Ferris1 project protocol

Host response to Influenza A (H1N1) in 8 inbred founder strains and 150+ emerging lines (pre-CC) of the Collaborative Cross   (2012)

Ferris MT, Bottomly D, McWeeney SK, Heise MT
With: Aicher LD, Rosenzweig E, Whitmore A, Aylor DL, Haagmans BL, Gralinski LE, Bradel-Tretheway BG, Bryan JT, Threadgill DW, de Villena FP, Baric RS, Katze MG

Ferris1 Protocol

Project protocol - Contents

Workflow and sampling

Data collected *
H1N1 viral preparation
Mice anesthetized and infected
3 Mice monitored daily Scales Morbidity (weight loss), mortality, clinical disease score
4 Mice euthanized and assessed for gross pathology - Lung hemorrhage score and edema score
5 TCID50 assay Incubator Lung viral titer
6 Histopathological analysis Microscope Extent and severity of immune cell infiltration and pathological damage (scores)

* Additional are data available, including data for mock-treated mice and males Ferris1_suppdata.xlxs

Equipment and supplies

  • Scales
  • Syringes and needles
  • 96-well plates
  • Tissue-culture incubator
  • Microscope

Reagents and solutions

  • Dulbecco's modified Eagle's medium (DMEM) (10% FBS and 1% Penicillin-Streptomycin)
  • Canine kidney (MDCK) cells
  • Isoflurane (Piramal, Bethlehem PA)
  • Phosphate-buffered saline (PBS)
  • Crystal Violet solution (1%)
  • Formalin (10% buffered; Fischer)
  • Paraffin
  • Protease (0.1%)
  • Hydrogen peroxide (3%)
  • Tween 20
  • Mouse anti-influenza virus nucleoprotein (clone Hb65, ATCC)
  • Horseradish peroxidase labeled goat anti-mouse IgG2a
  • 3-amino-9-ethylcarbozole (AEC, Sigma)
  • Hematoxylin and eosin

Procedure: H1N1 preparation

    1. Influenza A strain A/PR/8/34 (H1N1) stocks are made by infection of 10-day embryonated chicken eggs.
    2. Canine kidney (MDCK) cells are grown in high-glucose DMEM and used for titering H1N1 by plaque assay.

Procedure: H1N1 infection and monitoring mice

    1. Mice are lightly anesthetized via inhalation with Isoflurane.
    2. Mice are infected intranasally with 5x102 pfu of H1N1 in 50 µL of PBS.
    3. Mice are monitored daily for morbidity (weight loss) and given clinical disease scores.
    4. At day 4 post-infection, mice are euthanized via Isoflurane overdose.
    5. Mice are assessed for gross pathology (lung hemorrhage and edema) and lung tissue taken for other assays (below).

Procedure: Tissue Culture Infectious Dose (TCID50) assay

    1. A lung homogenate is prepared in DMEM.
    2. MDCK cells are seeded into 96-well plates at a density of 1.5x105 cells/well in DMEM and incubated at 37°C overnight.
    3. Cells are washed 2 times with PBS, and 100 µL of DMEM is added to each well.
    4. DMEM is removed from all wells in the first column of the plate and 146 µL of lung homogenate (in DMEM) is added to these wells (each biological sample is added to 4 wells).
    5. Serial dilutions of 46 µL (0.5 log dilutions) are carried out across the plate.
    6. Plates are incubated at 37°C for 1 h.
    7. Inoculum is removed, and 150 µL of serum-free DMEM with 1 µg/mL trypsin is added to each well.
    8. Plates are incubated at 37°C for 3 days.
    9. Media is removed and cells stained with Crystal Violet solution.
    10. Stain is washed away with water.
    11. Titer is determined (as defined below under Definitions).

Procedure: Lung histopathological analysis

    1. Right lung is removed and submerged in 10% buffered formalin without inflation for 1 wk.
    2. Two 5-micron thick Hematoxylin and Eosin stained lung sections (separated by 100 microns) are prepared and scored for a variety of metrics relating to the extent and severity of immune cell infiltration and pathological damage [slides were prepared and blind scored by the UNC Linberger Comprehensive Cancer Center Histopathology Core].

Definitions and calculations

  • TCID50: Log10 TCID100 = 2 x Log10TCID50 = 2 x (Xp + (0.5 x D) - (D x Sp)), where Xp is the last dilution where all replicates of a given sample are positive, D is the serial dilution log, and Sp is the sum of the proportion of replicates at all dilutions where positives are seen (starting with the Xp dilution)

Data collected by investigator

  • Body weight *
  • Clinical scores
  • Viral replication log titers
  • Virus-induced inflammation and damage to airways, vascular regions, and alveoli (scores) *
  • Immunohistochemical (IHC) analysis of viral replication (scores) *

* Additional data are available, including data for mock-treated mice and males Ferris1_suppdata.xlxs.

Susceptibility index was computed by MPD based on the summation of relative weight loss and clinical score at day 4 post-infection.