Lymphocystivirus type 1 (LCDV-1)
Ranavirus Frog virus Amphibia Frog Virus 3 (FV3)
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• Iridoviruses contain a single copy linear dsDNA
  genome that ranges in size from 150 to 280 kbp
  depending on viral species. The genomes appear
  unique within the eucaryotic viruses in that they
  are terminally redundant and cyclically permuted.
  This structure is a result of the resolution of
  genome concatamers during DNA replication (see
  replication).
• A simplistic view of terminal redundancy and
  cyclic permutation.
• During replication multiple copies of a
  hypothetical viral genome consisting of 10 genes
  (A) forms a long concatamer (B). The resolution
  of this concatamer (C) results in packaged DNA
  lengths that contain a complete genome as well as
  duplicated copies of some genes (terminal
  redundancy). The ends of each of these packaged
  DNAs differs from one virus particle to the next
  (cyclic permutation).

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• 1) Virus particles enter the cell by pinocytosis
  and uncoating occurs.
• 2) Viral DNA is transported to the cell nucleus
  where host macromolecular synthesis is rapidly
  shutdown. Transcription is initiated by virally
  modified host RNA polymerase II.
• 3) Parental DNA is used to produce genome and
  greater than genome length DNA. This becomes the
  template for cytoplasmic replication.
• 4) Progeny DNA is transported into the cytoplasm
  where large concatamers of viral DNA are formed
  by recombination. Transcription of very late
  transcripts may also take place in the cytoplasm.
• 5) Concatamers are resolved into packaged
  lengths, possibly by a headful packaging
  approach. Virions exit the cell by budding or
  cell lysis.

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• 1) Virus particles enter the cell by pinocytosis
  and uncoating occurs.
• 2) Viral DNA is transported to the cell nucleus
  where host macromolecular synthesis is rapidly
  shutdown. Transcription is initiated by virally
  modified host RNA polymerase II.
• 3) Parental DNA is used to produce genome and
  greater than genome length DNA. This becomes the
  template for cytoplasmic replication.
• 4) Progeny DNA is transported into the cytoplasm
  where large concatamers of viral DNA are formed
  by recombination. Transcription of very late
  transcripts may also take place in the cytoplasm.
• 5) Concatamers are resolved into packaged
  lengths, possibly by a headful packaging
  approach. Virions exit the cell by budding or
  cell lysis.

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Irido
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irido
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Irido
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Family Polydnaviridae Properties of Virion

• Morphology.
• Virions ovoid (or drop-shaped).
• Virions more than one type of particle
• different shape and size.
• Virions enveloped, or multiple enveloped
• 2 envelopes)
• Nucleocapsids rod-shaped, or ovoid (prolate
  ellipsoid, fusiform)
• 30-150 nm long, or 330 nm long 40 nm in
  diameter, or 85 nm in diameter.

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Family Polydnaviridae properties of Virion

• Genome.
• Supercoiled circular, or linear (chromosomal)
• double stranded DNA.
• Genome multipartite in 20-40 segments.
• Encapsidated nucleic acid
• solely genomic, or
• Both genomic and non-viral I
• including fragments of host DNA.
• virions contains multiple copies of genome.
• Total genome 2000-28000(-300000) nucleotides
  long.

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Family Polydnaviridae special tricks

• found only in parasitic wasps
• transmitted vertically within wasps
• part of chromosome provirus
• process
• replication of DNA and virion formation take
  place in oviduct of females
• During pupal development
• Doesnt seem to harm wasp
• wasps lay eggs in insects of other species
• Often larvae
• Many virus particles deposited too

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Family Polydnaviridae special tricks

• Virus counters insects defenses
• Knock out Hemocytes that usually encapsulate the
  egg
• Also lead to degeneration of prothoracic gland
• upsets host physiology
• wasp eggs hatch
• Larvae develop within host
• Protected by actions of virus
• Larval host often dies
• wasp pupates
• More viral replication

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