Group 1 Canine Coronavirus: Genotyping, Virus Isolation, and Sequence Analysis
Date Issued
2009
Date
2009
Author(s)
Chuang, Huei-Chu
Abstract
Coronaviruses (CoVs) cause a variety of different clinical forms of disease in a wide range of animal species. Group 1a Canine coronaviruses (CCoVs) can be divided into two genotypes, CCoV type I and CCoV type II. The viruses are known to be mild enteropathogen of dogs since their first recognition in early 1970s. Recently, CCoV type II strains with uncommon virulence, including a pantropic variant causing systemic disease in puppies, have been reported. In this study, 80 samples collected from canine distemper suspected dogs were tested for CCoV, and 47.5% were found positive by RT-nPCR. Among 30 CCoV positive dogs, 3 dogs were positive for CCoV type I, 8 dogs for CCoV type II and 3 dogs for both genotypes.e further tried to use a canine fibroma (A-72) cell line to isolate CCoV type II from two fecal samples. After co-incubation, cytopathy effects with the characteristic of shrinking and rounding up of cells can be observed. One of the sample was immunofluorescence assay (IFA) and RT-nPCR tests positive after 7 passages. However, active multiplication of the viruses were unable to be observed on the cell line. igh frequency of recombination of CoVs have been the reason of emerging of new disease. Full length of genomic sequence can provide the correct informations for the analysis of the evolution of recombined virus. Up to present, none of a whole genomic data concerning CCoV has been reported in the world. Here we reported the whole genomic RNA sequence of a field strain CCoV II. A consensus sequence of the feces-derived genomic RNA (CCoV II/NTU336/F/2008, NTU336) was determined from overlapping cDNA fragments produced by reverse transcriptase polymerase chain reaction (RT-PCR) amplification. RT-PCR products were sequenced by a reiterative sequencing strategy and the genomic RNA termini were determined using a rapid amplification of cDNA ends PCR strategy. The full length of NTU336 is 29226 nt, excluded poly-A tail and 5’ end. This local virus shows a higher genetic relatedness to TGEV-like CCoV type II bearing a recombination site in downstream of ORF 1b and upstream of S gene. Like other CCoV type II related viruses, CCoV type II, FCoV type II, TGEV and TGEV-like CCoV type II, which had ORF 3 residues after S gene. This is a evidence of CCoV type II related viruses are evolutionary from CCoV type I. Among group 1a CoVs, NTU336 has the largest M gene resulted from a 15-21 nt insertion. SimPlot and DNAstar analyses of the NTU336, TGEV, and FCoV type II, reveal NTU336 and TGEV sharing common ancestor on ORF 1a, downstream of ORF 1b, upstream of S gene. On the other parts, NTU336 is closely related to CCoV type II, and some parts are close related to NTU156, a FCoV type II virus of Taiwan.
Subjects
canine coronavirus
genotyping
sequence analysis
virus isolation
Type
thesis
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