Comparative Examination of Feline Coronavirus and Canine Coronavirus Effects on Extracellular Vesicles Acquired from A-72 Canine Fibrosarcoma Cell Line

Abstract

Introduction: Coronavirus (CoV) is an extremely contagious, enveloped positive-single-stranded RNA virus, which has become a global pandemic that causes several illnesses in humans and animals. Hence, it is necessary to investigate viral-induced reactions across diverse hosts. Herein, we propose utilizing naturally secreted extracellular vesicles (EVs), mainly focusing on exosomes to examine virus-host responses following CoV infection. Exosomes are small membrane-bound vesicles originating from the endosomal pathway, which play a pivotal role in intracellular communication and physiological and pathological processes. We suggested that CoV could impact EV formation, content, and diverse immune responses in vitro. Methods: In this study, we infected A-72, which is a canine fibroblast cell line, with a feline coronavirus (FCoV) and canine coronavirus (CCoV) independently in an exosome-free media at 0.001 multiplicity of infection (MOI), with incubation periods of 48 and 72 h. The cell viability was significantly downregulated with increased incubation time following FCoV and CCoV infection, which was identified by performing the 3-(4,5-dimethylthiazo-1-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After the infection, EVs were isolated through ultracentrifugation, and the subsequent analysis involved quantifying and characterizing the purified EVs using various techniques. Results: NanoSight particle tracking analysis (NTA) verified that EV dimensions fell between 100 and 200 nm at both incubation periods. At both periods, total protein and RNA levels were significantly upregulated in A-72-derived EVs following FCoV and CCoV infections. However, total DNA levels were gradually upregulated with increased incubation time. Dot blot analysis indicated that the expression levels of ACE2, IL-1β, Flotillin-1, CD63, caspase-8, and Hsp90 were modified in A-72-derived EVs following both CoV infections. Conclusions: Our results indicated that FCoV and CCoV infections could modulate the EV production and content, which could play a role in the development of viral diseases. Investigating diverse animal CoV will provide in-depth insight into host exosome biology during CoV infection. Hence, our findings contribute to the comprehension and characterization of EVs in virus-host interactions during CoV infection.

Keywords: A-72 cells; canine coronavirus; exosomes; extracellular vesicles; feline coronavirus; immunomodulation; pathogenesis.

Figure 1

Canine fibrosarcoma cell line (A-72), cell viability following FCoV and CCoV infection. (A) Bright-field microscopy images demonstrated A-72 cellular morphology at 48 h and 72 h time intervals. (B) A-72 cells were infected independently with FCoV and CCoV in exo-depleted RMPI media at 0.001 MOI at 48 h and (C) 72 h. Following the incubations, A-72 cells were incubated again with MTT assay dye for 4 h (37 °C), and absorbance was detected at 570 nm. Statistical analysis was executed utilizing a one-way analysis of variance (ANOVA) with Tukey post hoc analysis for collected data values. Statistical significance is exhibited by the mean ± standard deviation (SD) as listed in p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).

Figure 2

Characterization of A-72-originated EVs after FCoV and CCoV infection. (A) NTA analysis illustrated mean particle size distribution; (B) particle concentration distribution following FCoV and CCoV infection at 48 h and 72 h time markers. Statistical analysis was executed utilizing a one-way ANOVA with Tukey post hoc analysis. Statistical significance is exhibited by the mean ± SD as listed in p ≤ 0.05 (*) and p ≤ 0.01 (**).

Figure 3

The biomolecular content of FCoV-exposed and CCoV-exposed lysates. (A) total DNA; (B) total RNA; and (C) total protein concentration of post-FCoV and CCoV infection lysates at 48 h and 72 h time intervals. Statistical analysis was executed utilizing a one-way ANOVA with Tukey post hoc analysis. Statistical significance is exhibited by the mean ± SD as listed in p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).

Figure 4

The biological relevance of FCoV-exposed and CCoV-exposed A-72-originated EVs. (A) total DNA; (B) total RNA; and (C) total protein concentration of A-72-derived EVs after FCoV and CCoV infection at 48 h and 72 h time intervals. Statistical analysis was executed utilizing a one-way ANOVA with Tukey post hoc analysis. Statistical significance is exhibited by the mean ± SD as listed in p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).

Figure 5

The presence of classical exosome protein and membrane trafficking protein following FCoV and CCoV infection. Graphs demonstrate the quantitative densitometry examination of dot blot analysis of (A) CD63 after 48 h and 72 h infections, (B) flotillin-1 after 48 h and 72 h infections in control, and FCoV-infected and CCoV-infected EVs isolated from A-72 cells. Statistical analysis was executed utilizing a one-way ANOVA with Tukey post hoc analysis. Statistical significance is exhibited by the mean ± SD as listed in p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).

Figure 6

The impact of FCoV and CCoV infections on host receptor protein and pro-inflammatory response. Graphs demonstrate the quantitative densitometry examination of dot blot analysis of (A) ACE2 after 48 h and 72 h infections, (B) IL-1β after 48 h and 72 h infections in control, and FCoV-infected and CCoV-infected EVs isolated from A-72 cells. Statistical analysis was executed utilizing a one-way ANOVA with Tukey post hoc analysis. Statistical significance is exhibited by the mean ± SD as listed in p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).

Figure 7

The impact of FCoV and CCoV infections on heat shock protein and caspase. Graphs demonstrate the quantitative densitometry examination of dot blot analysis of (A) Hsp90 after 48 h and 72 h infections, (B) caspase-8 after 48 h and 72 h infections in control, and FCoV-infected and CCoV-infected EVs isolated from A-72 cells. Statistical analysis was executed utilizing a one-way ANOVA with Tukey post hoc analysis. Statistical significance is exhibited by the mean ± SD as listed in p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).