Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Study design
• Procedure: TMEV infection
• Procedure: Health assessment (body weight and clinical score)
• Procedure: Open field test
• Procedure: Gait analysis
• Procedure: Accelerating rotarod
• Procedure: Tissue collection, RNA extraction, and TMEV quantification
• Data

Workflow and sampling

	Procedure	Equipment	Data collected
	Mice anesthetized and infected with TMEV at 4 wks of age	-	-
	Body weight and health assessment (day 0 to day 87 post infection)	Scales	Body weights and clinical scores
	Open field test (3 wks post infection)	Open field arena	Locomotor activity
	Gait analysis (27 days post infection)	DigiGate	Many gait parameters
	Gait analysis (7-13 wks post infection)	Foot print analysis	Stride length, stride width, overall gait
	Rotarod (7 wks post infection) over 9 trials	Accelerating rotarod	Latency to fall
	Euthanasia and tissue collection	Dissecting kit	-
	RNA extraction and TMEV quantification (7 and 28 days post infection)	PCR machine	TMEV levels

Equipment and supplies

• Syringes and needles
• Scales
• DigiGate (Mouse Specifics, Boston MA)
• Foot print analysis using ImageJ software (NIH)
• Open field arena: Plexiglas arena 43 x 43 x 43 cm equipped with an activity monitoring system (Tru Scan, Coulbourn Instruments, Holliston MA)
• Accelerating rotarod (5 cm diameter) (Ugo Basile, Model 7650, Varese ITALY)
• -80°C freezer
• Nucleic acid purification: Maxwell 16 automated instrument (Promega, Sunnyvale CA)
• Quantification of RNA: Cytation 5 Imaging Multi-Mode Reader (BioTek)

Reagents and solutions

• Isoflurane (MWI, Meridian ID)
• Beuthanasia (Schering-Plough Animal Health)
• Theiler's murine encephalomyelitis virus (TMEV) BeAn strain (American Type Culture Collection VR995, Manasses VA)
• Phosphate buffered saline
• LEV simplyRNA Purification Kit (Promega, Sunnyvale CA)
• Reverse transcription: SuperScript II (Life Technologies, Grand Island NY)
• Power SYBR Green PCR Master Mix (Life Technologies)
• Primers (see Brinkmeyer-Langford et al., 2017 for sequences)

Procedure: Study design

1. Two groups of mice were evaluated:
   • Group A mice were monitored for 3 months post infection to evaluate long-term phenotypic differences in TMEV infection. Group A mice were evaluated for body weight, clinical score, open field test, rotarod, and foot print (gait) analysis.
   • Group B mice were monitored to identify phenotypic differences related to the acute phase of TMEV infection. Group B mice were evaluated for body weight, clinical score, gait analysis (via DigiGate), and TMEV quantification.

Procedure: TMEV infection

1. At 4 wks of age, mice are anesthetized by isoflurane inhalation.
2. Mice are injected intracerebrally with 5.0 x 10⁴ plaque-forming units (PFU) of TMEV in 20 µL of PBS placed into the right mid-parietal cortex at a depth of approximately 1.5 mm.

Procedure: Health assessment (body weight and clinical score)

1. Mice from both Groups are weighed daily for at least 7 days post infection, and at least once a week thereafter.
2. Mice are assessed daily for clinical signs of neurological disease using a scoring system based on established markers for evaluating TMEV disease, including ruffled fur, hunched posture, grooming, and limb weakness. The severity of each marker is scored on a scale of 1 to 6, with 0 being normal and 6 being moribund. An overall score is recorded for each day. Grooming is measured by recording videos of individual mice in an open field arena for 10 min.

Procedure: Open field test

1. Individual mice from Group A are placed in the arena.
2. Under ambient room light, mice are measured for horizontal and vertical movement for 30 min.

Procedure: Gait analysis

1. Mice in Groups A and B are evaluated separately:
   • Foot print analysis (Group A): Fore and rear paws of mice are painted red and blue, respectively, with non toxic paint. Mice are then allowed to walk along a strip of white paper inside a defined walkway approximately 7 x 90 cm with walls 10 cm on each side. Resulting footprints are scanned electronically and distances between prints are measured using ImageJ. Stride lengths and widths for both forelimbs and hind limbs are measured for a minimum of 6 sequential strides. These assessments are performed weekly for 7 consecutive wks starting at 7 wks post infection.
   • DigiGate (Group B): Measurements are taken at 27 days post infection. Mice first undergo an initial training period, after-which mice are recorded walking at a speed of 19 cm/s for a minimum of 6 consecutive strides. Many gait parameters are automatically assessed.

Procedure: Accelerating rotarod

1. Each mouse from Group A is placed on a rod rotating initially at 4 rpm.
2. At 30 s intervals, the rotation is increased by 4 rpm for a total of 300 s per trial. Latency to fall is recorded for 9 trials (15 min break between trials).

Procedure: Tissue collection, RNA extraction, and TMEV quantification

1. Group B mice are euthanized either 7 or 28 days post infection by intraperitoneal injection of a lethal dose of Beuthanasia special (150 mg/kg).
2. One cerebral hemisphere is snap-frozen on dry ice and then stored at -80°C.
3. Total RNA is extracted using the Maxwell 16 automated instrument with LEV simplyRNA Purification Kit. Quantification and quality checking are performed using a Cytation 5 Imaging Multi-Mode Reader.
4. Total RNA (500 ng) is reverse transcribed to cDNA in 8 µL reactions and then diluted 1:5 in nuclease-free water.
5. qPCR reactions are performed in duplicate using SYBR Green. Each 20 µL reaction contains 10 µL Power SYBR Green PCR Master Mix, 0.6 µL each forward and reverse primers (10 µM), 6.8 µL ultra-pure water, and 2.0 µL cDNA. (Primers designed using Primer 3 software; primer sequences available in Brinkmeyer-Langford et al., 2017.)

Data collected by investigator

• Body weights
• Clinical scores
• Locomotor activity parameters
• Gait analysis parameters
  
• Accelerating rotarod latency to fall
• TMEV quantification

Data for mock infected mice are available in the supplemental data file download.