Molecular characterization of the FCoV-like canine coronavirus HLJ-071 in China

Background According to the differences of antigen and genetic composition, canine coronavirus (CCoV) consists of two genotypes, CCoV-I and CCoV-II. Since 2004, CCoVs with point mutations or deletions of NSPs are contributing to the changes in tropism and virulence in dogs. Results In this study, we isolated a CCoV, designated HLJ-071, from a dead 5-week-old female Welsh Corgi with severe diarrhea and vomit. Sequence analysis suggested that HLJ-071 bearing a complete ORF3abc compared with classic CCoV isolates (1-71, K378 and S378). In addition, a variable region was located between S gene and ORF 3a gene, in which a deletion with 104 nts for HLJ-071 when compared with classic CCoV strains 1-71, S378 and K378. Phylogenetic analysis based on the S gene and complete sequences showed that HLJ-071 was closely related to FCoV II. Recombination analysis suggested that HLJ-071 originated from the recombination of FCoV 79-1683, FCoV DF2 and CCoV A76. Finally, according to cell tropism experiments, it suggested that HLJ-071 could replicate in canine macrophages/monocytes cells. Conclusion The present study involved the isolation and genetic characterization of a variant CCoV strain and spike protein and ORF3abc of CCoV might play a key role in viral tropism, which could affect the replication in monocyte/macrophage cells. It will provide essential information for further understanding the evolution in China. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-021-03073-8.

Currently, coronaviruses can be divided into 4 subfamily, named Alpha, Beta, Gamma and Delta [6]. Canine coronavirus is a member of alpha subfamily of coronavirus, which based on the spike protein gene was divided into two distinct genotypes, CCoV I and CCoV II [7,8], both of which were distributed widely [9][10][11][12][13][14]. There were two different subtypes, CCoV IIa and CCoV IIb, have been found in dogs. CCoV IIa was served as the classic CCoV strains, which caused mild enteritis in young dogs [15]. CCoV IIb was emerged because of the homologous recombination between the transmissible gastroenteritis virus of swine (TGEV) and CCoV IIa strains [16], which caused the acute gastroenteritis and the virus was detected in the guts and internal organs [16]. Intermediate viruses, CCoV-A76 possessing a distinct spike with pathogenicity, which is the result of a recombination between CCoV I and CCoV II, had been detected [17].
Since 2004, more virulent CCoV strains with systemic disease have been reported without obvious coinfections Open Access *Correspondence: congfeng521@126.com; 397030703@qq.com; liang679@163.com [18][19][20][21]. Among of these infections, CB/05 was identified as a pantropic, highly pathogenic variant of CCoV type II which could be detected in the internal organs and caused both enteric and systemic signs [21,22]. In addition, HLJ-073 causing gross multiple organ lesions and diarrhea had been isolated and identified as a pantropic strain in our laboratory. Sequence analysis suggested that the strain bearing a 350-nt deletion of ORF3abc and originated from the recombination of FCoV 79-1683 and CCoV A76. Cell tropism experiments suggested that HLJ-073 could effectively replicate in canine macrophages/ monocytes and human THP-1 cells [21]. Therefore, it needs to concern the switch mechanism of cell tropism for CCoV.
In this study, the isolation of a novel pantropic CCoV strain, HLJ-071, is reported in China, which genetically related to the prototype HLJ-073. To better understand the genetic characterization of a variant CCoV strain, the analysis of complete genome sequences, phylogenetic tree and cell tropism was obtained. It will provide essential information for further understanding the evolution in China.

Clinical case
During the summer of 2015, a dead 5-week-old female Welsh Corgi with severe diarrhea and vomit was submitted for laboratory investigation. Necropsy of the dog showed hemorrhagic enteritis and lung on their surfaces. At post-mortem examination, samples were from intestine, brain, lungs, spleen, liver, kidneys, heart and mesenteric lymph nodes for real-time RT-PCR investigation of CCoV RNA [23].
Crandell feline kidney (CrFK) cells were grown in D-MEM supplemented with 10% foetal calf serum (FCS). The fecal sample was homogenized in phosphate-buffered saline (PBS) and centrifuged at 3,000 g for 15 min. Then the supernatant was filtered through a 0.22-μm-pore-size filter and inoculated into CrFK cells, which were confluent into the monolayers. When the sample was passaged three times, cytopathic effects (CPE) was observed. Then, after three rounds of purification by plaque assay [25], the purified virus was titrated and harvested by one cycle of freezing and thawing, and aliquots were stored at -80°C.

Electron microscopy
The electron microscopy protocol for negative-stain and thin-section examination was described previously [26,27].

Isolation and culture of the canine blood monocytes
Canine blood monocytes were isolated following the previous described [28]. Briefly, canine blood monocytes were isolated from 5 five specific-pathogen-free (SPF) dogs. The blood mononuclear cells were purified on Histopaque-1077 (Sigma-Aldrich), and then seeded in a 24-well dish and cultured at 37 °C with 5% CO 2 . After 24 h, nonadherent cells were removed and washed twice with PBS buffer.

Growth curve and titrations of HLJ-071
The CrFK cells were infected at multiplicity of infection (MOI) of 0.1. After 1 h of adsorption at 37 °C, cells were washed twice with the PBS buffer and incubated at 37 °C with 5% CO 2 . To determine the growth kinetics of the virus in canine blood monocytes, cells were infected at an MOI of 1, and after 1 h of adsorption at 37 °C, monocytes were gently washed three times with PBS medium to remove residual virus and incubated at 37 °C with 5% CO 2 [28]. The titers are given as the means from triplicate experiments (log 10 TCID 50 /ml); error bars represent standard deviations.

Indirect immunofluorescence assay (IFA)
The IFA was conductive by a standard procedure. Briefly, mononuclear cells were inoculated with CCoV HLJ-071 isolates at a MOI of 1 for 48 h and removed the supernatant medium, after washing with PBS, the infected cells were fixed with paraformaldehyde (4%) for 30 min. After blocking with 2% BSA for 2 h at 37 °C, the cells were incubated with N protein polyclonal antibodies (1:400) for 1 h at 37 °C, then the fluorescein isothiocyanate-conjugated goat anti-mouse antibody against immunoglobulin G (1:1000; Abcam, British). The CCoV-N polyclonal antibody was prepared in our laboratory [21].

Genome sequencing and phylogeny analysis
Fourteen pairs of primers were designed based on the conserved regions of CCoV strain HLJ-073 [21]. The RNA extraction and cDNA synthesis were performed as previously [26].
Sequence data were assembled and analyzed using Clustal X software (1.83), Vector 10 and DNASTAR. Phylogenetic trees based on the complete sequences and the spike proteins were performed using Neighbor-joining (NJ) method with Kimura 2-parameter model in molecular evolutionary genetics analysis software (version 4.0). The support for the tree nodes was calculated with 1,000 replicates. Simplot 3.5.1 was conductive for evaluating the recombination events between the reference CCoV and FCoV strains. The HLJ-071 sequence obtained in this study was assembled and submitted to the GenBank database under accession number KY063616.

Viral isolation and identification
The results of Colloidal gold diagnostic reagent and PCR confirmed that the fecal sample was CCoV-positive; the sample was negtive for CPV, CAV, CDV and major canine bacterials (data not shown). All of the organs except the heart were found to be positive for CCoV (Table S2), indicating that this strain is a pantropic CCoV strain. After inoculation of CrFK cells with samples and three serial passages, one CCoV isolate designated that HLJ-071 was successfully obtained from the fecal samples and CPE were found in the CrFK cells at 3-5 days post-inoculation with rounding and the detachment of the cells into the medium (Fig. 1B). The titre of HLJ-071 was 10 7.5 TCID 50 / mL in CrFK cells. Electron microscopy observed that the virus displayed a circular shape with petal-shaped, which had diameters of about 150 nm. Ultra-thin sections of infected CrFK cells displayed typical virus particles in the cytoplasm (Fig. 1D).

Full-length nucleotide sequence and phylogenetic analysis
The complete genome sequence of HLJ-071 was assembled and comparative analysis with other canine coronavirus was performed. The full genome of HLJ-071 was amplified using the 14   Sequence analysis suggested that there was an entire ORF3abc with 1,149 nts when compared to classic CCoV strains 1-71 and K378. Interestingly, comparing with classic CCoV strains 1-71, S378 and K378, there is a variable region was located between S gene and ORF 3a gene in varient CCoV strains (HLJ-071, A76 and CB/05). For HLJ-071, there is a 104-nt deletion in that region (Fig. 2). A highly conserved core sequence transcription regulatory sequences (TRS) is 5'-CTA AAC -3' in CCoV. However, a unique mutant TRS, 5'-UUA AAC -3' , was presents in classic 1-71, K378, S378 and located between gene of S and ORF3a, comparing with HLJ-071, varients CCoV, FECV, FIPV and TGEV strains (Fig. 2), which may influence the stability of RNA transcription and expression of subgenome.
By phylogenetic analysis, HLJ-071 based on complete sequences show that divided into FCoVs cluster, closed with TN-449, HLJ-073 and A76. The complete spike protein did not cluster with either type I or type II CCoVs, and related to FCoV WSU 79-1683. In addition, analysis of S1 (receptor-binding) domian showed that it clustered closely with FCoV 79-1683 and HLJ-073, while S2 (fusion) domain clustered with CCoV IIb 174/06 and WSU79-1683 (Fig. 3). The occurrence of recombination of HLJ-071 between CCoV A76 and FCoV 79-1683 and FCoV DF2 was detected, which had led to a new genetype emergence of the FCoV-like CCoVs (Fig. 4).

Cell tropism of HLJ-071
Previous studies showed that the ORF3abc deletion of canine and feline coronavirus alter the cell tropism [21,28]. To further investigate the in vitro growth characteristics of HLJ-071, canine monocytes cells were inoculated with HLJ-071 and HLJ-073 at an MOI of 1. The results showed that HLJ-073 could efficiently replicate in canine monocytes, however, HLJ-071 could poorly replicate in that cells (Fig. 5). Furthmore, the titers of HLJ-071 and HLJ-073 were determined at 24 h p. i. in CrFK cell lines and canine monocytes cells. The results showed that growth characteristics of HLJ-071 was similar to HLJ-073 and reached the 10 7.5 TCID 50 /mL at 24 h p.i. in CrFK cell lines (Fig. 6A). However, the titers of HLJ-071 peaked at 2×10 1.6 TCID 50 /mL at 6 h p.i. significantly slower growth characteristics compared to titers of HLJ-073 reached at 10 3.5 TCID 50 /mL at 36 h p.i. (Fig. 6).

Discussion
Generally, CCoV mainly causes intestinal infections, resulting in the viral enteritis and diarrhea in dog polulation. In the last decades, an increasing number of pantropic strains had been reported with system infections, causing multiple organ damage [29]. However, the reason   The cell tropism of FCoVs, so far, has been well investigated. Previous studies indicated that the spike protein and ORF3abc played a crucial part in macrophae infection in FCoVs [28,30]. For feline infectious peritonitis viruses (FIPV), the virus could replicate in the monocyte/macrophage lineage cells and then disseminate that to the organs causing system infections [31]. On the country, feline enteric coronavirus (FECV) was primary replicated in enterocytes and could not replicate in the monocyte/macrophage lineage cells. For CCoV, a system infection with CCoV CB/05 with a partial detetion of ORF3b and a FCoV-like spike protein was found in 2005 [32]. We speculated that stain could replicate in monocyte/macrophage cells, although no available data in viral tropism experiment. Furthmore, we have isolated and reported a CCoV strain, HLJ-073 bearing a 350nt deletion in ORF3abc and a FCoV-like spike protein, could efficient replicate in canine monocyte/macrophage cells and human THP-1 cells [21]. In this study, HLJ-071 bearing entire ORF3abc and FCoV spike protein could weakly replicate in canine monocyte/macrophage cells comparing with HLJ-073, which was distinct with the cell tropism of FECV. These suggested that 1) both of HLJ-071 and HLJ-073 had a common feature, that were the acquissiton of macrophage tropism; 2) ORF3abc could enhance the replication in monocyte/macrophage cells, when compared with HLJ-071 and HLJ-073.
Previous studies indicated that 5' and 3' flanks of the TRSs of TGEV and MHV influenced transcription levels (accumulation) and protein expression [33,34]. Besides, the regions flanking the TRS also have a profound impact on the production of subgenomic RNA [35,36]. In this study, a mutant TRS was found in classic CCoVs 1-71, S378, K378 and a variable region was discoved in varient CCoVs HLJ-071. Both of these suggested that 1) the transcription levels and protein expression of ORF3abc were affected, and 2) ORF3abc might be associated with an alternation in virus tropism. Further research was needed to investigate the relationship between transcription of ORF3abc or the pathogenicity of CCoV with these variable regions and mutant TRS.

Conclusion
In this present, we indicated that 1) CCoV HLJ-071 was closely related to FCoVs and recombinated with CCoVs and FCoVs, indicated that CCoVs underwent a rapid evolution in China. 2) The cell tropism of CCoVs may be correlated with the function ORF3abc and transcription of subgenome.
Additional file 1: Table S1. Primers used for identifying and completely sequencing the strains Additional file 2: Table S2. RNA copies (log 10 (CoV genome copies per 10 3 GAPDH copies)) of template in the samples of dead puppy, tested by specific real time RT-PCR