Protocols


Screen (pdf version)
ScreenIn Vivo RVFV Susceptibility Screen
Posted On02/18/2010 12:24 PM
AuthorSungyong Won
Science WriterEva Marie Y. Moresco
Background
Rift Valley Fever Virus (RVFV; genus Phlebovirus, family Bunyaviridae) is an important zoonotic pathogen transmitted by mosquitoes that causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients typically develop an acute febrile illness, which may be followed by fatal hemorrhagic fever, encephalitis or ocular disease (1;2).  RVFV is known to be sensitive to the interferons (IFNs) (3;4), and an antibody response is critical for protection against the virus (5;6).  In a rodent model, wild type RVFV infection causes acute death with fatal hepatic disease at 2 to 3 days post-infection (dpi), whereas infection with a mutant RVFV strain lacking NSm protein expression results in delayed death with fatal neurological disease as well as hepatic failure (7).
 
This screen is designed to identify genes required for the control of RVFV infection in vivo.  Since wild type RVFV is categorized as an enhanced animal biosafety level 3 (ABSL-3) containment agent, a mutagen-attenuated recombinant strain (arMP-12) that can be handled in ABSL-2 containment is used here (8).  Preliminary experiments indicate that C57BL/6J mice injected intraperitoneally with 1x105 pfu of arMP-12 control the infection with no signs of illness, but Ifnar-/- and Stat1domino/domino mice succumb to death with high viremia and viral loads in the liver and spleen within 3 dpi (Figure 1). Mutagenized G3 mice that display severe sickness or death after intraperitoneal challenge with 1x105 pfu of arMP-12 are identified as potential mutants in this screen. The Ex Vivo Macrophage Screen for Control of Viral Infection is also employed to discover components important for the control of RVFV infection, but utilizes thioglycolate-elicited peritoneal macrophages, thus saving the animal from infection-induced death.
 
Reagents and Solutions
Vero E6 medium
Dulbecco’s modified eagle medium (DMEM) (GIBCO Invitrogen, Carlsbad, CA)
10% (v/v) heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA)
100 IU/ml of penicillin and 100 μg/ml of streptomycin (GIBCO Invitrogen, Carlsbad, CA)
 
Virus stock diluent
Hank’s Balanced Salt Solution (HBSS) (HyClone, Logan, UT)
2% (v/v) FBS
100 IU/ml of penicillin and 100 μg/ml of streptomycin
 
Tissue diluent
HBSS
5% (v/v) FBS
200 IU/ml of penicillin and 200 μg/ml of streptomycin
 
Plaque assay medium
Modified Eagle Medium (MEM) (GIBCO Invitrogen, Carlsbad, CA)
0.6% (w/v) tragacanth gum (MP Biomedicals, Solon, OH)
2.5% (v/v) FBS
5% (w/v) tryptose phosphate broth (TBP) (BD Biosciences, San Jose, CA)
 
Cell fixation solution
Phosphate-buffered saline (PBS)
10% (w/w) formaldehyde
 
Cell staining solution
distilled H2O
0.25% (w/v) crystal violet
2.5% (v/v) ethanol in distilled water
Method
RVFV stock (arMP-12) preparation
1. Infect VeroE6 cells with a seed stock of arMP-12.
2. Collect culture supernatant at 2 to 3 dpi when most cells show severe cytopathic effect (CPE).
3. Centrifuge supernatant at 1500g for 5 min to remove cell debris.
4. Store clear supernatant at -80C° until use for titration and infection.
 
RVFV titration
5. Prepare confluent Vero E6 cells in 24- or 6-well plates. For each virus dilution, plan to infect triplicate wells.
6. Serially dilute (from 1:10 to 1:107) RVFV stock in Vero E6 medium.
7. Infect Vero E6 cells in triplicate wells with each virus dilution. (Use 100 μl for each well of a 24-well plate; 400 μl for each well of a 6-well plate).  Incubate at 37oC/5%CO2 in a humidified incubator for 1 hour.
8. Wash infected cells with PBS three times.
9. Add plaque assay medium to each well and incubate at 37oC/5%CO2 in a humidified incubator for 4 days.
10. At 4 dpi, remove plaque assay medium and fix cells with cell fixation solution for at least 10 minutes at room temperature (RT).
11. Remove cell fixation solution and stain cells with cell staining solution for 5 to 10 min.
12. Remove cell staining solution and wash cells with water.
13. Count plaques and calculate titers according to dilution factors.
 
In vivo screening
14. Prepare the virus inoculum (5x105 pfu/ml) by diluting the titrated virus stock with virus stock diluent.
15. Challenge each G3 mouse intraperitoneally with 200 μl of the inoculum (1x105 pfu/mouse). Save leftover inoculum for re-titration.
16. Check mice daily for sickness and death up to 2 to 3 weeks.
17. Once affected mice are identified, collect blood, liver, spleen and brain from the mice.
18. Make 10% (w/v) tissue homogenates in tissue diluent and determine virus titers as described in RVFV titration (steps 5-13).
19. Check for RVFV susceptibility in siblings of affected mice.
20. Fix and map mutations using general genetic mapping protocol.
Critical Parameters and Troubleshooting

Leftover inoculum from step 15 may be re-titrated in case unexpected results are observed after infection, such as widespread death of wild type control mice.

Alleles Identified
domino
macro-2
maladaptive
tortellini
References
   1. Schmaljohn, C. S., and Nichol, S. T. (2007) Bunyaviruses, in Fields Virology (B. N. Fields, D. M. Knipe, P. M. Howley, and D. E. Griffin, Eds.) pp 1741-1789, Lippincott Williams and Wilkins, Philadephia, PA.

   8. Won, S., Ikegami, T., Peters, C. J., and Makino, S. (2006) NSm and 78-Kilodalton Proteins of Rift Valley Fever Virus are Nonessential for Viral Replication in Cell Culture. J. Virol. 80, 8274-8278.