Graham2 project protocol

SARS-CoV infection in females of 117 F1 hybrid lines of Collaborative Cross strains of mice (CC RIX)   (2021)

Graham JB, Baric RS, Lund JM
With: Swarts JL, Leist SR, Schäfer A, Menachery VD, Gralinski LE, Jeng S, Miller DR, Mooney MA, McWeeney SK, Ferris MT, Pardo-Manuel de Villena F, Heise MT

Project protocol - Contents


General Information for All Procedures: F1 hybrid mice (CC RIX) were derived from intercrossing Collaborative Cross strains at UNC. Six to eight week old CC RIX male mice were transferred to the University of Washington. Two weeks later, these mice were used in the baseline flow cytometry studies for T cell subsets (see Graham3 for these data). Concurrently CC RIX female mice were transferred internally at UNC to a BSL-3 facility for SARS-CoV infection (Graham2, this study).

Procedure 1: Strain selection criteria


Introductory Comments: F1 hybrid mice (CC RIX) were generated based on the following criteria.

  1. Each CC strain used in an F1 cross had to be certified distributable.
  2. The UNC Systems Genetics Core Facility had to be able to provide a sufficient number of breeding animals.
  3. Each CC RIX had to have one parent with an H2Bb haplotype from either C57BL/6J or 129S1/SvImJ, and one parent with a haplotype from one of the other six CC founder strains. This design was selected such that antigen-specific T cell responses could be examined.
  4. Each CC had to be used at least once (preferably twice) as a dam, and once (preferably twice) as a sire in the relevant CC RIX.
  5. Two CC RIX lines were included which appeared twice in the screen, once measured in the beginning and once towards the end of the five year period to specifically assess and control for batch and seasonal effects.

Procedure 2: Virus preparation, administration, and quantification from infected mice and clinical outcomes

Reagents and solutions

  • Vero cells
  • SARS-CoV MA15 (mouse adapted)


  1. Mouse adapted SARS-CoV MA15 is propagated and titered on Vero cells as previously described (Gralinski et al., 2015; Gralinski et al., 2018).
  2. Mice are intranasally infected with 5x103 PFU of SARS-CoV MA15. The virus inoculum dose is selected to result in a range of susceptibility phenotypes in the 8 CC founder strains. The SARS-CoV dose is chosen to maximize phenotypic diversity while still maintaining sufficient survival such that immune phenotypes can be assessed post-infection.
  3. Mice are measured daily for weight loss. Mice exhibiting extreme weight loss or signs of clinical disease are observed three times a day and euthanized if necessary based on humane endpoints.
  4. Mice are scored according to the following criteria: 0-5% weight loss, 5-15% weight loss, greater than 15% weight loss and death.
  5. For virus quantification, plaque assays are performed on lung (post-caval lobe) tissue homogenates as previously described (Gralinski et al., 2017). Viral titers are determined post-infection on days 2 and 4. CC RIX with an average lung viral load of less than 105 at day 2 post infection are considered to be "low titer", and CC RIX with load of greater than 107 are considered "high titer".

Investigator Notes: SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. Our study serves as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection.


Primary References

Graham JB, Swarts JL, Leist SR, Schäfer A, Menachery VD, Gralinski LE, Jeng S, Miller DR, Mooney MA, McWeeney SK, Ferris MT, Pardo-Manuel de Villena F, Heise MT, Baric RS, Lund JM. Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection in Collaborative Cross mice. PLoS Pathog. 2021 Jan;17(1):e1009287. doi: 10.1371/journal.ppat.1009287. Epub 2021 Jan 29.   PubMed 33513210     FullText

Protocol References

Gralinski LE, Ferris MT, Aylor DL, Whitmore AC, Green R, Frieman MB, Deming D, Menachery VD, Miller DR, Buus RJ, Bell TA, Churchill GA, Threadgill DW, Katze MG, McMillan L, Valdar W, Heise MT, Pardo-Manuel de Villena F, Baric RS. Genome Wide Identification of SARS-CoV Susceptibility Loci Using the Collaborative Cross. PLoS Genet. 2015 Oct;11(10):e1005504. doi: 10.1371/journal.pgen.1005504. Epub 2015 Oct 9.   PubMed 26452100     FullText

Gralinski LE, Menachery VD, Morgan AP, Totura AL, Beall A, Kocher J, Plante J, Harrison-Shostak DC, Schäfer A, Pardo-Manuel de Villena F, Ferris MT, Baric RS. Allelic Variation in the Toll-Like Receptor Adaptor Protein Ticam2 Contributes to SARS-Coronavirus Pathogenesis in Mice. G3 (Bethesda). 2017 Jun 7;7(6):1653-1663. doi: 10.1534/g3.117.041434.   PubMed 28592648     FullText

Gralinski LE, Sheahan TP, Morrison TE, Menachery VD, Jensen K, Leist SR, Whitmore A, Heise MT, Baric RS. Complement Activation Contributes to Severe Acute Respiratory Syndrome Coronavirus Pathogenesis. mBio. pii: e01753-18. doi: 10.1128/mBio.01753-18.   PubMed 30301856     FullText