Coronaviruses (CoVs) are animal and human pathogens that can cause respiratory and enteric diseases (V’Kovski et al. 2021). CoVs are divided into four genera termed Alpha-, Beta-, Gamma- and Delta-coronavirus based on phylogenetic analyses and genomic structures (Terada et al. 2019). Canine coronavirus (CCoV) is an enveloped, single-stranded positive-sense RNA virus belonging to the genus Alphacoronavirus and is considered as the main pathogen responsible for enteritis in dogs (Decaro and Buonavoglia 2008). CCoV genome is approximately 30 kb in length. Two large overlapping open reading frames (ORFs) are presented at the 5′-terminal genome region, namely, ORF1a and ORF1b, which encode two replicase polyproteins. A key enzyme required for both genome replication and transcription is RNA-dependent RNA polymerase (RdRp). Four kinds of structural proteins (including S, E, M and N) and several nonstructural proteins are encoded by ORFs downstream of the replicase gene (Decaro and Buonavoglia 2008; Ntafis et al. 2011). These structural proteins are essential for the assembly and infectivity of viral particle. S glycoprotein is associated with tropism, binding to cell-surface receptors, fusion and entry of the virus into cells (Timurkan et al. 2021). M and E proteins are involved in virus assembly. The nucleocapsid protein forms complexes with genomic RNA and plays a critical role in enhancing the efficiency of virus transcription, translation and assembly (Liu et al., 2021).
CCoV is divided into two different genotypes, namely, CCoV type I (CCoV I) and CCoV type II (CCoV II). The typical CCoV reference strain is CCoV II (Decaro and Buonavoglia 2008; Le Poder et al. 2013). According to amino acid sequence in the N-terminal region of S protein, CCoV II is further classified into two different subtypes, namely, CCoV IIa (typical strains) and CCoV IIb (TGEV-like strains). In the N-terminus of S protein of CCoV IIb, amino acid sequence is highly similar to that of infectious gastroenteritis virus (TGEV). The appearance of CCoV IIb may be due to the recombination of CCoV II and TGEV (Decaro et al. 2010; Licitra et al. 2014).
Virus isolation and cell culture adaptation are crucial for better understanding about biological characteristics, disease diagnosis and prevention of CCoV. However, CCoVs are not easily propagated in cell culture (Pratelli et al. 2000), and only a few CCoV strains are available. Genetic information concerning CCoVs detected in recent years is scarce. In this study, we successfully isolated two strains of CCoV IIa that had stable cellular adaptability and high viral titers.
Isolation of CCoV from clinical samples
From 2016 to 2019, CCoV isolations from clinical samples were conducted continuously in our laboratory. A total of 53 fecal samples from dogs infected with CCoV were collected in the animal hospital of Yangzhou University in China. From these dogs, CCoV was isolated from only two samples and named CCoV JS1706 and CCoV JS1712. Sample 1 was collected on 20 June 2017 from the feces of a 3-month-old male Labrador. Sample 2 was collected on 28 December 2017 from the feces of a 2-year-old male Welsh corgi. Neither dog had ever been vaccinated. Viral isolation was achieved at the 2nd to 3rd passage from the fecal sample. CPE (cell cytopathic effect) was characterized by cell rounding and lysis of the infected monolayer. Viral growth was confirmed by IFA (indirect immunofluorescence assay) using 2B8, a CCoV-specific monoclonal antibody. CCoV protein stained by the monoclonal antibody was distributed in cytoplasm but not nucleus. Viral titers (measured as TCID50: 50% tissue culture infective dose) of A-72 cells were 2 × 103 TCID50/ mL (CCoV JS1706) and 1 × 106 TCID50/ mL (CCoV JS1712) at the 4th passage. The two isolates were serially propagated in cell culture for more than 100 generations (data not shown). In this study, we verified that only a few strains were suitable for growth in vitro.
Two virus isolates showed different biological characteristics
Plaque morphology and size of the 4th generation CCoV JS1706 and CCoV JS1712 isolates are showed in Fig. 1. plaques of CCoV JS1706 strain measured 1–2 mm (Fig. 1a), while JS1712 strain produced larger uniform plaques of 3–4 mm in 3 days post infected A-72 cells (Fig. 1b). Previous studies have shown that animal coronaviruses with different plaque sizes may have different pathogenicity. For example, FIPV (feline infectious peritonitis virus) is macrophage tropic and is believed to cause aberrant cytokine and/or chemokine expression and lymphocyte depletion, resulting in lethal disease. Small plaques (1 mm) of FIPV are more virulent than the virus generated large plaques (3 mm) (Mochizuki et al. 1997). Whether the pathogenicity of CCoV JS1706 and CCoV JS1712 strains differs in vivo requires further study.
After CCoV JS1706 (12th passage of virus) and CCoV JS1712 (5th passage of virus) were fully adapted to cells, virus growth kinetics were evaluated using a standard infection time course. After 24 h, partial cell round-reduced CPE was observed. After 48 h of inoculation, the cytopathic rate was 80–90%, and after 72 h of inoculation, most cells detached from flask surface. CPE induced by the two strains did not significantly differed (Fig. 2). TCID50 determination showed that they both replicated in A-72 cells, reaching maximum titers of TCID50 > 106/mL in 24 h post infection (Fig. 3). This result demonstrated that infected A-72 cells were able to produce CCoV rapidly.
Electron microscopy of virus supernatant showed a large number of coronavirus like particles (Fig. 4a), with most 80–120 nm in diameter. In CCoV-infected cells, multiple inclusion bodies and mature coronavirus particles were found (Fig. 4b). Virus are released by exocytosis, the secretory pathway of cell, thereby infecting new host cell.
Two virus isolates are of the CCoV IIa subtype
A 758 bp product was amplified by using the primer pairs 20179 and NS-R-dg, which selectively recognized the CCoV IIa subtype. In addition, PCR assays with primers 20179 and 174–268 specifically for CCoV IIb subtype did not generate any amplicons. Therefore, CCoV JS1706 and CCoV JS1712 are CCoV IIa subtype viruses.
Sequencing and phylogenetic analysis of CCoV
Sequencing results showed that the full-length M gene of CCoV JS1706 and CCoV JS1712 was 792 bp in length, encoding 263 amino acids (GenBank accession number: MN078152 and MN078151). The full length of N gene was 1149 bp, encoding 382 amino acids (GenBank accession number: MN163040 and MN163039). RdRp protein was found to be 316 amino acids in length, similar to most CCoV reference strains. S gene of both strains was 4362 nucleotides in length, encoding a protein of 1453 amino acids.
Sequence comparison of RdRp of two isolates revealed 100% amino acid identity and high amino acid identity (96.5–100%) with that of Alphacoronavirus reference strains. Amino acid sequences of S protein of the them showed 99.6% homology and 84.0–95.2% identity with reference strains of CCoV IIa and FCoV II. The complete M and N proteins of two isolates revealed 99.2 and 100% amino acid identity, respectively. They showed 88.5–99.2% and 91.9–99.7% amino acid sequence identity with CCoV II reference strains. They had lower amino acid sequence identity (88.5–89.3%) of M protein and N protein (89.1–91.9%) with CCoV II vaccine strain INSAVC. Protective efficacy of the vaccine strain against Chinese epidemic strains requires further investigation.
To explore the evolution of CCoV, Four phylogenetic trees were reconstructed using RdRp, S, M and N protein (Fig. 5). Relationship of different CCoV strains was basically consistent with the results of amino acid homology analysis. In RdRp trees, various FCoV II, CCoV II and TGEV strains clustered together. Since RdRp domain is conserved across all groups, it will most likely reflect the true coronavirus phylogeny (Koçhan et al. 2021). Phylogenetic tree from the deduced amino acid sequence of S protein showed that two CCoV isolates of this research were most closely related to CCoV IIa but had a significant distance from FCoV I and CCoV I. Phylogenetic tree based on M and N proteins showed that they were closest to strain CCoV IIb NTU366/F/2008 (99.2 and 98.5%) which identified in Taiwan and CCoV IIb dog/HCM47/2015 (99.7%) which identified in Vietnam. These three strains clearly clustered into a single clade, suggesting that they may have a common ancestor. Cluster structures of M and N proteins were very similar but differed from those of S protein. In the phylogenetic tree constructed by partial S protein sequence, CCoV IIa and CCoV IIb formed two independent branches; therefore, phylogenetic analysis of S protein can provide more accurate and meaningful subtype classification.
In summary, two strains of CCoV IIa with different biological characteristics were isolated and stably propagated with high virus titers. These CCoV strains isolated in China are important tools for further studies on their pathogenesis, evolution and development of diagnostics and vaccines development.