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Variation of the sequence in the gene encoding for transmembrane protein M of canine coronavirus (CCV)
A Pratelli1, V Martella, G Elia
1Department of Health and Animal Well-being, Faculty of Veterinary Medicine, 70010 Valenzano, Bari, Italy. a.pratelli@veterinaria.uniba.it
Abstract:
A nucleotide variability in the sequence of the gene encoding for the transmembrane protein M of canine coronavirus (CCV) is described. A total of 177 faecal samples from pups with enteritis were analysed by a PCR and n-PCR specific for CCV. Four samples, collected from a dog presenting a long-duration shedding of CCV, and a sample from another diarrhoeic dog, were found positive by PCR but negative by n-PCR. Sequence analysis of the samples revealed silent nucleotide substitutions in the binding site of the internal primer used for the n-PCR. Moreover, the nucleotide substitutions occurring over the whole fragment of the five samples analysed were similar.
Insights
Nucleotide variability in canine coronavirus (CCV) M gene sequences can affect diagnostic PCR tests. Silent substitutions in primer binding sites caused false negatives in nested PCR, highlighting the need for sequence-aware diagnostic strategies for canine enteric disease.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Canine Infectious Diseases
Background:
- Canine coronavirus (CCV) is a significant cause of enteritis in puppies.
- Accurate molecular detection of CCV is crucial for diagnosis and disease management.
- The transmembrane protein M gene is a target for CCV genetic analysis.
Purpose of the Study:
- To investigate nucleotide variability within the CCV M gene.
- To assess the impact of genetic variations on the performance of PCR-based diagnostic assays.
- To identify the cause of discrepancies between PCR and nested PCR (n-PCR) results.
Main Methods:
- Analysis of 177 fecal samples from puppies with enteritis using PCR and n-PCR specific for CCV.
- Nucleotide sequencing of the M gene fragment from positive samples.
- Comparative sequence analysis to identify genetic variations.
Main Results:
- Five samples tested positive by PCR but negative by n-PCR.
- Sequence analysis revealed silent nucleotide substitutions within the internal primer binding site of the n-PCR.
- The nucleotide substitutions observed across the analyzed fragment were similar in the discrepant samples.
Conclusions:
- Nucleotide variability in the CCV M gene can lead to false-negative results in specific PCR assays.
- Silent substitutions affecting primer binding sites are a potential source of diagnostic error.
- Understanding genetic diversity is essential for developing robust and reliable molecular diagnostic tools for canine coronavirus.