Protocols


Screen (pdf version)
ScreenInfluenza Resistance Screen
Posted On02/18/2010 12:24 PM
AuthorAmanda L. Blasius
Science WriterNora G. Smart
Background
Influenza, commonly known as the flu, is an infectious disease that affects birds and mammals, and is caused by RNA viruses of the family Orthomyxoviridae. Influenza infections are extremely common and can result in serious disease depending on the strain. Typically, influenza is transmitted from infected mammals through the air by coughs or sneezes, creating aerosols containing the virus, and from infected birds through their droppings. Influenza can also be transmitted by saliva, nasal secretions, faeces and blood. Influenza A viruses are the most virulent of these viruses, and are the cause of flu pandemics in humans. 
 
The Influenza A viral genome is made up of seven or eight pieces of segmented negative-sense RNA that encodes 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2 (NEP), PA, PB1, PB1-F2 and PB2. Influenza viruses bind through HA onto sialic acid sugars on the surfaces of epithelial cells; typically in the nose, throat and lungs of mammals and intestines of birds. The cell imports the virus by endocytosis, where the virus is then replicated, assembled and released. The single-stranded, segmented nature of the influenza viral genome, plus the lack of RNA proofreading during replication, allows these viruses to rapidly evolve and recombine with other strains of influenza virus, leading to the generation of new strains of virus every year (1)
 
Relatively little is known about host genetic risk factors for susceptibility to influenza, but the study of such factors in humans is difficult. Inbred strains of laboratory mice are ideal for studying genetic factors that may cause susceptibility or resistance to a certain disease. Mice are not naturally susceptible to human influenza viruses because they lack the predominant receptor to which it binds. The HA of human type A viruses binds preferentially to sialic acid (SA)-α2,6-Gal-terminated saccharides, while mice predominantly have the SA-2,3-Gal receptor. However, human viruses can be adapted to mice by repeated passages through mice, which selects for a viral variant that can bind the appropriate receptor (2). In mice, infection with high enough dose of influenza results in death, although this dose varies considerably in different strains of laboratory mice (3). The influenza resistance screen was designed to probe for mutations resulting in resistance to lethal doses of the virus. Examples of proteins and pathways that may be important in controlling influenza infection include the type I interferon (IFN) pathway, the IFN-induced MX proteins, and many chemokines/cytokines including IFNγ and TNFα [for a review see (2)]. It is likely that influenza infections are sensed by the toll-like receptor 7 (TLR7) and retinoic acid inducible gene I (RIG-I), which both bind single-stranded RNA (4).  Other studies suggest that TLR3, which senses double-stranded RNA, may contribute to the influenza response in humans (5;6)

 

Reagents and Solutions
2 dozen low viral, 10 day embryonated chicken eggs (McIntyre Poultry, Lakeside, CA)
 
PR8 influenza stock (1700 HAU/mL; made 1996; 67μL aliquots). 1 HAU = 5x105 PFU.
 
Penicillin/Streptomycin
Sterile PBS
200 IU/mL penicillin (Gibco)
200 mg/mL streptomycin (Gibco)
 
Hot plate
 
Paraffin
 
0.5mL syringes
 
 
28.5 gauge 1 inch needles
 
70% ethanol
 
Craftman drill (Craftsman Hobby Tool Model 572.251711 Ser.C)
 
Sterile Q-tips
 
15mL screw-cap tubes
 
Tri-R egg punch
 
Medium tweezers
 
Fine tweezers
 
Plastic bulbed pipets
 
10% bleach
 
P20 pipetter
 
Multi-channel pipetter
 
Sterile pipette tips
 
marked pipette tips
 
Chicken red blood cells (RBC), 30mL (Colorado Serum Co., Denver, Co; Catalogue No. 1151). Good for up to 2 weeks.
 
Alsever’s Solution (Colorado Serum Co.; special order). Can substitute with PBS. 
 
200mL sterile disposable flask
 
Ketamine/Xylazine cocktail
Ketamine stock (100mg/mL) 
Xylazine stock (20mg/mL)
900μL ketamine to 500 μL xylazine (20mg/mL)
Mix 2.8mL drug to 9mL sterile PBS
 
Heat lamp

 

Method
Generation of influenza stock
  1. Day 1: Place eggs in a 36o C incubator with the CO2 off (or in a warm room). Make humidifying chambers by adding 100-150 mL distilled water to each of 3 Rubbermaid containers. Place four T-25 flasks in the bottom of each container to support an 8-place egg carton without the cartons getting wet. Place eggs in the cartons with large sides up. Cover loosely and incubate 24 hours. 
  2. Day 2: Infection of eggs (2 hours). Place hot plate in hood. Melt paraffin over low heat and keep warm. 
  3. Dilute 4/96 PR8 influenza stock 1:10,000 (20μL to 2mL PBS with 20x penicilin/streptomycin, twice). Return the remainder of the stock aliquot to the freezer and use it for the first titration on Day 5. 
  4. Load syringes with diluted PR8 50μL at a time, using one syringe for up to 4 eggs. 
  5. Take 4 eggs out of the incubator. Lay one egg on its side, spray with 70% ethanol and let dry. 
  6. Wipe one end of the drill with ethanol. Drill must be wiped down after every 4 eggs. Very carefully drill a hole in the middle of the shell without breaking the membrane. The sound the drill makes will change just as it breaks through the shell.
  7. Holding the syringe horizontally with the needle bevel up, slide the needle just under the shell about a ¼ inch. Slowly inject PR8. 
  8. Spray the next egg with ethanol and let dry while you finish with the first egg. Using a Q-tip dipped in the melted paraffin, cover the hole with 2-3 drops and let dry. 
  9. Return eggs to the humidified containers and incubate 48 hours, checking water levels each day. 
  10. Day 4: Harvesting influenza (3 hours). Put eggs in the refrigerator for a minimum of 2 hours to kill the embryos. 
  11. While hood is being set up, place eggs at room temperature. Label two sets of 15mL screw-cap tubes 1-24. Place an absorbent pad in the hood. 
  12. Wipe each egg with a paper towel before placing it in the hood. Place 8 eggs in an egg carton holder, large end up. Squirt with 70% ethanol.
  13. Wipe the Tri-R egg punch with ethanol and punch a 1 inch hole in the top of each egg. The punch occurs on the release of the lever. If the break is not complete, rotate the egg and punch again. Do not press down too firmLy or the egg will collapse. 
  14. Keep both pairs of tweezers in a beaker of 70% ethanol. Use medium tweezers to remove the punched out egg shell. Use fine tweezers to tease apart the first white, paper-like membrane, exposing the allantoic fluid cavity. 
  15. Using a sterile plastic bulb pipette, harvest the clear fluid to a 15mL tube. Change pipettes for each egg. Be careful to not harvest any yolk, and avoid rupturing blood vessels. Good eggs will yield 5-8mL of clear, straw-colored fluid. 
  16. Centrifuge all 24 tubes 10 minutes at 2,000 rpm to get rid of the RBC and other debris. Pour the fluid into the second set of labeled tubes. Record the volume of each tube and any color or cloudiness. May store at 4 o C overnight.
  17. Soak tweezers in 10% bleach for 20 minutes to decontaminate. 
  18. Day 5: Titration. Titrate each of PR8 against the 4/96 stock virus in duplicate (see Table 1). Use the remainder of the stock aliquot used on Day 2. Use 7 round-bottom 96-well plates (7th plate will have only one sample in duplicate).
  19. Vortex all PR8 tubes 5 seconds. 
  20. Use a P20 pipetter wiped with 70% ethanol to pipette virus. Pipette 180μL/well PBS into row 1 of each plate, 100μL/well in rows 2-12. 
  21. Add 20μL virus to each well in column 1 using sterile pipette tips.
  22. Using 8 non-sterile tips on a multi-channel pipetter, mix up and down 5x in column 1 and transfer 100μL to column 2. Mix column 2 up and down 5x.  
  23. Change tips. Transfer 100μL from column 2 to column 3 and mix 5x. Repeat for all columns, changing tips for each dilution. Discard the extra 100μL from column 12.  The first column is a 1:10 dilution, the second 1:20, third 1:40, fourth 1:80 etc.
  24. Cut the tips off marked pipette tips at the 10μL mark, 56 tips or use wide bore tips.  
  25. Thaw 4 tubes of Alsever’s Solution (or use PBS).
  26. Rock the bottle of chicken blood gently. Do not shake. Pipette 10mL into a 15mL tube.
  27. Spin 1000 rpm for 5 minutes.
  28. If the supernatant is not clear, aspirate, add PBS and continue to wash until the supernatant is straw-colored.
  29. Aspirate off the supernatant and add an equal volume of Alsever’s solution.
  30. From the volume of the RBC pellet, determine the % RBC and dilute to 2% with Alsever’s. Example: 2.7ML RBC out of 10mL = 27%. Dilute 1:13.5 to get 2% = 2.5mL diluted to 33.8mL.
  31. Rock gently to resuspend and take out 2.5mL. Add 31.3mL of Alsever’s. About 30mL will be needed for both titrations, roughly 4mL/plate.
  32. Keep RBC on ice until needed.
  33. Move the plates with the diluted samples to a bench top or a clean cabinet with the blower off. Drip 1 drop of the RBC dilution per well using a plastic disposable pipette held vertically (50-60μL). Do two plates at a time.
  34. Mix up and down 2x after every 2 plates using cut off pipette tips to avoid damaging the RBC. Start at column 12 and go to column 1 on each plate, changing tips between plates. 
  35. Rock the RBC solution very gently 3-4x and repeat with the next two plates, etc.
  36. Cover the plates with blue pads to protect from the light. Incubate for 30 minutes, undisturbed.
  37. Lower dilutions will have a complete agglutination lattice. The highest dilutions will have a RBC pellet. Read the titration at 50% hemagglutination where there is some agglutination lattice and a ring or halo of RBC, but no pellet. Multiply the dilution by 5 to give HAU/mL. The titer is not absolute and will vary depending on the age and quality of the chicken RBC.
  38. Select the tubes that have a titer at least as good as the 4/96 stock virus. Discard the filter on a 200mL sterile flask and pipette each tube directly into the flask. Swirl to mix well.
  39. Repeat the titration in triplicate using additional 4/96 stock and the new pool of virus (1 plate).
  40. Make 12 aliquots of 67ul and aliquot the remainder of the batch in 0.3 mL aliquots. Label the top of each tube with virus name and batch #/year.  
Table 1 | 96 well plate layout.
 
 Dilution
 
 
 
 
 
 
 
 
 
1
2
3
4
5
6
7
8
9
10
11
12
sample 1
A
 
 
 
 
 
 
 
 
 
 
 
 
B
 
 
 
 
 
 
 
 
 
 
 
 
sample 2
C
 
 
 
 
 
 
 
 
 
 
 
 
D
 
 
 
 
 
 
 
 
 
 
 
 
sample 3
E
 
 
 
 
 
 
 
 
 
 
 
 
F
 
 
 
 
 
 
 
 
 
 
 
 
sample 4 or 4/96 stock
G
 
 
 
 
 
 
 
 
 
 
 
 
H
 
 
 
 
 
 
 
 
 
 
 
 
 
Titer virus in vivo
  1. Prepare dilutions of virus 0.03 HAU, 0.1 HAU, 0.3 HAU, 1 HAU, 3 HAU.
  2. Challenge 4-5 mice at each dose. 
  3. Anesthetize mice with ketamine/xylazine cocktail 100-200μL/mouse depending on size  
  4. Once unconscious, lay mouse on back under heat lamp. Wait 3-5 minutes. 
  5. Inject 10μL of influenza in each nostril. 
  6. Leave mouse under heat lamp for an additional 3-5 minutes, or until breathing is stable. 
  7. Mice should be left on backs for a total of 5-10 minutes after inoculation.  Subsequently, mouse is placed on belly in cage for remainder of recovery.
  8. Check mice every day for signs of sickness and death for 14 days. Mice become sick as early as day 4 and generally die 6-8 days following influenza infection.
  9. Identify the lowest dose where all mice die. Then choose one dose higher and infect 75 mice to test variability. Make any necessary adjustments.
In vivo influenza screening
  1. Anesthetize mice with ketamine/xylazine cocktail 100-200μL/mouse depending on size  
  2. Once unconscious, lay mouse on back under heat lamp. Wait 3-5 minutes. 
  3. Inject 10μL of influenza in each nostril.  Total dose per mouse is 0.3 HAU in 20μL corresponding to a 1:100 dilution of 1500 HAU/mL stock. This should be a lethal dose in C57BL/6J mice when using the June 2007 stock.
  4. Leave mouse under heat lamp for an additional 3-5 minutes, or until breathing is stable. 
  5. Mice should be left on backs for a total of 5-10 minutes after inoculation.  Subsequently, mouse is placed on belly in cage for remainder of recovery.
  6. Check mice every day for signs of sickness and death for 14 days. Mice become sick as early as day 4 and generally die 6-7 days following influenza infection.
  7. Breed mice that survive infection without serious signs of illness. Wait for 31 days after initial infection before breeding. 
  8. Retest subsequent generations and breed survivors to generate homozygous stock. 
  9. For mapping, please see the Genetic Mapping protocol.
Critical Parameters and Troubleshooting
Influenza stock
When dealing with influenza, make sure biohazard waste cans are available to collect any waste.
 
Virus stock is made as single use aliquots. Do not freeze/thaw.
 
Infection of mice
Mice have difficulty with thermoregulation while anesthetized, which is why they must be warmed. This compounds with difficulty breathing following insertion of liquid into their respiratory pathway. Continued heating and raising the paws above the head is helpful for mice with breathing difficulties.

 

References