Eight to ten week old BALB/c mice (Division of Cancer Therapeutics, National Cancer Institute, MD) were used in all experiments. Mice received intranasal inoculations (50 µL) while under light anaesthesia (20% halothane in mineral oil) and were likewise anaesthetized prior to sacrifice via cervical dislocation. All protocols were evaluated and approved as per the National Institute of Allergy and Infectious Diseases Animal Study Protocol LAD-8E and carried out in accordance with the Institute’s Animal Care and Use Committee Guidelines.
PVM strain J3666 (105
units / mL) was maintained by mouse passage. CnPnV from tissue culture supernatant from infected canine fibroblast A72 cells ((Renshaw et al., 2010
; 3 × 106
units / mL) was used for initial intranasal inoculation of BALB/c mice to prepare mouse-passaged stocks. Virus replication was detected in lung tissue from mice inoculated with unmanipulated tissue culture supernatant; no replication was detected in mice receiving heat-inactivated tissue culture supernatant, i.e., three serial freeze (dry-ice) and heat (95°C) cycles, a process previously shown to inactivate PVM (Gabryszewski et al., 2011
). Stocks of mouse-passaged CnPnV were generated by blade homogenization of excised mouse lung tissue in complete tissue culture medium (Iscove’s Modified Dulbecco’s medium with 10% fetal calf serum) followed by centrifugation and storage of the virion-containing clarified supernatant in liquid nitrogen. The titer of mouse-passaged CnPnV stock, assayed as described below, was 4 × 105
units / mL.
Virus quantification by TCID50 analysis
TCID50 analysis was used to evaluate PVM J3666 as well as CnPnV. Briefly, A-72 (canine tumor fibroblast ATCC CRL1542) maintained in standard culture, were plated at 1.6 × 105 cells/mL, 0.2 mL / well in 2% FBS, and incubated overnight at 37°C, 5%CO2. Lung homogenates from PVM J3666 or CnPnV-infected mice were dialyzed (50K MWCO tubing) against DMEM to remove cell-activating and cytotoxic mediators. Serial 10-fold dilutions were added to cells, which were then incubated at 37°C; cytopathic effect (CPE), which including rounding and piling of cells, was recorded at day 3 after inoculation.
Virus detection by quantitative PCR
We have developed a dual standard curve method for determining virus recovery in absolute copies from infected mouse lung tissue (PVMSH
/GAPDH; Percopo et al., 2009
; Gabryszewski et al., 2011
). This assay has been altered to target the sequence of the CnPnV SH
gene, including cpv-F-primer 5’-GCT GTT ATC AAC ACA GTG TGT G-3’, cpv-R-primer 5’-GCC TGA TGT AGC AAT GCT C-3’and probe: 6FAM-CGC TGA TAA TGG CCT GCA GCA-TAMRA. The GAPDH standard curve includes serial dilutions (107
molecules / reaction) of mouse GAPDH coding sequence in pCMV Sport 6 (American Type Culture Collection cat no. 10539385). The CnPnV SH
standard curve includes serial dilutions (106,
molecules per reaction) of the full-length CnPnV SH
gene cloned into pCR 2.1. Experimental triplicate data points are interpolated to linear standard curves over the concentration ranges indicated.
Preparation of anti-PVM N protein polyclonal antibody and Western blot
Recombinant glutathione-S-transferase-PVM N fusion protein was generated using the vector pGEX-5X-3. GST-PVM-N protein was purified on glutathione agarose and used to inoculate rabbits via a standard protocol with Freund’s complete and incomplete adjuvants (Spring Valley Laboratories, Sykesville, MD). The antibody fraction was purified by ammonium sulfate precipitation followed by affinity-isolation on protein A agarose and stored at 1.2 mg/mL in phosphate buffered saline (PBS) with 0.1% BSA. Clarified homogenates from infected mouse lung tissue were diluted 1:1 with reducing sample buffer (Invitrogen), heated to 65°C and run on 14% trisglycine acrylamide gels, blotted to nitrocellulose and blocked with 5% non-fat dry milk in tris-buffered saline with 0.1% Tween (T-TBS). The anti-PVM N primary antibody was used at a concentration of 12 µg / mL (1:100) in T-TBS with 1% gelatin. Secondary antibody was a 1:1000 dilution of alkaline-phosphatase conjugated goat anti-rabbit IgG, followed by NBT/BCIP developing reagents (BioRad, Richmond, CA). Rabbit anti-beta actin was purchased from Cell Signaling (Millipore Corporation) and used at a 1:500 dilution.
Anti-PVM antibodies in mouse sera were detected by ELISA (Biotech Trading Partners, catalog #SMART-M12, El Cerrito, CA).
Detection of Proinflammatory Cytokines
Lungs from control (uninfected) mice, mice challenged with heat-inactivated CnPnV and mice infected with actively-replicating CnPnV were blade homogenized into 1 mL IMDM with 10% heat-inactivated fetal calf serum and the supernatants clarified by centrifugation. Clarified homogenates were evaluated by ELISA (R&D Systems, Minneapolis, MN) and values obtained were normalized for total protein (BCA assay, Pierce, Rockford, IL). Values were controlled for matrix effects by diluting 5 µL of various concentrations of standards in 45 µL of buffer provided or 45 µL of lung tissue from an uninfected mouse, blade homogenized and clarified as above.
Lung histopathology and immunohistochemical detection of virus antigens
Prior to excision from the chest cavity, excess blood was eliminated by perfusion via the right ventricle, and the lungs were inflated trans-tracheally with 10% phosphate buffered formalin. The heart and lungs were excised, fixed in 10% phosphate buffered formalin, and mounted in butterfly formation. Hematoxylin and eosin stained tissue was prepared by Histoserv, Inc. (Germantown, MD). Tissue sections were also probed with purified anti-PVM N antibody (1:200 dilution) or control rabbit IgG followed by peroxidase-conjugated anti-Ig and developing reagents for immunohistochemical localization of virus antigen (Histoserv).
All experiments were repeated at least twice with number of mice (n) as indicated. Data were evaluated by Mann-Whitney U test (non-parametric, for small samples) and Kruskal–Wallis one-way analysis of variance for multiple samples.