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General Virology- Exam 3 > Insect Viruses > Flashcards

Insect Viruses Flashcards

1
Q

Viruses pathogenic to insects: dsDNA

A 
Baculoviridae
Iridoviridae
Poxviridae
Ascoviridae
Polydnaviridae (not really a virus)
Hytrosaviridae (flies)
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2
Q

Viruses pathogenic to insects: ssDNA

A

Parvoviridae (Densovirus of crickets and mosquitos)

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3
Q

Viruses pathogenic to insects: dsRNA

A
  • Cypovirus (cytoplasmic polyhedrosis virus) (genus of reoviridae which replicates in cyptoplasm)
  • Birnaviridae (drosphilia X virus)

unique!

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4
Q

Viruses pathogenic to insects: (+) ssRNA

A

Picornaviridae
Tetraviridae
Nodaviridae

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5
Q

Viruses pathogenic to insects: (-) ssRNA

A

Rhabdoviridae

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6
Q

where does iridoviridae replicate?

A

cytoplasm (shown in arrays)

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7
Q

Entomopoxvirus Entry

A
  • in the family of poxviridae
  • fusion, endosome, endocytosis
  • entomopox virions and occlusions replicate in the cytoplasm (just like the vertebrate poxviruses)
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8
Q

Types of insect poxviruses

A

Coleopteran (beetle) Poxvirus
Dipteran (mosquito, flies) Poxvirus
Lepidopteran (caterpillar) poxvirus

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9
Q

Poxvirus in larvae

A

Expression of egt enables the virus to inhibit molting of its infected host

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10
Q

Polydnavirus in wasp injected into caterpillar

A

The full genome of the virus is integrated into the genome of the wasp and the virus only replicates in specific cells in the female wasp’s reproductive system
- Plasmatocytes blocked from encapsidating egg
by polydnavirus

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11
Q

Hytrosaviridae

A

-viruses of flies

they produce non-occluded, enveloped, rod-shaped virions

they possess a large circular dsDNA genome

they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility

Transmission is either horizontally (per os) through feeding or vertically (transovarially) from mother to offspring

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12
Q

Small RNA virus infections in honeybees

A

Chronic bee paralysis virus (note swollen, shiny abdomen)

Acute bee paralysis virus infection (note disjointed mouthparts)

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13
Q

Chronic Bee Paralysis

A

ssRNA positive-strand viruses, Picornavirales; Dicistroviridae (Genus: Aparavirus & Cripavirus)

Syndrome 1: Abnormal trembling of the wings and body. The bees cannot fly and often crawl on the ground and up plant stems.
Syndrome 2: Affected bees are able to fly but are almost hairless. They appear dark or black and look smaller.

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14
Q

Navel orangeworm Picornavirus

A

pathology includes small larvae and small abnormal adults

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15
Q

Baculoviruses- Structure

A

Covalently closed dsDNA + capsid = Nucleocapsid
Nucleocapsids are enveloped
Protein matrix = Occlusion Body (OB) or Polyhedra
- Major component: polyhedrin protein coded by the virus
- Soluble at alkaline pH (>10) in insect gut

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16
Q

Baculovirus- Taxonomy

A

Alphabaculovirus: 33 Species: multiple nucleopolyhedrovirus
Betabaculovirus: 14 Species: granulovirus
Deltabaculovirus: 1 Species: nucleopolyhedrovirus
Gammabaculovirus: 2 Species: nucleopolyhedrovirus

Phenotype:
Nucleopolyhedrovirus= many enveloped nucleocapsids per occlusion body
- Single NPV: one nucleocapsid per envelope
- Multiple NPV: various numbers of nucleocapsids per envelope

Phenotype:
Granulovirus: one enveloped nucleocapsid per occlusion body

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17
Q

Baculoviridae

A
  • alkali released virus
  • budded virus
  • uncoating of nucleocapsid at nuclear pore
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18
Q

sucrose gradient

A

purification of baculovirus

bands correspond to various #s of enveloped nucleocapsids purified from MNPV

19
Q

Application of Baculovirus

A

used instead of pesticides

ex: 26 Baculoviruses developed as microbial pesticides to control Lepidoptera

20
Q

Baculovirus replication cycle in larvae

A
  • ingestion of virus on leaf surface
  • alkaline pH in gut dissolves polyhedrin releasing enveloped nucleocapsid
  • columnar gut cells infected or virus passes through
  • virus replication
  • budded virus
  • occluded enveloped nucleocapsid virus
21
Q

Soybean Yield with Baculovirus and Chemical Treatments

A 
diflubenzuron= highest seed yield & lowest # of caterpillars
methonyl= lowest seed yield
22
Q

Baculovirus Infection

A

Non-occluded budded virus -> infected cell -> budded virus -> infected cell -> budded virus
(passage 1= 0-24 hrs//cell to cell spread//passage 2=24-48 hrs)
infected cells-> occluded virus -> insect to insect spread (degraded cell)
(>60 hrs)

23
Q

Entry of Infectious Virus into Cells (Baculovirus)

A

Attachment- to columnar cells in midgut (next slide)
Penetration- fusion or viropexis
Uncoating
- Granuloviruses release DNA at nuclear pore
- Nucleopolyhedroviruses uncoat inside nucleus

24
Q

Replication and Metabolic Events (Baculovirus)

A

Transcription- Baculovirus Ie1, immediate early gene, is transcribed as soon as the DNA is uncoated

Protein synthesis- cascade IE (immediate early), E (early), L (late), VL (very late)

DNA synthesis- 4-9 hours peak occurs in nucleus

Late genes are transcribed- 9 to 48 hours

25
Q

which gene does the baculovirus regulate early?

A

early transcriptional regulation of the gp64 gene

IE1= baculovirus transcription factor

26
Q

Baculovirus infected cell protein synthesis

A

DNA synthesis occurs at 5 hours
polyhedrin protein (24 hrs)
glycoprotein (15 hrs)
200 = MOI

27
Q

Baculovirus glycoproteins radioactively labeled then detected by fluorography and x-ray film

A

3H labeled
budded virus= 64 -> Gal, Glu, Man
alkali released virus= 45 -> Gal, Glu
37 -> Man

28
Q

Baculovirusb budded virus growth curve

A

Budded virus can be titered before polyhedra are formed

29
Q

Glycosylation of proteins

A

Asparagine amino acid

Insects are fine with:
Mannose (mannosidase)
Glucose (glucosidase)
GlcNAc transferase

Insects need help with:
Galactose (galactosyltransferase)
Silica acid (sialyltransferase) (on membranes)

30
Q

Envelope acquisition- Baculovirus

A
  • De novo assembly in nucleus

- Nuclear and cytoplasmic budding

31
Q

Polyhedrin protein formation- Baculovirus

A

occludes enveloped nucleocapsids

32
Q

How is Infectious Virus Determined?

A 
plaque assay (PFU)
end point dilution (TCID50)
bioassay (LD50)
33
Q

plaque assay

A 
Plaque assay (PFU- plaque forming units) of serially diluted virus in cell monolayers with agar overlay. 
Plaque count times the reciprocal of the last dilution times dilution (if less than 1 mL) equals PFU/mL.
34
Q

end point dilution

A

End point dilution (TCID50 -mean tissue culture infectious dose) in microtiter plates. Serial dilute inoculum, add same dilution to 10 wells. Count number of infected wells with susceptible cells times reciprocal of the last dilution, 1/10-x, times dilution, times 100.5 equals TCID50

The baculovirus uses beta-galactoside as indicator that the virus is a recombinant with the foreign gene.

35
Q

bioassay

A

Bioassay (LD50 -mean lethal dose) inject live larvae with serial dilutions of virus. Determine 50% of larval deaths at last serial dilution.

36
Q

Baculovirus proteins

A

they’re eukaryotic proteins

Phosphoproteins- are labeled with 32PO4

Glycoproteins- label with carbohydrate precursors (3H-N-Acetyl- Glucosamine, 3H-Mannose, 3H-N-Acetyl Galactosamine)

Infected cell proteins- can be labeled with 35S-methionine

37
Q

Genetic Engineering of Baculoviruses

A
  • Determination of gene on the physical map by hybrid selection of RNA to DNA fragments followed by in vitro translation of RNAs to make protein corresponding to the size of the polyhedrin protein.
  • Site directed mutagenesis deletes (knocks out) coding region of the gene where foreign genes insert.
  • Plasmid vector transfer of foreign gene into genome.
  • Construction of Baculovirus protein expression vectors.
38
Q

Physical Map

A

Locates DNA fragments and genes on the dsDNA genome cut with multiple restriction enzymes

BC Envelope glycoprotein (64-67 kD)
IE-1 (67 kd) (46 kd)
polyhedrin (29 kd)
DNA polymerase (114 kd)

39
Q

Site Directed Mutagenesis of genes

A

cut with restriction enzyme (plasmid) –(exoIII)–> S1 nuclease ( digests sand) –(dna ligase)–> plasmid with mutated gene + viral or bacterial genome DNA (for co-transfection with virus genome) OR to transform bacteria to grow lots of recombinant plasmid for transfection

40
Q

Baculovirus Transfer Vector with Beta- Interferon

A

For transforming bacteria to make plasmid copies for transfecting insect cells.
*Transfer vectors do not infect insect cells.

41
Q

Commercial Baculovirus transfer vector

A

Beta- galactosidase

To get recombinant gene in the genome- need recombination sequences
Promoters (early and late)

42
Q

Baculovirus protein expression system

A
  1. After picking recombinant plaque, grow virus plaque in 24 cluster well -16 mm diameter
  2. Infect 25 cm2 flask with Sf9 cells.
  3. Grow 2 days, harvest, titer supernatant.
  4. Infect Sf9 cells in shaker flask.
  5. Harvest supernatant and infected cells at 4 days post-infection.
  6. Column purify recombinant protein.
43
Q

ELISA or Enzyme Immunoassay

A

Testing for antibodies in a person’s serum by coating microtiter wells with baculovirus expressed antigen

Enzyme linked to anti-IgM then add substrate

Enzyme linked to antibody to virus then add enzyme substrate

44
Q

Baculovirus Expression Vector system

A

It has become a core technology for:

1) the cloning and expression of genes for study of protein structure, processing and function;
2) the production of biochemical reagents;
3) the study of regulation of gene expression;
4) the commercial exploration, development and production of vaccines, therapeutics and diagnostics;
5) drug discovery research;
6) the exploration and development of safe, selective and environmentally compatible bio-pesticides consistent with sustainable agriculture.

General Virology- Exam 3 (6 decks)

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