Patent Application: US-52251303-A

Abstract:
a canine respiratory coronavirus that is present in the respiratory tract of dogs with canine infectious respiratory disease and which has a low level of homology to the enteric canine coronavirus , but which has a high level of homology to all bovine coronavirus strains and human coronavirus strain oc43 . the crcv spike , polymerase and hemagglutinin / esterase cdna and protein partial sequences are listed in figs . to , and . ctcagatgaa tttgaaatat gctattagtg ctaagaatag agcccgcact gttgctggtg 60 tttccatact tagtactatg actggcagaa tgtttcatca aaaatgtttg aaaagtatag 120 cagctacacg tggtgttcct gttgttatag gcaccactaa attttatggc ggctgggatg 180 atatgttacg tcgccttatt aaagatgttg acaatcctgt acttatgggt tgggattatc 240 ctaagtgtga 250

Description:
detection of a novel coronavirus associated with canine infectious respiratory disease an investigation into the causes of canine infectious respiratory disease ( cird ) was carried out in a large re - homing kennel . tissue samples taken from the respiratory tract of diseased dogs were tested for the presence of coronaviruses using rt - pcr with conserved primers for the polymerase gene . sequence analysis of four positive samples showed the presence of a novel coronavirus with high similarity to both bovine and human coronavirus ( strain oc43 ) in their polymerase and spike genes whereas there was a low similarity to comparable genes in the enteric canine coronavirus . this canine respiratory coronavirus ( crcv ) was detected by rt - pcr in 32 / 119 tracheal and 20 / 119 lung samples with the highest prevalence being detected in dogs with mild clinical symptoms . serological analysis showed that the presence of antibodies against crcv on the day of entry into the kennel decreased the risk of developing respiratory disease . dogs from a well - established re - homing kennel with a history of endemic respiratory disease were monitored for this study . on entry into the kennel , all dogs were vaccinated with kavak da 2 pip69 ( fort dodge ) a live attenuated vaccine for distemper virus , canine adenovirus type 2 , canine parainfluenzavirus and canine parvovirus . also , a killed leptospirosis vaccine was used ( fort dodge ). the health status of each dog was assessed twice a day by a veterinary clinician and the respiratory symptoms were graded as follows : 1 : no respiratory signs , 2 : mild cough , 3 : cough and nasal discharge , 4 : cough , nasal discharge and inappetence , 5 : bronchopneumonia . the overall health status of the dogs was graded as follows : 1 : good health , 2 : poor health , 3 : very poor health . the age , breed and sex of the dogs were recorded . for 119 dogs a full post mortem examination was performed . the tissue samples were stored at − 70 ° c . until further use . serum samples were collected from 111 dogs on day of entry into the re - homing kennel . for 81 dogs a follow - up serum was available on day 7 and for 111 dogs a serum was available on day 21 after entry . of the 111 dogs , 30 remained healthy during the 21 days between the first and the last serum sample whereas 81 dogs developed respiratory disease . sera from 35 dogs housed elsewhere were obtained from the diagnostic service of the royal veterinary college . these sera had been submitted for biochemical analysis for various reasons . five of these sera were from 18 - month - old beagles with no history of respiratory disease . sera were routinely stored at − 20 ° c . rna was extracted from tracheal and lung tissue of 119 dogs using trireagent ( sigma ). approximately 25 - 50 mg of homogenised tissue was used and rna was extracted as recommended by the manufacturer . synthesis of cdna was performed using random hexamers ( roche ) and impromii reverse transcriptase ( promega ). the polymerase gene of coronaviruses is known to be highly conserved , and has previously been used for phylogenetic analysis of this virus family ( stephensen et al ., 1999 ). for the detection of coronaviruses a modification of the primers 2 bp and 4bm directed against the polymerase gene as described by stephensen et al . ( 1999 ) were used ( conscoro5 : 5 ′- act - car - atg - aat - ttg - aaa - tat - gc ( seq id no : 31 ); and conscoro6 : 5 ′- tca - cac - tta - gga - tar - tcc - ca ( seq id no : 32 )). pcr was performed using taq polymerase ( promega ) in the provided reaction buffer containing a final concentration of 2 . 5 mm mgcl 2 and 0 . 5 μm of primers . for pcr with the primers conscoro5 and conscoro6 the following temperature profile was used : after denaturation at 95 ° c . for 5 min , 10 cycles were carried out at 95 ° c . for 1 min , annealing at 37 ° c . for 1 min and extension at 72 ° c . for 1 min . this was followed by 10 cycles using an annealing temperature of 45 ° c ., 10 cycles at an annealing temperature of 50 ° c . and 10 cycles at an annealing temperature of 53 ° c . followed by a final extension at 72 ° c . for 10 min . a 20 μl fraction of the pcr product was analysed on a 1 . 5 % agarose gel and blotted onto a nylon membrane ( roche ) after electrophoresis . the nylon membrane was hybridised with an oligonucleotide probe specific for the pcr product at 37 ° c . overnight ( probe conscoro : aag - ttt - tat - ggy - ggy - tgg - ga ( seq id no : 33 )). the probe was 3 ′ a - tailed with digoxigenin - dutp and was detected using anti - digoxigenin conjugate and cspd chemoluminescent substrate ( roche ). primer sequences specific for the spike gene were derived from an alignment of the spike region of bovine coronavirus strain ly - 138 ( af058942 ) and human coronavirus strain oc43 ( l14643 ). a pcr was performed with the primers spike 1 and spike 2 , followed by a nested pcr using the primers spike 3 and spike 4 and 2 μl of the product of the first amplification . the numbers in brackets refer to the nucleotide position in the bovine coronavirus genome . oligonucleotide spike 1 has seq id no : 34 , spike 2 has seq id no : 35 , spike 3 has seq id no : 36 , spike 4 has seq id no : 37 . the temperature profile used was denaturation at 95 ° c . for 5 min , followed by 35 cycles of denaturation at 95 ° c . for 1 min , annealing at 55 ° c . for 40 sec and elongation at 72 ° c . for 1 min . the final extension was performed at 72 ° c . for 10 min . the nested pcr produced a 442 bp fragment . pcr products were cloned into the pgem - t - easy vector ( promega ) and sequenced using the thermo sequenase fluorescent labelled primer cycle sequencing kit with 7 - deaza - dgtp ( amersham pharmacia ) using cy5 labelled primers . an alignment of the 250 bp cdna sequence from the polymerase gene to the corresponding sequences of 11 coronaviruses was performed using clustalx ( thompson et al ., 1997 ). the phylogenetic relationship to known coronaviruses was analysed using the phylip 3 . 6 package ( felsenstein , 1989 ). the alignments were followed by a bootstrap analysis using the seqboot programme . the obtained data sets were used for a maximum parsimony analysis using the dnapars programme and a consensus tree was calculated using consense . the resulting trees were drawn using the treeview programme ( page , 1996 ). elisa antigen for bovine coronavirus or enteric canine coronavirus ( cecv ) ( the antigens are a preparation from virus infected cell cultures obtained from churchill applied biosciences , huntingdon , uk ) was resuspended in pbs at the concentration recommended by the manufacturer and incubated on 96 well plates ( falcon ) overnight at 37 ° c . the plates were washed with pbs and blocked with pbs containing 5 % skimmed milk powder for 30 min . the sera were diluted 1 : 100 in blocking buffer and incubated on the plates for 1 h . after washing with pbs / 0 . 05 % tween 20 ( sigma ), a peroxidase labelled rabbit anti - dog igg conjugate ( sigma ) was added ( 1 : 5000 in pbs / 0 . 05 % tween 20 ) for 1 h . the plates were incubated with colour substrate ( opd , sigma ) for 10 min and the reaction was stopped by adding 2m h 2 so 4 . the adsorption was determined in an elisa photometer at 492 nm . virus isolation is performed on canine adult lung fibroblasts ( passage 3 to 7 ), mdck and a72 cells . ( it is appreciated , however , that virus isolation could be performed using primary cells or cell lines such as mdck or a72 ( canine ), mdbk ( bovine ), hrt - 18 ( human rectal tumour cell line ) and vero ( african green monkey ). the lung fibroblasts are maintained in mem with 20 % fetal calf serum ( fcs ), mdck and a72 cells are maintained in mem with 5 % fcs . tracheal tissue samples ( approx . 25 mg ) are homogenised using a scalpel and mixed vigorously in 1 ml mem containing penicillin ( 100 u / ml ), streptomycin ( 0 . 1 mg / ml ), amphotericin b ( 2 . 5 μg / ml ) and trypsin ( 1 μg / ml ). the samples are centrifuged at 13000 rpm for 10 min . and the supernatant is used to inoculate cell cultures . after 30 min . at 37 ° c . the supernatant is removed and maintenance medium added to the cultures . the cultures are passaged three times in the absence of a cytopathic effect . then , rna is extracted from the cells and rt - pcr to detect the presence of crcv is performed . the data were analysed using the chi - square test or fisher &# 39 ; s exact test and p values below 0 . 05 were considered statistically significant . using the primers conscoro5 and conscoro6 , cdna obtained from 40 tracheal samples was analysed by rt - pcr . out of these , seven were found to be positive by pcr and subsequent hybridisation ( 17 . 5 %). the pcr products were cloned and sequenced ( fig1 and 2 ) and the sequence data were compared to available viral sequences using the fasta search program ( pearson , 1990 ). comparison of the coronavirus cdna polymerase sequence obtained from four of the canine tracheal samples to other coronavirus sequences revealed that they were most similar to sequence data from bcv strain quebec and ly138 ( genbank accession nos . af220295 and af058942 , respectively ) and human coronavirus strain oc43 ( genbank accession no . af124989 ). the similarity in the analysed 250 bp sequence was 98 . 8 % for bcv quebec , and 98 . 4 % for bcv ly138 and the hcv pol genes , whereas it was only 68 . 53 % for ccv strain 1 - 71 pol gene ( fig6 and 7 ). an alignment of the novel sequence with the corresponding sequences of 11 coronaviruses and phylogenetic analysis using the maximum parsimony method resulted in the consensus tree shown in fig5 . the cdna sequence obtained from a tracheal sample ( t101 ) was found on a common branch with bovine coronavirus , human coronavirus - oc43 and hemagglutinating encephalomyelitis virus . for further analysis of the rna sequence of crcv , an alignment of the rna for the spike gene of the bovine coronavirus ly 138 strain ( af058942 ) and the human coronavirus oc43 strain ( l14643 ) was performed using clustal x ( thompson et al ., 1997 ). consensus regions were chosen for the selection of the nested primer sets spike 1 - 2 and spike 3 - 4 ( fig1 ). pcr analysis was performed with the cdna obtained from 119 tracheal and lung samples using these nested primers . in total 32 tracheal samples ( 26 . 9 %) and 20 lung samples ( 16 . 8 %) were found positive by nested pcr . for eight dogs a positive pcr result was obtained for both , trachea and lung . sequence analysis of the pcr products obtained from tissues of six different dogs showed identical dna sequences for these cdnas ( fig3 and 4 ). a comparison to known coronavirus spike sequences using the fasta program revealed a 98 . 1 % similarity to bovine coronavirus and a 97 . 8 % similarity to human coronavirus oc43 ( fig9 and 10 ). bovine coronavirus and other group ii coronaviruses contain an additional structural protein , the hemagglutinin / esterase ( he ). because of the high similarity of crcv with bcv , we analysed the presence of an he gene in crcv . an alignment of the he genes sequences of bcv and hcv oc43 was used to design the primers he1 and he2 ( table 2 ). four tracheal samples that had previously been identified as positive for coronavirus rna by rt - pcr with primers for the s gene were tested by rt - pcr with the primer set for the he gene . all four samples showed a pcr band of the expected size after agarose gel electrophoresis ( fig1 ). primer he1 has seq id no : 38 and he2 has seq id no : 39 . the sequence of the crcv pcr product obtained using primers he 1 and he 2 is given in fig1 ( seq id no : 21 ), and its predicted amino acid sequence is listed in fig1 ( seq id no : 22 ). a comparison of these nucleotide and amino acid sequences with the corresponding fragments of other related coronaviruses is shown in fig1 and 16 . three amino acids were shown to be unique to crcv , as shown in table 3 . the amino acid positions in bcv , hecv , hcv and hev are numbered from the initial m ( which is number 1 ) at the start of the bcv and hcv oc43 he proteins ( genbank accession nos . m84486 and m76373 , respectively ). using primers for the spike gene , tracheal and lung samples from 119 dogs were analysed by rt - pcr for crcv . of these 42 were from dogs with no respiratory signs ( grade 1 ), 18 dogs had shown mild respiratory signs ( grade 2 ), 46 had shown moderate ( grade 3 ) and 13 severe respiratory signs ( grades 4 and 5 ). grades 4 and 5 were merged due to the low case numbers in these groups . table 4 shows the pcr results for coronavirus in dogs with different grades of respiratory disease . specifically , table 4 shows the rt - pcr results from tracheal and lung samples of 119 dogs with different respiratory signs ( none to severe ) using a nested pcr directed against the coronavirus spike gene as well as the number of positive samples out of total sample number and the percentage of positive samples ( in brackets ). because of the homology of the spike cdna of crcv to the spike region of bovine coronavirus , an elisa antigen for bcv was used for serological analysis of crcv . sera from five dogs with no history of infectious respiratory disease that had not been housed in the investigated kennel were tested . the od values ranged from − 0 . 013 to 0 . 39 with an average od value of 0 . 154 . furthermore , sera from 30 dogs admitted to a veterinary clinic for various reasons were tested for antibodies to coronavirus . of these , 20 samples showed an od of & lt ; 0 . 4 (− 0 . 46 to 0 . 396 ) and 10 samples showed an od of & gt ; 1 . 0 ( 1 . 012 to 1 . 949 ). samples with an od of 0 . 6 or above were subsequently considered positive . comparison of the immune response to crcv of dogs with and without respiratory disease the bcv - antigen elisa was performed using paired sera of 111 dogs from the study kennel . of these , 81 dogs had shown symptoms of respiratory disease during a period of 21 days and 30 had remained healthy . of the group of dogs with respiratory disease , 17 were positive for antibodies to crcv on the day of entry into the kennel and 64 were negative . of the 64 dogs with no detectable antibodies to bcv on day one , 63 tested positive on day 21 . all 46 dogs out of these 63 for which a sample on day 7 was available tested negative on day 7 . therefore 63 dogs showed a seroconversion during the study - period whereas only one dog remained negative . of the 31 dogs that had remained healthy , 17 had antibodies to crcv on the day of entry . all of the 13 dogs that were negative on day 1 tested negative on day 7 but showed a seroconversion by day 21 . thus , of 34 dogs that were positive for antibodies to crcv on arrival in the kennel , 17 developed respiratory disease ( 50 %) whereas of 77 dogs that were negative on arrival , 64 developed respiratory signs during the study - period ( 83 . 1 %), ( fig1 ). therefore dogs that had no antibodies to crcv on entry into the kennel had an increased probability of developing respiratory disease ( p & lt ; 0 . 001 ). only one out of the 77 dogs that were negative on arrival remained negative during the study period of 21 days whereas 76 dogs showed a seroconversion . an elisa assay using a canine coronavirus antigen was performed to investigate whether crcv showed a serological cross reaction to canine enteric coronavirus . sera from 27 dogs , previously tested for antibodies to crcv using the bcv antigen were selected . it was found that eight dogs had antibodies to cecv on the day of entry into the kennel , of these four also had antibodies to crcv . nineteen dogs were found to be negative for cecv on day 1 , 17 of these were also negative for crcv . of the 19 negative dogs , five showed a seroconversion to cecv during the 21 - day period of the investigation and 17 showed a seroconversion to crcv . analysis of the prevalence of respiratory disease in this group showed that six out of the eight dogs ( 75 %) that were positive for antibodies to cecv on day 1 developed respiratory disease . out of the group of 19 dogs that had no detectable antibodies to cecv on day 1 , 15 showed signs of respiratory disease ( 78 . 9 %), ( p = 0 . 594 ). tracheal tissue samples from dogs that are identified as positive for crcv rna by rt - pcr are inoculated on cell cultures of canine adult lung fibroblasts and mdck cells . for some samples , virus isolation is also performed on a72 cells . the cultures show no signs of a cytopathic effect during three passages . after several passage , rna is extracted from the cultures and tested for the presence of crcv rna by rt - pcr . this study reports the detection of a novel coronavirus , crcv , in kenneled dogs with respiratory disease . coronaviruses have been reported to cause respiratory disease of man , cattle , swine and poultry , but their presence in the respiratory tract of dogs and a possible association with canine infectious respiratory disease ( cird ) has not been determined . dogs were investigated from a kennel in which cird was endemic and could not be controlled by the use of vaccines recommended against cird . samples taken from the respiratory tract of these dogs were examined using rt - pcr primers directed to the conserved polymerase gene of coronaviruses ( stephensen et al ., 1999 ). initially , seven tracheal samples were found to be positive ; the sequence of the rt - pcr products was determined and compared to all available coronavirus polymerase gene sequences . this analysis revealed that the cdna sequence obtained from the canine samples had the highest similarity to the polymerase gene of bovine coronavirus ( 98 . 8 %) and human coronavirus oc43 ( 98 . 4 %) but only a very low similarity to the polymerase gene of the enteric canine coronavirus ( strain 1 - 71 , 68 . 53 % similarity ). a phylogenetic analysis was performed using the polymerase sequences of eleven additional coronaviruses . the coronavirus detected in the respiratory tract of dogs ( crcv ) was located on a common branch with three group 2 viruses : bcv , hcv strain oc43 and hev . however , canine enteric coronavirus , a group 1 coronavirus , was shown to be only distantly related . canine respiratory coronavirus therefore is a novel coronavirus of dogs that is most closely related to bcv and hcv - oc43 , both of which are known to cause respiratory disease . to obtain more sequence information and to further determine the relationship to other coronaviruses using a more variable gene , a part of the spike gene was analysed . since crcv bad been shown to be most similar to bcv and hcv - oc43 , an alignment of the sequences of their spike genes was used to design a nested set of primers . nested primers were chosen to achieve a more sensitive assay . sequencing of the products of this rt - pcr confirmed the high similarity of crcv with bcv and hcv - oc43 . the presence of antibodies to crcv was analysed using an elisa based on a bcv antigen because of the high sequence similarity of the two viruses in the spike cdna . the elisa results confirmed the presence of a virus similar to bcv in the study population . the prevalence of antibodies was 30 % at the time of entry into the kennel and 99 % after 21 days . interestingly and unexpectedly , serological analysis revealed that dogs with antibodies to crcv on day of entry into the kennel developed respiratory disease less frequently than dogs without antibodies ( p & lt ; 0 . 001 ). therefore the presence of antibodies to crcv bad a protective effect against respiratory disease in this population . almost all dogs negative on day of entry into the kennel showed a seroconversion to crcv within three weeks , indicating that the virus is highly contagious . serology using an antigen for canine enteric coronavirus ( cecv ) showed a much lower prevalence of antibodies to cecv on day 21 . therefore the bcv - elisa results did not reflect an infection with canine enteric coronavirus and the cross - reactivity between the two antigens seems to be low . serum antibodies to crcv were present in about 30 % of dogs of various origins including dogs entering a re - homing kennel as well as pet dogs . the presence of crcv is therefore not limited to the investigated kennel and the virus seems to be established in the dog population . by pcr , crcv was detected in tracheal tissue and lung tissue and therefore appears to infect the upper and lower respiratory tract of dogs . within the kenneled population , crcv - rna was detected in 27 . 3 % of dogs with all grades of respiratory disease as well as in 26 . 2 % of dogs that were apparently healthy at the time of euthanasia . crcv - rna was most frequently found in the trachea of dogs with mild cough ( 55 %). studies using the human coronavirus strain 229e have shown , that coronaviruses can cause disruption of the respiratory epithelium and ciliary dyskinesia ( chilvers et al ., 2001 ). without being bound by theory , we believe that an infection with crcv has a similar effect , and that the virus plays an important role in the early stages of the pathogenesis of cird . by damaging the respiratory epithelium and disrupting ciliary clearance crcv facilitates the entry of other viral or bacterial pathogens . therefore while crcv infection on its own may cause only mild respiratory symptoms , in conjunction with other pathogenic agents it could lead to severe respiratory disease . the pathogenesis of cird has not been thoroughly investigated since the 1970s when bordetella bronchiseptica , canine adenovirus type 2 and canine parainfluenza were determined to be the main causes of the disease . however the vaccination of all dogs against cpiv , cav - 2 and distemper virus did not help to control the disease in this kennel despite evidence that the majority of dogs responded to the vaccine within 21 days ( data not shown ). this study shows an association of a novel canine respiratory coronavirus with cird . the aetiology of cird therefore needs to be re - evaluated and the role of novel microorganisms or microorganisms previously not associated with the disease has to be established . appel , m ., and binn l . n . 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( 1998 ). viruses and bacteria in the etiology of the common cold . j clin microbiol . 36 , 539 - 42 . page , r . d . m . treeview : an application to display phylogenetic trees on personal computers . computer applications in the biosciences 1996 12 : 357 - 358 pearson w r . rapid and sensitive sequence comparison with fastp and fasta . methods enzymol . 1990 ; 183 : 63 - 98 . pensaert m , callebaut p , vergote j . isolation of a porcine respiratory , non - enteric coronavirus related to transmissible gastroenteritis . vet q . 1986 july ; 8 ( 3 ): 257 - 61 . randolph j f , moise n s , scarlett j m , shin s j , blue j t , bookbinder p r . prevalence of mycoplasmal and ureaplasmal recovery from tracheobronchial lavages and prevalence of mycoplasmal recovery from pharyngeal swab specimens in dogs with or without pulmonary disease . am j vet res . 1993 march ; 54 ( 3 ): 387 - 91 . spaan w , cavanagh d , horzinek m c . coronaviruses : structure and genome expression . j gen virol . 1988 december ; 69 ( pt 12 ): 2939 - 52 . stephensen c b , casebolt d b , gangopadhyay n n . phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay . virus res . april ; 60 ( 2 ): 181 - 9 . storz j , purdy c w , lin x , burrell m , truax r e , briggs r e , frank g h , loan r w isolation of respiratory bovine coronavirus , other cytocidal viruses , and pasteurella spp from cattle involved in two natural outbreaks of shipping fever . j am vet med assoc . 2000 may 15 ; 216 ( 10 ): 1599 - 604 . tennant b j , gaskell r m , jones r c , gaskell c j . studies on the epizootiology of canine coronavirus . vet rec . 1993 jan . 2 ; 132 ( 1 ): 7 - 11 . thompson j d , gibson t j , plewniak f , jeanmougin f , higgins d g the clustalx windows interface : flexible strategies for multiple sequence alignment aided by quality analysis tools . nucleic acids res . 1997 dec . 15 ; 25 ( 24 ): 4876 - 82 . the crcv spike gene was cloned using the primers listed in table 5 and using the following cloning strategy , which is illustrated in fig1 . 1 . the spike gene was amplified in four overlapping fragments ( a , b , c , d ). 2 . the pcr product sp5 - sp2 ( b ) was joined to the product sp1 - sp8 ( c ) using the pvuii site in the overlap . 3 . this fragment was cloned into the pt7blue2 vector ( novagen ) using the restriction sites ncoi and bstxi . 4 . the pcr fragment spfxho - sp6 ( a ) was joined to bc using the restriction site bstxi in the overlap and the xhoi site that had been incorporated into the primer spf - xho . 5 . fragment abc was moved into the baculovirus transfer vector pmelbacb ( invitrogen ) using the restriction sites xhoi and ncoi . 6 . the pcr fragment sp7 - spr - histag - eco ( d ) was joined to abc using the restriction site ncoi in the overlap and the ecori site that had been incorporated into the primer spr - eco - histag resulting in the complete spike gene in pmelbacb ( spike melbac ). this construct contains a histag ( 6 × his ) at the c terminus of the expressed protein . 7 . for mammalian expression the complete gene was moved to psectaga ( invitrogen ) using the bamhi site in pmelbacb and the ecori site at the end of abcd resulting in the plasmid spikesectag . a co - transfection was performed in sf9 cells using the bac - n - blue baculovirus dna ( invitrogen ) and spike melbac . the resulting baculovirus ( acspcrcv 1 - 11 ) was shown to contain a full - length insert by pcr using primers ( invitrogen ) located upstream and downstream of the recombination site . the plasmid spike sectag was transfected into bhk - 21 cells using lipofectamine ( invitrogen ). expression of the spike protein was analysed using a serum sample from a dog that had been shown to be positive for antibodies to crcv using elisa ( bcv antigen obtained from churchill ) and a positive control serum for bcv obtained from churchill ( chicken anti bcv ). the transfected cells showed a positive signal in an immunofluorescence assay using the canine or the chicken serum and a fitc labelled conjugate ( fitc anti - dog igg or fitc anti chicken igg ).