key: cord-0865262-8awrzba8
authors: Pratelli, A.; Martella, V.; Elia, G.; Decaro, N.; Aliberti, A.; Buonavoglia, D.; Tempesta, M.; Buonavoglia, C.
title: Variation of the sequence in the gene encoding for transmembrane protein M of canine coronavirus (CCV)
date: 2001-08-31
journal: Molecular and Cellular Probes
DOI: 10.1006/mcpr.2001.0364
sha: d0067a3d2448fee1313c821e9638d0ee542336a9
doc_id: 865262
cord_uid: 8awrzba8

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.

loss of appetite, lethargy, and occasionally sudden death of younger pups. Canine coronavirus (CCV), a member of the Corona-

The isolation of CCV from infected dogs is often difficult and, frequently, coronavirus-like particles viridae family, was first isolated in 1971 during an epizootic in Germany from a case of canine enteritis. 1 seen by electron microscopy (EM) in stools have been difficult to propagate. These problems in isolating CCV is serologically and genetically related to other coronaviruses: feline infectious peritonitis CCV still hinder studies on their epidemiology and pathogenesis. virus (FIPV), feline enteric coronavirus (FeCV), transmissible gastroenteritis virus (TGEV) and porcine res-Recently a PCR and n-PCR have been developed for the diagnosis of CCV to overcame the difficulties piratory coronavirus (PRCV). [2] [3] [4] In naturally occurring infections, pups between six in virus isolation in vitro. 11, 12 Our original PCR and n-PCR assays are based on a hemi-nested strategy. and 12 weeks old appear to be more susceptible to disease [5] [6] [7] and this may be related to the decline of The pair of primers used for the PCR were chosen in a well-conserved region of the M gene of CCV and maternal antibodies. 8 In young pups CCV appears to replicate primarily in enterocytes on the villus tips of are able to amplify also FIPV and TGEV strains. The primer pair used in the n-PCR was able to amplify the small intestine, but also in the epithelium of the large intestine, 7 and was excreted in the faeces. 1, 9, 10 only CCV and TGEV strains. By using this heminested strategy we were able to detect CCV RNA with a CCV may cause diarrhoea, vomiting and dehydration, higher sensitivity (approximately of a 100-fold vestigating these occasional findings, the sequence of the samples displaying unusual response to the n-PCR assay was determined and analysed. Germany) from 1 ml of the supernatant fraction of each sample. The target sequence for amplification is a segment MATERIALS AND METHODS (nucleotides 337-746) of the gene encoding for transmembrane protein M of CCV.

The reverse transcriptase (RT) was performed in a total reaction volume of 20 l containing 1×PCR A total of 177 faecal samples of dogs were examined.

buffer (KCl 50 mM; Tris-HCl 10 mM; pH 8·3), MgCl 2 The samples were grouped as follows: A: 71 samples 5 mM, 1 mM of each deoxynucleotide (dATP, dCTP, were collected during a period of about 12 months dGTP, dTTP), Rnase inhibitor 1 U, RT 2·5 U, 50 pmol and stored at −20°C; B: 15 samples were fresh rectal primer antisense CCV2. The cDNA syntheses was swabs collected from pups with enteritis; C: 91 faecal carried out at 37°C for 30 min, followed by a desamples were collected, approximately every three naturation step at 94°C for 5 min. days, from a dog recovered from CCV enteritis and

The PCR reaction, in a total volume of 100 l kept under observation for a period of nine months.

containing 1×PCR buffer, MgCl 2 2 mM, 0·25 mM of each deoxynucleotide, Amplitaq Gold DNA polymerase 1·25 U, 50 pmol primer sense CCV1, was Virological examinations carried out for 34 cycles in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, USA), with the fol-Only the rectal swabs of group B and C (n=106) lowing reaction parameters: template denaturation were subjected to viral isolation. The samples were 94°C for 1 min, primer annealing 55°C for 1 min, and homogenized in Dulbecco-Minimal Essential extension at 72°C for 3 min. A single final extension Medium (D-MEM) and clarified by centrifugation at step was done at 72°C for 10 min to complete the 4000 g for 20 min at 4°C. The supernatant of each amplification reaction. sample was treated with antibiotics (5000 IU/ml peni-For the n-PCR, a 20 l aliquot of the 1 : 100 dilution cillin, 2500 g/ml streptomycin, 10 g/ml amof the first amplicon was subjected to a second round photericin) for 30 min at 37°C, inoculated onto of amplification using the CCV2 and CCV3 primers partially confluent monolayer of canine cell culture and the same PCR cycling procedures. (A-72), and then incubated at 37°C in a 5% (v/v) CO 2 Amplified products were electrophoresed on 2% incubator. Each sample was considered negative for (w/v) agarose gel at 50 V for about 120 min and CCV if, after three passages, no cytopathic effect (cpe) stained with the ethidium bromide. The sequence was observed. In the presence of cpe, the isolate was and position of the primers are shown in Table 1 . identified by indirect immunofluorescence assay (IFA) using monoclonal antibodies (MoAbs) to coronaviruses.

The samples examined were from group A (A-32/99) and from group C (C-5/4, C-10/4, C-11/7 and C-PCR and nested-PCR (n-PCR) 15/7). The samples from group C were collected, respectively, two months (C-5/4 and C-10/4) and The faecal samples were tested for CCV by PCR and n-PCR as previously reported. 11 Briefly, specimens five months (C-11/7 and C-15/7) after the presumed primary infection of the dog. All these samples were were diluted 1 : 100 in phosphate-buffered saline (PBS) and homogenised by vigorous vortexing. In-negative by the n-PCR. Sequence analysis was also performed on the PCR amplicon of sample C-22/2, soluble components were removed by centrifugation for 5 min at 8000 g, and genomic RNA was extracted collected from the pup at the onset of enteritis and resulted, as expected, positive by n-PCR. using the Rneasy Total RNA kit (Qiagen GmbH, which were collected from the dog with long shedding and between samples C-11/7 and C-15/7, collected, respectively, two and five months after the onset of the enteritis. Moreover, samples C-5/4 and C-10/4 were slightly different from samples C-11/7 and C-The RT-PCR amplicons, obtained with the CCV1-CCV2 primer pair, were purified on Ultrafree-DA 15/7 ( Figure 1 ). Columns (Amicon, Millipore) and then sequenced directly with ABI-PRISM 377 (Perkin Elmer, Applied Biosystem Instruments). Each sample was sequenced DISCUSSION twice. Sequence analysis was performed with NCBI's and EMBL's analysis tools. The alignment of the se-

The M glycoprotein of coronaviruses is a transmembrane molecule deeply embedded in the viral quences was performed with CLUSTAL W. 13 For alignment and comparative analysis, published sequence envelope so that only a small region is exposed on the outer surface of the lipid bylayer. Its function of the M gene of CCV strain Insavc (accession number D 13096) was used.

is essential for coronaviruses budding from rough endoplasmic reticulum (RER) and Golgi membranes. Antibodies to M glycoprotein can neutralize viral infectivity, but only in the presence of comple-RESULTS ment. 14, 15 At the moment, the significance of the variations Four CCV strains out of the 106 faecal samples examined from groups B and C, were isolated on A-72 observed in the gene of the M glycoprotein of CCV is not completely clear. The leader-primed replication cells. Two isolates were made from group B, while two isolates were made from the pup kept under and transcription of coronavirus RNA result in a high frequency of recombination and also of mutation observation (group C), from samples collected three and five days after the onset of enteritis. The isolates because most RNA polymerases would probably introduce one or more mutations during every round induced the typical lytic cpe of coronavirus after three days of incubation and the A-72 cells resulted positive of replication of the long genome of coronaviruses, generating, as consequence, a great deal of genetic by the IFI test using MoAbs.

In Table 2 are reported the results of PCR and n-variations in the progeny of coronaviruses from one infected individual. 16 A high degree of genetic vari-PCR analysis for CCV. In group A, 14/71 samples resulted positive by the PCR assay and 30/71 were ation in FeCV RNA has been already revealed by sequence analysis of the 7b and S genes and by positive by the n-PCR assay. All the 15 fresh samples from pups with enteritis (group B) resulted positive denaturing gradient gel electrophoresis of PCR amplicons of the S and N genes, suggesting that each both by the PCR and n-PCR assays. Twenty-two samples collected from the pup kept under ob-feline coronavirus exists as a quasispecies. 17- 19 The findings of our study, though based on a limited servation (group C) resulted positive by PCR, and 28 samples by n-PCR. Interestingly, shedding of CCV number of samples and on a short fragment of CCV RNA, suggest that a high degree of variation in the occurred intermittently over a period of nine months.

Five rectal swabs, one (A-32/99) from group A and genome of CCV also exists. In our study, five samples were found to be positive four (C-5/4, C-10/4, C-11/7 and C-15/7) from the pup kept under observation (group C) resulted positive by by PCR, but, unexpectedly, negative by n-PCR, though the n-PCR is more sensitive than the PCR assay. 11 PCR but negative by n-PCR. Sequence analysis of these five samples revealed repetitive nucleotide sub-All these samples exhibited similar variations in the binding site of the internal primer CCV3 used in the stitutions in the binding site of the internal primer CCV3, though these nucleotide variations do not n-PCR, with one of the nucleotide variations occurring at the very 3′ terminus of the primer binding site, determine amino acid changes (data not shown).

The sequences of the five PCR amplicons resulted which is important for the stability of primer annealing. In regard to sample A-32/99, there are only highly similar to each other. Several mutations observed in sample A-32/99 occurred in the same po-three substitutions in this region and they are not in the 3′ terminus. In this case, it may be supposed that sitions as samples C-5/4, C-10/4, C-11/7 and C-15/7, 

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