Molecular characterization of HLJ-073, a recombinant canine coronavirus strain from China with an ORF3abc deletion

Abstract

Canine enteric coronaviruses (CCoVs) are important enteric pathogens of dogs. CCoVs with different variations are typically pantropic and pathogenic in dogs. In this study, we isolated a CCoV, designated HLJ-073, from a dead 6-week-old male Pekingese with gross lesions and diarrhea. Interestingly, sequence analysis suggested that HLJ-073 contained a 350-nt deletion in ORF3abc compared with reference CCoV isolates, resulting in the loss of portions of ORF3a and ORF3c and the complete loss of ORF3b. Phylogenetic analysis based on the S gene showed that HLJ-073 was more closely related to members of the FCoV II cluster than to members of the CCoV I or CCoV II cluster. Furthermore, recombination analysis suggested that HLJ-073 originated from the recombination of FCoV 79-1683 and CCoV A76, which were both isolated in the United States. Cell tropism experiments suggested that HLJ-073 could effectively replicate in canine macrophages/monocytes and human THP-1 cells. This is the first report of the isolation of strain HLJ-073 in China, and this virus has biological characteristics that are different from those of other reported CCoVs.

Keywords: Canine coronavirus; ORF3abc; Pantropic; Recombination; Viral cell tropism.

Fig. 1

(A) Multiple sequence alignment of the ORF3abc region of CCoV strains (B) Schematic diagram of ORF3a, 3b and 3c of CCoVs. (C) Identification of mutant strains by RT-PCR

Fig. 2

(A-G) Phylogenetic analysis of the spike protein (S), S1, S1-NTD, S1-C-domain, S2, membrane (M) and envelope (E) genome regions of HLJ-073 and the most closely related strains in GenBank for which whole genome sequences were available. Neighbor-joining was used for the construction of the phylogenetic tree with bootstrap values of 1000 replicates shown at the branches. The scale bar represents the p-distance. (H) Sequence alignment of the N-terminal membrane region of CCoV strains analyzed in the present study with CCoV strains A76, 23-03, 1-71, CB-05, 341-05, and NTU336. (I) The crossover region in the HLJ-073 genome was detected by Simplot 3.5.1. The y-axis shows the percentage of permuted trees employing a sliding window of 200 nucleotides (nt) and a step size of 20 nt. The other parameters used included the Kimura (2-parameter) distance model, 2.0 Ts/Tv ratio, neighbor-joining tree model, and 1000 bootstrap replicates

Fig. 3

Indirect immunofluorescence detection of CCoVs in canine macrophage/monocyte (A) and THP-1 cells (B). The cells were infected with HLJ-073 or DF2 (MOI = 1) and detected using CCoV N protein-positive serum