Dirección

2.2.1. Viruses

CHPV strain I653514 (GenBank accession number KF468775) and temperature sensitive derivatives from it (Gadkari & Pringle, 1980) (GenBank accession numbers KF468772, KF468773, KF468774 for tdCE mutants CH112, CH157 and CH256 respectively) were obtained from Prof. A.J. Easton. Recombinant viruses CVeGFP (which contains the gene for enhanced green fluorescent protein inserted between the M and G genes of CHPV) and CVPRFP (in which the red fluorescent protein ORF mRFP1 (Campbell et al., 2002) was fused in-frame within a putative hinge region of the CHPV phosphoprotein, between residues 213 and 214 of P) were made by Dr A.C. Marriott.

2.2.2. Growth of CHPV stocks

BSC-1 cells were grown to confluence in glass roller bottles and infected with CHPV at a M.O.I. of 0.01 pfu/cell in complete GMEM with 2% FBS. Cells were incubated at 37°C and virus harvested when all cells exhibited CPE, approximately 24-48 hours after infection. Sterile glass beads were used to break open any remaining cells that had not lysed. Cell debris was removed by low speed centrifugation and aliquots of the virus stocks were stored at -70°C.

2.2.3. Determination of virus titre by plaque assay

Cells were grown to 90-100% confluence in 6 well plates. Serial, 10-fold dilutions of virus were made up to the 10-9 dilution and 200 µl of the virus dilutions (or mock) were used to inoculate each well, with each dilution assayed in duplicate. The cells were incubated with the inoculum at the required temperature for 1 hour. Following

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this, the inoculum was removed and 1% carboxymethyl cellulose (CMC)-GMEM (supplemented with 2% FBS, 0.2 mM L-glutamine and 100 µg/ml

penicillin/streptomycin) overlay was added to each well. Cells were then incubated at the required temperature. Once plaques were clearly visible cells were fixed using 4% gluteraldehyde in PBS for 2 hours at room temperature followed by staining with crystal violet for 15 minutes. The plates were then washed, dried and plaques

counted.

2.2.4. Microplaque assay

BSC-1 cells were grown to 90% confluence in 96 well tissue culture plates and infected in triplicate with 10-fold serial dilutions of wild-type CHPV for 1 hour at 37°C. Following this, the inoculum was replaced with complete GMEM with 2% FBS and incubated for 6 hours. The cells were then fixed with 1:1 acetone: methanol for 1 hour, washed once in TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) and blocked in 1% (w/v) BSA in TBS for 1 hour at room temperature. Cells were washed once in TBS before being incubated with 50 µl of polyclonal sheep anti- CHPV antibody (diluted 1 in 100 in 1% BSA (w/v) in TBS) for 1 hour at room temperature. The cells were washed three times in TBS prior to incubation with 50 µl of donkey anti-sheep biotin (Amersham LS) diluted at 1 in 400 in 1% BSA (w/v) in TBS for 1 hour. After three washing steps, 50 µl of alkaline phosphatase-

conjugated streptavidin at 1 in 3000 in 1% BSA (w/v) in TBS was added to the cells and incubated for 1 hour. The cells were washed three times in TBS and alkaline phosphatase activity was detected using the BCIP/NBT (5-bromo-4-chloro-3-indolyl- phosphate/nitro blue tetrazolium) detection system, according to the manufacturer’s instructions (Sigma).

61 2.2.5. Virus growth curves

BSC-1 or CE cells were grown to 90-100% confluence in 6 well plates and infected with the appropriate virus at an M.O.I. of 10 pfu/cell. The inoculum was incubated at 33°C for 1 hour, after which the cells were washed three times in PBS and 3 ml of complete medium with 2% FBS was added. Medium was removed at 0, 1, 2, 3, 4, 5, 6, 7, and 8 hours post infection and the virus titre at each time point deduced by plaque assay (section 2.2.3). The assays were performed in duplicate.

2.2.6. Semi-purifying virus

Virus was underlaid with a 20% sucrose cushion in PBS and centrifuged at 120,000 x g for 45 minutes in a Beckman Coulter optima L-90K ultracentrifuge, SW50.1 rotor at 4°C. The pellet was resuspended in PBS.

2.3. Mice

All animal experiments were approved by the University of Warwick’s Ethical Review Committee and the UK Home Office.

2.3.1. In vivo infection with CHPV

8 day old ICR (CD1) mice (bred in-house) of both sexes were used to investigate the neuropathogenesis of CHPV. Litters of 6 mice (housed with their mothers) were used in the study. Animals were kept at 19-23°C and 45-65% relative humidity. Mice were inoculated intraperitoneally with wild-type or recombinant CHPV at the required dose (or PBS as a control) in a volume of 50µl. After infection, mice were monitored, given a clinical score and each group of mice was weighed daily (or twice

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daily for titration experiments, see section 2.3.2). The quantitative clinical scoring system used in all experiments required individual mice to be scored as follows: 1 point for healthy, 2 points for displaying the first signs of clinical disease (abnormal gait), 3 points for unilateral hind limb paralysis (at the side adjacent to the site of inoculation), 4 points for paralysis in two limbs or showing little activity, 5 points for dead. The animals were monitored for a maximum of 10 days. Any animals

presenting with bilateral paralysis (score of 4) were immediately culled using a schedule 1 method. All surviving animals were culled using a schedule 1 method at the end of the 10 day observation period.

2.3.2. Titration of CHPV

In order to determine the dose of the strains of CHPV required to cause encephalitis groups of 3 animals were infected by the intraperitoneal route with various doses of CHPV, starting at a dose of 1.0 x 104 pfu (informed by an earlier study). If no disease was seen a further group of three mice was infected with 3.1 x 104 pfu of virus and observed. Conversely the dose was lowered if the disease onset was too rapid and a further group of three animals was tested. The process was repeated (increasing by half log increments or lowered appropriately) until a dose of virus was identified that caused observable disease in all three animals with bilateral paralysis seen on or near day three after infection. The animals were monitored twice daily from the time of infection for signs of disease and the weight of the group was measured daily.

2.3.3. Timecourse of infection with CHPV

Using the information from the procedure described in section 2.3.2. 5 groups of 6 mice were infected with a dose of wild type or mutant CHPV sufficient to cause

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signs of clinical disease (as described in section 2.3.1.) and one group of 6 mice was inoculated with PBS. All infected and control mice were observed at daily intervals for signs of disease and the weight of the animals in each group was recorded. At daily intervals 1 mouse from each experimental group was sacrificed using a

schedule 1 method. The process concluded when the remaining mice presented with bilateral paralysis or at ten days after infection, whichever was sooner, at which point all animals affected were sacrificed. The brains were removed from all of the

sacrificed animals and divided into two (down the midsagittal plane). One half of each brain was fixed in 4% paraformaldehyde in PBS for pathological examination and the other frozen at -70°C (for storage).

2.3.4. Homogenising tissue

Brain samples (section 2.3.3.) were thawed and homogenised in 500 µl PBS using a 21 gauge needle. Lungs, kidneys, liver samples were homogenized with sterile sand in a pestle and mortar in 1 ml PBS and centrifuged to remove debris before titrating (section 2.2.3.).

2.3.5. Sectioning of fresh brain tissue

Immediately after harvesting, brains were transferred in ice-cold ACSF (low sodium Artificial Cerebrospinal Fluid; 1mM calcium chloride, 10mM D-glucose, 4mM potassium chloride, 5mM magnesium chloride, 26mM sodium bicarbonate, 246mM sucrose, pH 7.3) previously bubbled with carbogen (95% oxygen, 5% CO2). 300 µM coronal sections or parasagittal cerebellar sections were then made using a

vibrotome. The sections were fixed in 4% paraformaldedyde in PBS overnight, in preparation for immunohistochemistry (section 2.8.3.).

64 2.3.6. Deparaffinisation and rehydration of paraffin-embedded sections

Paraffin-embedded brain sections were incubated in clearene solvent, a xylene substitute (Leica Biosystems) and washed in decreasing concentrations of ethanol (100, 95, 70, 50 and 30%).

2.3.7. Heat-induced antigen retrieval

Brain sections were microwaved (750 W) in sodium citrate buffer (0.1M Citric acid monohydrate, 0.1M Tri-sodium citrate) for 12 minutes and allowed to cool for 20 minutes. The sections were incubated with 20 µg/ml proteinase K solution at 37°C in a humid chamber for 15 minutes. Immunohistochemical staining was then performed (section 2.8.3).