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Canine pneumovirus (CnPnV) was recently isolated from the respiratory tracts of shelter dogs and shares sequence similarity with the rodent pathogen, pneumonia virus of mice (PVM). We show here that CnPnV replicates in and can elicit local proinflammatory cytokine production and neutrophil recruitment to lung tissue and the airways. In contrast to PVM J3666 infection, fatal CnPnV infections are observed only in response to high titer intranasal inocula (>67 TCID50 units). Sera from mice that recover from CnPnV infection contain antibodies that cross-react with PVM antigens; these mice are protected against lethal PVM infection. Given these findings, it will be intriguing to determine the relative role(s) of CnPnV and PVM in eliciting respiratory symptoms in susceptible canine species.
Canine pneumovirus (CnPnV) was identified by Renshaw et al. (2010) as part of a search for novel respiratory virus pathogens in dogs confined to animal shelters. The novel infectious agent was detected by a unique cytopathic effect induced in A72 fibroblast cultures, and was subsequently identified with pooled monoclonal antibodies against the human pneumovirus pathogen, respiratory syncytial virus (RSV). Sequence fragments of the virus L polymerase and N nucleoprotein genes initially suggested a close relationship between CnPnV and the murine pneumovirus pathogen, pneumonia virus of mice (PVM; Easton et al., 2006; Krempl et al., 2005). Recent publication of virus genome sequence indicates substantial homology with some distinct differences between CnPnV and the characterized PVM J3666 and 15 strains; these include an extended CnPnV G (attachment) protein amino-terminus and sequence divergence among the SH (small hydrophobic) genes (Renshaw et al. 2011).
PVM is a natural rodent pathogen (family Paramyxoviridae, genus Pneumovirus) that was first isolated and characterized by Horsfall and Curnen (1946). While PVM is among the viruses recognized as contaminant pathogens of research and commercial rodent colonies (Miyata et al., 1995; Zenner, Regnault, 2000), little is known regarding its prevalence among wild rodents. PVM has been used to model of severe respiratory virus infection in inbred strains of mice (Domachowske et al., 2000; Domachowske et al., 2002; Garvey et al., 2005; Anh et al., 2006; Gabryszewski et al. 2011; Rosenberg and Domachowske, 2008). In response to a minimal inoculum, the PVM J3666 strain undergoes robust replication and elicits profound granulocyte recruitment to the airways and lung tissue in response to soluble proinflammatory mediators; this condition can progress to pulmonary edema and acute respiratory failure, similar to the more severe forms of RSV experienced by hospitalized human infants (Tregoning, Schwarze, 2010; Hall et al., 2009). The PVM model of acute respiratory virus infection has been used for the in vivo exploration of both therapeutic and preventive antiviral strategies (Bonville et al., 2004; Gabryszewski et al., 2011).
Given its recognized similarity to the PVM pathogen, we explored the BALB/c mouse as a target host species for CnPnV infection in vivo.
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 TCID50 units / mL) was maintained by mouse passage. CnPnV from tissue culture supernatant from infected canine fibroblast A72 cells ((Renshaw et al., 2010; 3 × 106 TCID50 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 TCID50 units / mL.
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.
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, 106, 105, 104, 103 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, 105, 104, 103, 102 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.
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).
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.
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.
We demonstrate that CnPnV, a novel pneumovirus with sequence similarity to PVM, replicates in lung tissue and can cause a lethal infection in wild-type BALB/c mice. We evaluated survival in response to intranasal inoculation with CnPnV [Figure 1A]. All mice inoculated with 67 TCID50 units / 50 µL survived through day 30, as did 40% of the mice inoculated with 200 TCID50 units / 50 µL. All surviving mice seroconverted, documenting recovery from active infection. All mice inoculated with 400 TCID50 units / 50 µL died between days 7 and 14 (t1/2 = 8.7 days). In contrast, these mice are much more susceptible to the lethal sequelae of PVM J3666 infection [Figure 1B]. There were no survivors in response to intranasal inocula as low as 1.7, 0.5 or 0.17 TCID50 units / 50 µL (t1/2 = 5.9, 8.0, and 9.9 days, respectively). If one interpolates from the inverse correlation between TCID50 units administered vs. t1/2 for PVM J3666 infection, the survival (t1/2) of 8.7 days in response to 400 TCID50 units CnPnV is similar to the response one would anticipate from 0.4 TCID50 units of PVM J3666, or a difference of approximately 1000-fold.
Virus replication was assessed in response to a non-lethal inoculum (35 TCID50 units / 50 µL; [Figure 2]). CnPnV genome copies, measured on a log scale via a dual standard curve qRT-PCR assay targeting the virus SH gene (CnPnVSH/GAPDH), was detected in lung tissue as early as day 3 after initial challenge, with increases in virus recovery measured at all time points thereafter. No CnPnV was detected in uninfected mice, nor was CnPnV detected in lung tissue in mice challenged with an equivalent inoculum of heat-inactivated virions (data not shown).
At a survival inoculum (35 TCID50 units / 50 µL), CnPnV infection results in local production of proinflammatory mediators [Figure 3; Supplemental Figure 2], including CCL3 (MIP-1α), CCL2 (MCP-1), CXCL10 (IP10), CXCL1 (KC), and IFNγ, all cytokines associated with disease pathology in the PVM infection model (Gabryszewski et al., 2011; Bonville et al., 2006). Interestingly, despite the presence of these proinflammatory mediators, we observe only minimal histopathology in response to CnPnV at this inoculum [Figures 4A and 4B]. The neutrophils that are recruited are found primarily in focal perivascular infiltrates [Figure 3C and 3D] rather than dispersed throughout the parenchyma, as is observed in severe pneumovirus infection (Rosenberg, Domachowske, 2008); neutrophils can also be detected in bronchoalveolar lavage (BAL) fluid [Figure 4E] but not as prominently or in as substantial numbers as in the acute, fatal PVM J3666 infection (Bonville et al., 2006). It is intriguing to find limited neutrophil recruitment and dispersion in the presence of prominent proinflammatory cytokine expression. Interestingly, we have observed similar limited neutrophil recruitment despite relatively normal levels of proinflammatory cytokine expression in PVM-infected interferon alpha-beta receptor (IFNαβR−/−) mice (Garvey et al. 2005) and likewise in the absence of interferon-gamma-mediated signaling (Bonville et al. 2009). It will be intriguing to determine if there are any connections between these findings and what we have observed here.
Renshaw et al. (2010, 2011) reported high sequence similarity between CnPnV and PVM strains 15 and J3666. In Figure 5A, a Western blot with clarified lung-tissue homogenates from CnPnV-infected and naïve mice were probed with polyclonal anti-PVM N (nucleoprotein) antibody. A 43 kDa PVM N protein was detected clarified homogenates from CnPnV-infected mouse lungs; a protein of indistinguishable electrophoretic mobility was detected in the homogenate from PVM-infected mouse lungs [Supplemental Figure 2]. Virus was detected in epithelial cells lining the bronchioles in lungs of CnPnV infected mice [Figure 5B], similar to what was observed previously for mice infected with PVM strain J3666 (Bonville et al., Virology 2006). Similarly, we found that sera from CnPnV-inoculated mice that had recovered from infection (day 21) contain antibodies that cross-react with PVM antigens by ELISA [Figure 5C]; no cross-reacting antibodies were detected in control mice, nor were any cross-reacting antibodies detected in sera from mice that were inoculated with heat-inactivated CnPnV. Furthermore, mice that recovered from CnPnV infection were protected against subsequent lethal PVM J3666 infection; no protection is observed in mice previously inoculated with heat-inactivated, non-replicating CnPnV [Figure 5D]. Claassen et al. (2007) have explored the role of CD4+ and CD8+T cell PVM G protein protective epitopes in promoting immunity against PVM infection in vivo; Frey et al. (2008) likewise reported noted the contributions of both T cell subsets in promoting virus clearance. This is an intriguing and important field for continued study, as human pneumovirus infection (respiratory syncytial virus) is notorious for its inability to elicit long-term immunity in vivo. Models such as these will assist with the examination of long-lasting immunity to pneumovirus infection. The full nature of potential protective responses (or lack thereof) generated in response to acute infection can be determined via experiments such as those in Figure 5D, carried out in both in B, T cell and subset deficient, as well as various appropriate gene-deleted mouse models, with the second pneumovirus challenge delayed for weeks to months.
Of note, while heat-inactivated CnPnV is a foreign antigen that will likely irritate lung tissue and elicit a cytokine response if inoculated repeatedly at high concentration, it is not an effective immunogen when administered at this dosage via the intranasal route when compared to the active virus. We observed similar results when C57BL/6 mice that recovered from infection with attenuated PVM strain 15 were then challenged with PVM J3666 (Ellis et al., 2007)
Shown in Figure 5 is a phylogenetic tree featuring the SH sequences of human RSV, bovine RSV, two isolates of PVM (strain J3666 and strain 15) and CnPnV (Renshaw et al. 2011). CnPnV shares significant sequence homology with both strains of PVM, including 97% amino acid sequence homology shared with both F (fusion) proteins. In contrast, the SH (small hydrophobic) protein is the most divergent among the three viruses. SH is a pneumovirus structural protein found on the virus surface; recently Fuentes et al. (2007) found that the SH protein from RSV had a protective, immunomodulatory function, and could inhibit proinflammatory signaling by tumor necrosis factor (TNF). The CnPnV SH gene encodes a protein with 90 and 96% amino acid sequence identity to its counterparts in PVM strain J3666 (highly virulent, mouse passaged) and PVM strain 15 (less virulent, tissue culture maintained), although the highly virulent J3666 SH sequence is somewhat longer than that of either strain 15 or CnPnV (114 vs. 92 amino acids). Another point of significant divergence is the G protein; by inference to other pneumoviruses, this protein is essential for virus attachment (reviewed in Easton et al., 2006) although this has not been formally evaluated for PVM. CnPnV G protein is 92% identical to the G proteins from both PVM J3666 and PVM strain 15, but includes an amino terminal cytoplasmic extension that may have an impact on function and virulence.
Although pneumoviruses are species-limited pathogens, bovine and human RSV are similar to one another and maintain numerous cross-reactive epitopes (Pastey, Samal, 1997); likewise, antibodies cross-reactive to bovine RSV have been identified in numerous non-bovine species, including cats and dogs (van der Poel, et al., 1995). As such, given the findings presented here, it will ultimately be important to determine the extent to which this emerging mammalian pneumovirus pathogen can replicate and elicit respiratory symptoms in the canine population. Likewise, it will be intriguing to determine whether or not the previously-identified PVM pathogens can do likewise.
In conclusion, we have shown that CnPnV, a novel pneumovirus isolated from domesticated dogs, replicates effectively in mouse lung tissue and can lead severe respiratory sequelae, although mice can survive in response to a 1000-fold increased virus inoculum compared to PVM J3666. Virus replication leads to cytokine production but only minimal recruitment of neutrophils; as such, this virus strain may ultimately provide important insights into which proinflammatory mediators are truly crucial, and likewise, which mediators are supportive or redundant, for progression to irreversible inflammatory pathology.
The authors thank Ricardo Dreyfuss (NIAID, NIH) for preparation of the photomicrographs. Work presented in this manuscript is supported by funds from the NIAID Division of Intramural Research Project Z01-AI000943 (HFR), Children's Miracle Network of Central New York (JBD), and discretionary funds of the Animal Health Diagnostic Center (EJD, RWR).
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