Screen (pdf version)
ScreenMCMV Susceptibility and Resistance Screen
Common NameMCMV
Posted On02/18/2010 12:24 PM
AuthorCeline Eidenschenk, Duanwu Zhang
Science WriterNora G. Smart
Mouse cytomegalovirus (MCMV) is a β-herpes virus that is contained by the host through the action of natural killer (NK) cells of the innate immune system before the onset of the adaptive immune response.  Mice of the C57BL/6 or C57BL/10 backgrounds do not become sick in response to MCMV inoculated at a dose of 1x105 PFU.  The virally encoded m157 protein is detected by NK cell–activating receptor Ly49H, and as a consequence, infected cells are lysed (1;2).  In BALB/c mice, which lack the Ly49H-encoding gene, MCMV is frequently lethal within one week following inoculation of 1 x 105 PFU.
Although C57BL/6 mice are completely resistant to challenge with 1x105 PFU of MCMV, higher doses cause sickness and death in these animals.  A few mice die when MCMV is inoculated at a dose of 2 x 105 PFU, and nearly all mice appear sick; ~90% of mice die when inoculated with 4 x 105 PFU (Figure 1).  The exceptionally sharp dose-lethality characteristics of the virus permit sensitive screens for enhanced susceptibility or resistance.  Binary screens for MCMV susceptibility and resistance are carried out by inoculating G3 germline mutants on a C57BL/6 background with 1x105 and 1x106 PFU of MCMV, and either severe illness and death, or lack of illness and survival, are taken as endpoints, respectively.  Wild type C57BL/6 and BALB/c mice are used as controls (3). Quantification screen for MCMV susceptibility is carried out by inoculating G3 mice with 1x105 PFU of MCMV, and MCMV titers in the tissues (spleen and liver) are determined by quantitative real-time PCR.  High MCMV titers represent susceptible mice.
Mice susceptible to MCMV may have mutations in genes required to sense virally encoded nucleic acids and proteins, in genes encoding cytokine mediators along with their receptors and transducers, and in genes encoding components required for proper NK cell granule exocytosis (4;5).  In addition, mutations may affect proteins that support homeostasis during the innate immune response (6), or genes required for the ontogeny of NK cells or other cells that support the innate immune response.  Many of the mutations causing MCMV susceptibility or resistance can be distinguished by testing mutants for standard cytokine responses and NK cell function (see NK Cytotoxicity Protocol).
Resistance to MCMV may be caused by mutations affecting host proteins that are needed by the invading virus for infection and replication. MCMV has evolved evasion mechanisms to avoid detection by the host by targeting virus-recognition mechanisms, and antigen processing and presentation (5).  MCMV resistance mutants may also have mutations that allow the host to overcome evasion.
Reagents and Solutions

MCMV medium

  • DMEM
  • 3% (v/v) heat-inactivated calf bovine serum
  • 100 IU/mL penicillin
  • 100 mg/mL streptomycin

3T3 medium

  • DMEM
  • 10% (v/v) heat-inactivated calf bovine serum
  • 100 IU/mL penicillin
  • 100 mg/mL streptomycin

Agar medium

  • DMEM
  • 10% (v/v) heat-inactivated calf bovine serum
  • 100 IU/mL penicillin
  • 100 mg/mL streptomycin
  • 100 μg/mL gentamycin
  • 2% carboxymethyl cellulose

To make 500 mL:

  • 50 mL calf bovine serum
  • 5 mL penicillin/streptomycin stock (100x)
  • 1 mL gentamycin (50 mg/mL)
  • 10 g carboxymethyl cellulose (Sigma, Cat.# 419311), autoclaved
  • 440 mL DMEM
  • Shake every day for one week until the medium is homogenized.

Cell staining solution

  • 1% crystal violet (w/v) in 20% ethanol.
  • Before use, dilute 1 part with 9 parts Milli-Q water.


IFNγ, IL-6, IL-12 p70 and TNFα ELISA kits (eBioscience).

Tissue tearor is cleaned with 10% bleach and rinsed with ethanol and sterile PBS.



Generation of MCMV stock

  1. Inject 3-week old BALB/c females with 1-5x103 PFU of MCMV stock.
  2. After two weeks, harvest salivary gland and homogenize in 1-2 mL of MCMV medium per mouse using a tissue tearor, spin at 200 x g for 3 min to pellet cellular debris, save supernatant to a new tube.
  3. Spin supernatant at 800 x g for 5 min, eliminate upper lipidic layer by aspiration, aliquote and freeze the rest at -80 oC.

In vitro MCMV titration (Plaque assay)

  1. Day 0: Plate 3T3 cells in a 24 well plate at 40,000 cells/well in 1 mL of 3T3 medium.
  2. Day 1: Make a series of dilutions of organ supernatants from step 3 (i.e. 1/10, 1/100, 1/1000...).
  3. Infect cells with MCMV by first removing cell media, and then adding 200 μL of the undiluted and diluted samples. Incubate for 2 hours at 37 oC.
  4. Remove the samples, and add 1 mL of agar medium to each well, and incubate for 4 to 5 days at 37 oC.
  5. Day 5: Fix the cells by adding 200 μL of 10% formalin to each well, and incubate for 6 hours or overnight at RT.
  6. Remove fix and add 200 μL of cell staining solution per well. Incubate 2 minutes at RT.
  7. Wash the plate well for 2-3 times. Let dry and count the number of plaques.

In vivo MCMV titration

  1. Inject 8-week old mice of each strain (Balb/c and C57BL/6) with 1x105, 2x105, 5x105, and 1x106 PFU of MCMV (in vitro titer) using 5 mice per dose.
  2. Check mice every day for appearance and death. Discard cages by day 7.
  3. Adjust in vitro titer if needed.

MCMV binary screen

  1. Use in vivo titer to inoculate ENU-mutagenized G3 C57BL/6 mice at 1x105 PFU and 1x106 PFU. As described above, wild type C57BL/6 mice are completely resistant to the low dose of MCMV, but die at day 5 post-infection with a dose of 1x106 PFU.
  2. Check mice every day for sickness or death (both of which are scorable outcomes of infection). Discard cages by day 7.
  3. Breed and fix mutants, and confirm the MCMV phenotype. Please see the Genetic Mapping protocol for breeding schemes and mapping strategies.  

MCMV quantification screen

  1. Inoculate ENU-mutagenized G3 C57BL/6 mice at 1.5x105 PFU per 20 gram of body weight.
  2. Check mice every day for sickness or death.
  3. Harvest spleen and liver at day 5 post-infection.
  4. Extract total DNA from the tissues.
  5. Perform quantitative real-time PCR to determine the copy numbers of MCMV genome (i.e. MCMV Immediate-Early 1 gene) and mouse genome (i.e. β-actin gene) in tissue DNA samples. The standard curves are generated using their own plasmids.
  6. The viral titer is represented as the copy number ratio of MCMV genome to mouse genome.

Analysis of MCMV mutants

  1. Test mutant tissues at 5 days post-MCMV infection for viral titers.
  2. Test mutants for standard cytokine responses using ELISA kits 36 hours after inoculation with 1x105 PFU of MCMV.
  3. Test mutants for NK cell function. For an example of an in vivo NK cell cytotoxicity protocol, see the In Vivo NK Cell and CD8+ Cell Cytotoxicity ScreenIn vitro methods are listed in the In Vitro NK Cell Cytotoxicity Protocols.
Alleles Identified