Quantification of mRNA encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (CRCoV)

Abstract

One of the first lines of defence against viral infection is the innate immune response and the induction of antiviral type I interferons (IFNs). However some viruses, including the group 2 coronaviruses, have evolved mechanisms to overcome or circumvent the host antiviral response. Canine respiratory coronavirus (CRCoV) has previously been shown to have a widespread international presence and has been implicated in outbreaks of canine infectious respiratory disease (CIRD). This study aimed to quantify pro-inflammatory cytokine mRNAs following infection of canine air-interface tracheal cultures with CRCoV. Within this system, immunohistochemistry identified ciliated epithelial and goblet cells as positive for CRCoV, identical to naturally infected cases, thus the data obtained would be fully transferable to the situation in vivo. An assay of ciliary function was used to assess potential effects of CRCoV on the mucociliary system. CRCoV was shown to reduce the mRNA levels of the pro-inflammatory cytokines TNF-alpha and IL-6 and the chemokine IL-8 during the 72 h post-inoculation. The mechanism for this is unknown, however the suppression of a key antiviral strategy during a period of physiologic and immunological stress, such as on entry to a kennel, could potentially predispose a dog to further pathogenic challenge and the development of respiratory disease.

Fig. 1

Mean log₁₀ fold change in TNF-α mRNA copies in CRCoV and LPS-inoculated, relative to control-inoculated, tracheal cultures at 24, 48, 72 and 96 h post-inoculation. Error bars show standard error of the mean. No data was available for LPS at 96 h.

Fig. 2

Mean log₁₀ fold change in IL-6 mRNA copies in CRCoV and LPS-inoculated, relative to control-inoculated tracheal cultures at 24, 48, 72 and 96 h post-inoculation. Error bars show standard error of the mean. No data was available for LPS at 96 h.

Fig. 3

Mean log₁₀ fold change in IL-8 mRNA copies in CRCoV and LPS-inoculated, relative to control-inoculated tracheal cultures at 24, 48, 72 and 96 h post-inoculation. Error bars show standard error of the mean. No data was available for LPS at 96 h.

Fig. 4

Mean CRCoV nucleocapsid gene RNA copies (ng cDNA⁻¹) in canine tracheal cultures at 24, 48, 72 and 96 h post-inoculation with CRCoV. Error bars show standard error of the mean.

Fig. 5

Tracheal culture latex clearance scores after 30 min at 24 h intervals, given as a percentage of the maximal clearance of each tracheal piece recorded pre-inoculation (0 h) and recorded over 96 h. Error bars show standard error of the mean.

Fig. 6

Immunohistochemical staining of CRCoV-inoculated canine tracheal cultures (A–C) and naturally infected canine trachea, positive for CRCoV by RT-PCR (D). (A–D) Trachea—coronaviral-antigen positive epithelial cells and corresponding surface/ciliary layer antigen accumulation (arrows). Chromogen, Vector VIP; counterstain, methyl green.