Recombinant canine coronaviruses related to transmissible gastroenteritis virus of Swine are circulating in dogs

Abstract

Four canine coronavirus type II (CCoV-II) strains were identified in the guts and internal organs of pups which had died of acute gastroenteritis. The CCoV-II strains were strictly related to porcine transmissible gastroenteritis virus (TGEV) in the N-terminal domain of the spike protein, whereas in the other parts of the genome, a higher genetic relatedness to recent CCoV-II isolates was observed. Experimental infection of dogs with a TGEV-like isolate induced mild gastroenteritis without any systemic involvement. By virus neutralization tests, antigenic differences between reference and TGEV-like CCoVs were found. Our data support the potential recombinant origin of the TGEV-like CCoVs.

FIG. 1.

Phylogenetic analysis of group 1 CoVs. Neighbor-joining trees based on the spike protein full-length (a), N-terminal (b), and C-terminal (c) sequences and the envelope (d), membrane (e), and nucleocapsid (f) proteins of group 1 CoVs. For phylogenetic tree construction, the group 1a CoVs listed in Table 1 and the following CoV strains were used (GenBank accession numbers are in parentheses): human CoV (HCoV) 229E (NC_002645), porcine epidemic diarrhea virus (PDEV) CV777 (NC_003436), and HCoV-NL63 (NC_005831). The tree is rooted on the group 2 CoV canine respiratory CoV 240/05 (EU999954). Statistical support was provided by bootstrapping more than 1,000 replicates. The scale bars indicate the estimated numbers of amino acid substitutions per site.

FIG. 2.

Recombinant origin of the TGEV-like CCoVs. (a) SimPlot analysis of the nucleotide sequences of the nearly full-length genome of the TGEV-like CCoVs. Each point plotted is the percent genetic similarity within a 200-nt-wide sliding window centered on the position plotted with a step size of 20 nt, and Hamming correction on each curve represents a comparison of the sequence data of TGEV-like CCoVs 341/05 and 174/06 and CCoV-II-CB/05 to the reference sequence data of TGEV-Purdue. (b) Nucleotide sequence alignment of TGEV, TGEV-like CCoV, and CCoV-II genomes at the 5′ end of the S-encoding gene. Indicated are the S-gene transcription-regulating sequence (circled number 1), the S-gene initiation translation codon (circled number 2), and the ORF1b termination codon (circled number 3). The potential acceptor and donor sequences involved in the recombination event are boxed or underlined. Blue, red, and green letters indicate nucleotides conserved among all viruses, nucleotides conserved among TGEV and TGEV-like CCoV genomes, and typical nucleotides of CCoV origin, respectively. A putative location of the acceptor site is the sequence 22-TTACTAAAC-30, which has high identity (seven out of nine nucleotides) with that present in the proposed donor site (1061-TTACTACAA-1069). Alternatively, small sequence domains present in the TGEV-like CCoVs 341/05 (1021-CTAAAT-1026) and 174/06 (1054-CTTAAT-1059) are observed in the potential donor sites that could act as a slow-down signal and promote recombination between these sequences and others with high identity that are present at the acceptor sites, such as the sequence 25-CTAAAC-30.

FIG. 3.

Results of experimental infection of dogs with a TGEV-like CCoV. Three 12-week-old beagle pups were inoculated oronasally with 2 ml of strain 341/05 (titer of 10^6.25 50% tissue culture infective doses ml⁻¹) and monitored for up to 21 days for total white blood cell (WBC), lymphocyte, and polymorphocyte counts (top graph). In addition, fever, viral RNA shed in feces, and clinical scores were determined (bottom graphs). Total WBC, lymphocyte, and polymorphocyte counts are presented as percentages of the cell counts determined at day 0. Viral RNA titers as determined by real-time RT-PCR are expressed as copy numbers per microliter of template. Clinical scores were calculated as previously described (4) and are reported for each day with reference to the temperature curves.