Insect Viruses

Question 1

Viruses pathogenic to insects: dsDNA

Answer 1

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

Question 2

Viruses pathogenic to insects: ssDNA

Answer 2

Parvoviridae (Densovirus of crickets and mosquitos)

Question 3

Viruses pathogenic to insects: dsRNA

Answer 3

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

unique!

Question 4

Viruses pathogenic to insects: (+) ssRNA

Answer 4

Picornaviridae
Tetraviridae
Nodaviridae

Question 5

Viruses pathogenic to insects: (-) ssRNA

Answer 5

Rhabdoviridae

Question 6

where does iridoviridae replicate?

Answer 6

cytoplasm (shown in arrays)

Question 7

Entomopoxvirus Entry

Answer 7

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

Question 8

Types of insect poxviruses

Answer 8

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

Question 9

Poxvirus in larvae

Answer 9

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

Question 10

Polydnavirus in wasp injected into caterpillar

Answer 10

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

Question 11

Hytrosaviridae

Answer 11

-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

Question 12

Small RNA virus infections in honeybees

Answer 12

Chronic bee paralysis virus (note swollen, shiny abdomen)

Acute bee paralysis virus infection (note disjointed mouthparts)

Question 13

Chronic Bee Paralysis

Answer 13

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.

Question 14

Navel orangeworm Picornavirus

Answer 14

pathology includes small larvae and small abnormal adults

Question 15

Baculoviruses- Structure

Answer 15

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

Question 16

Baculovirus- Taxonomy

Answer 16

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

Question 17

Baculoviridae

Answer 17

• alkali released virus
• budded virus
• uncoating of nucleocapsid at nuclear pore

Question 18

sucrose gradient

Answer 18

purification of baculovirus

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

Question 19

Application of Baculovirus

Answer 19

used instead of pesticides

ex: 26 Baculoviruses developed as microbial pesticides to control Lepidoptera

Question 20

Baculovirus replication cycle in larvae

Answer 20

• 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

Question 21

Soybean Yield with Baculovirus and Chemical Treatments

Answer 21

diflubenzuron= highest seed yield & lowest # of caterpillars
methonyl= lowest seed yield

Question 22

Baculovirus Infection

Answer 22

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)

Question 23

Entry of Infectious Virus into Cells (Baculovirus)

Answer 23

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

Question 24

Replication and Metabolic Events (Baculovirus)

Answer 24

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

Question 25

which gene does the baculovirus regulate early?

Answer 25

early transcriptional regulation of the gp64 gene | IE1= baculovirus transcription factor

Question 26

Baculovirus infected cell protein synthesis

Answer 26

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

Question 27

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

Answer 27

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

Question 28

Baculovirusb budded virus growth curve

Answer 28

Budded virus can be titered before polyhedra are formed

Question 29

Glycosylation of proteins

Answer 29

Asparagine amino acid Insects are fine with: Mannose (mannosidase) Glucose (glucosidase) GlcNAc transferase Insects need help with: Galactose (galactosyltransferase) Silica acid (sialyltransferase) (on membranes)

Question 30

Envelope acquisition- Baculovirus

Answer 30

- De novo assembly in nucleus | - Nuclear and cytoplasmic budding

Question 31

Polyhedrin protein formation- Baculovirus

Answer 31

occludes enveloped nucleocapsids

Question 32

How is Infectious Virus Determined?

Answer 32

``` plaque assay (PFU) end point dilution (TCID50) bioassay (LD50) ```

Question 33

plaque assay

Answer 33

``` 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. ```

Question 34

end point dilution

Answer 34

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.

Question 35

bioassay

Answer 35

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

Question 36

Baculovirus proteins

Answer 36

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

Question 37

Genetic Engineering of Baculoviruses

Answer 37

- 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.

Question 38

Physical Map

Answer 38

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)

Question 39

Site Directed Mutagenesis of genes

Answer 39

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

Question 40

Baculovirus Transfer Vector with Beta- Interferon

Answer 40

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

Question 41

Commercial Baculovirus transfer vector

Answer 41

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

Question 42

Baculovirus protein expression system

Answer 42

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.

Question 43

ELISA or Enzyme Immunoassay

Answer 43

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

Question 44

Baculovirus Expression Vector system

Answer 44

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.