Patent Application: US-48088295-A

Abstract:
this invention provides a recombinant raccoon poxvirus that expresses the nucleocapsid and transmembrane proteins of feline infectious peritionitis virus . the recombinant viruses are useful as vaccines , either alone or in combination with carriers and adjuvants .

Description:
the vaccine of the present invention may be prepared by creating recombinant raccoon poxviruses ( rrpvs ) containing the genes encoding the n or e1 proteins of fipv or immunogenic fragments thereof . these genes are first inserted into a transfer plasmid , which is then introduced into appropriate host cells that have been previously infected with a raccoon poxvirus . as a result , the dna from the transfer plasmid is incorporated into the poxvirus dna by homologous recombination , producing the rrpvs that are released from the cells . dna encoding the fipv n or e1 proteins is inserted into the transfer plasmid immediately downstream of a poxvirus promoter . in a preferred embodiment , the early / late 7 . 5 kd protein promoter of vaccinia virus is used ; however , alternate promoter elements that are functional in poxviruses can also be used . the preferred transfer plasmid also contains a beta - galactosidase marker gene , which allows for selection and detection of the plasmid dna sequences in recombinant viruses . it will be obvious to one skilled in the art that alternative selectable marker genes , such as the neomycin resistance gene or the e . coli gpt gene or others , can be used to practice the invention . flanking the foreign gene of interest and the selectable marker gene are thymidine kinase dna sequences , which facilitate recombinatorial integration of the plasmid dna sequences into the raccoon poxvirus dna . recombinant viruses expressing the fipv n or e1 genes are prepared by first infecting a susceptible cell line such as vero ( atcc ccl 81 ), bsc - 1 ( atcc ccl 26 ), rat - 2 ( atcc crl 1764 ), or crfk ( atcc ccl 941 ) with wild type raccoon poxvirus ( atcc vr - 838 or similar isolates , such as , for example , rcnv71 - i - 85a ). transfer plasmid dna containing the e1 or n gene is then transfected into the infected cells using cationic liposome - mediated transfection , or other suitable techniques such as electroporation or calcium phosphate precipitation . virus replication is allowed to proceed until cytopathic effects are noted in all cells . incorporation of the fipv e1 or n genes into poxvirus dna is accompanied by disruption of the viral thymidine kinase gene . therefore , virus harvested from this infection may be isolated by selecting for the absence of a thymidine kinase gene ; this is achieved by growth on tk - rat - 2 cells ( atcc crl 1764 ) in the presence of 5 - bromodeoxyuridine . viruses containing a gene insert from the transfer plasmid are further identified by the appearance of a blue plaque color when grown in the presence of a chromogenic substrate for beta - galactosidase such as x - gal . viral plaques that survive these selection and screening procedures are then subjected to several cycles of plaque purification . subsequently , the presence of the e1 or n genes is confirmed by polymerase chain reaction technology , and the presence of e1 or n protein is confirmed by immunoblot analysis using specific antibodies . these viruses are designated rrpv - fipv e1 and rrpv - fipv n , respectively . in a further embodiment of the present invention , the genes encoding n and e1 were inserted into a single transfer plasmid . introduction of this plasmid into cells previously infected with wild - type raccoon poxvirus results in the production of recombinant viruses that express both proteins simultaneously ( rrpv - fipv e1 / n ). in a still further embodiment , rrpvs can be produced that express less - than - full - length segments of the fipv e and n proteins . the techniques used to engineer transfer plasmids encoding partial sequences of e1 and n are well - known and widely used in the art , as are the methods for production and screening of rrpvs as detailed in this specification . for example , introduction of oligonucleotides containing a stop codon at various points along e1 or n dna will produce a nested set of carboyxterminal - truncated versions of that gene , which can then be incorporated into rrpvs . it will be apparent to one of ordinary skill in the art that systematic screening of such recombinant rrpvs can establish whether the intact protein , or subfragments thereof , are most preferred in practicing the present invention . furthermore , as stated above , dna encoding different fragments of e1 and n can be used in a combination vaccine after incorporation into the same , or different , rrpvs . for vaccine preparation , susceptible cells such as those listed above are infected with rrpvs at a multiplicity of infection ( moi ) of 0 . 1 infectious units / cell or less . in this specification , an infectious unit is defined as a tissue culture infectious dose ( tcid 50 ), an amount of virus yielding 50 % infection under defined conditions . a method for tcid 50 determination is detailed in example 1 below . when cytopathology is noted in & gt ; 90 % of the cells , the infected cells and extracellular fluids ( both of which contain viruses ) are harvested as a single virus - cell lysate . the highly concentrated virus stock to be used as a vaccine may be stored frozen (- 50 ° c . or colder ) or lyophilized until the time of use . compounds such as nz - amine , dextrose , gelatin or others designed to stabilize the virus during freezing and lyophilization may be added . the virus initially present in the virus - cell lysate may be further concentrated using commercially available equipment . typically , the concentration of virus in the vaccine formulation will be a minimum of 10 6 . 5 tcid 50 per dose , but will typically be in the range of 10 7 . 0 to 10 9 . 0 tcid 50 per dose . at the time of vaccination , the virus is thawed ( if frozen ) or , if lyophilized , is reconstituted with a physiologically - acceptable carrier such as deionized water , saline , phosphate buffered saline , or the like . the present invention is not limited to native ( i . e . replication - competent ) rrpvs . the virus - cell lysate can be subjected to treatments commonly used in the art to inactivate viruses . a composition comprising inactivated virus and expressed protein will be effective in eliciting protective immunity against fipv if it contains a sufficient quantity of fipv protein . this type of vaccine would provide added assurance that recipient felines will not be exposed to infectious fipv as a consequence of vaccination . in addition , a physiologically - acceptable adjuvant may be added to the virus , such as ema 31 ( ethylene maleic anhydride 31 ) ( monsanto co ., st . louis , mo . ), neocryl ( polyvinyl chemical industries , wilmington , mass . ), mvp ( modern veterinary products , omaha , nebr . ), squalene , pluronic l121 ( polyoxypropylene - polyoxyethylene block copolymer ) or the like . individual raccoon poxviruses expressing the n or e1 genes may be mixed together for vaccination . furthermore , the virus may be mixed with additional inactivated or attenuated viruses , bacteria , or fungi such as feline leukemia virus , feline panleukopenia virus , feline rhinotracheitis virus , feline calicivirus , feline immunodeficiency virus , feline herpesvirus , feline enteric coronavirus , feline chlamydia psittaci , microsporum canis , or others . in addition , antigens from the above - cited organisms may be incorporated into combination vaccines . these antigens may be purified from natural sources or from recombinant expression systems , or may comprise individual subunits of the antigen or synthetic peptides derived therefrom . in a further embodiment of the present invention , live or inactivated rrpv virus - cell lysates can be incorporated into liposomes , or encapsulated in peptide -, protein -, or polysaccharide - based microcapsules prior to administration , using means that are known in the art . the final vaccine is administered to cats in a volume that may range from about 0 . 5 to about 5 ml . the vaccine can be administered by subcutaneous , intramuscular , oral intradermal , or intranasal routes . the number of injections and their temporal spacing may be varied . one to three vaccinations administered at intervals of one to three weeks are usually effective . the following examples are intended to further illustrate the invention without limiting its scope . the techniques used to infect and transfect cells , plaque purify virus , perform immunoblot analysis are widely practiced in the art . generation of recombinant raccoon pox viruses expressing fipv n and e1 genes 1 . cloning of fipv n and e1 genes and preparation of transfer plasmids the sequences of the e1 seq . id . no . 1 and n seq . id . no . 2 genes used in the present invention are shown in fig1 a to 1e and 2a to 2g , respectively , of the specification . the methods for cloning of the n and e1 genes of fipv and their insertion into a psc11 transfer vector are detailed in european patent application 0 , 376 , 744 , which is incorporated by reference . the plasmid used to clone the cdna for the e1 and n genes is shown in fig3 a and 3b . the psc11 plasmids carrying the e1 and n genes are shown in fig4 b and 4 b , respectively . the sequences of these plasmids are shown in fig5 a to 5f ( seq id no : 3 ) and fig6 a to 6f ( seq id no : 4 ). to construct a psc11 transfer plasmid containing both n and e1 genes , a 1 . 0 kb dna fragment containing the vaccinia 7 . 5 promoter and the e1 gene was inserted downstream of the n gene in psc11 - fipv n . the resulting plasmid was designated psc11 - fipv n / e1 . monolayers of vero cells ( atcc ccl 81 ) that were 80 % confluent ( approximately 5 × 10 6 cells / 100 mm tissue culture dish ) were infected for 30 - 60 minutes at 37 ° c . with wild - type raccoon pox virus ( atcc vr - 338 ) at a multiplicity of infection moi ) of 0 . 1 tcid 50 / cell . the medium ( 2 ml ) consisted of eagle &# 39 ; s minimum essential medium (&# 34 ; mem &# 34 ;, gibco brl # 410 - 1500 ) containing 0 . 05 % lactalbumin hydrolysate and 15 μg / ml gentamicin sulfate and adjusted to ph 7 . 2 with sodium bicarbonate . after infection , the medium was removed and the cells were transfected with the psc11 - fipv n , psc11 - fipv e1 , or psc11 n / e1 transfer plasmid by cationic liposome - mediated transfection using dioctadecylamidoglycyclsperimine - 4 - trifluoroacetic acid , ( transfectam ) ( promega corporation , madison , wis .) and dioleoyloxy ) propyl - n , n , n - trimethylammonium methyl sulfate ( dotap ) ( boehringer mannheim , indianapolis , ind . ), respectively , per manufacturer &# 39 ; s instructions . the cells were incubated with the dna - liposomes mixture in 3 ml of mem containing 5 % fetal bovine serum ( fbs ) overnight at 37 ° c . ( 5 % co 2 ), after which the medium was replaced with 8 ml of fresh mem - 5 % fbs . the transfected cells were incubated at 37 ° c . ( 5 % co 2 ) until greater than 80 % showed cytopathic effects ( cpe ), which took approximately 3 - 4 days . the virus - cell lysates were then removed from the plates and subjected to two cycles of freeze - thawing before storage at - 70 ° c . 3 . isolation of recombinant raccoon pox virus carrying the fipv n gene rrpvs carrying the fipv n gene ( rrpv - fipv n ) were isolated and purified from the psc11 - fipv n vero cell transfection by standard viral plaque purification methods . monolayers of vero cells ( 50 - 80 % confluent ) were infected with 2 ml of ten - fold serial dilutions ( 10 - 1 to 10 - 3 ) of the viral - cell lysates for 1 hour at 37 ° c . after incubation , the media was removed and the infected cells were overlaid with 8 - 10 ml of 1 . 25 % noble agar containing mem / 5 % fbs . the infected cells were then incubated for 3 - 4 days at 37 ° c . ( 5 % co 2 ), and overlaid again with 4 ml of 1 . 25 % nobel agar containing 0 . 5 x pbs and 600 μg / ml 5 - bromo - 4 - chloro - 3 - indolyl - β - d - galactopyranoside ( x - gal , states biochemical cleveland , ohio ). the plates were incubated at 37 ° c . ( 5 % co 2 ) for 4 - 16 hours , until blue ( i . e . β - galactosidase positive ) viral plaques were observed . the recombinant viral plaques were picked with sterile blunt needles attached to a 1 cc syringe , suspended in 0 . 5 ml of 0 . 25 μg / ml trypsin , vortexed vigorously , and incubated at 37 ° c . for 15 - 30 min . the disrupted viral plaques were then inoculated onto 5 × 10 5 vero cells in 25 cm 2 flasks and incubated at 37 ° c . ( 5 % co 2 ) until greater than 80 % cpe was observed . the viral - cell lysates containing rrpv - fipv n were subjected to two cycles of freeze - thawing and stored at - 70 ° c . six individual rrpv - fipv n clones were selected and plaque - purified five times as described above . isolation of recombinant raccoon pox virus containing the fipv e1 gene rrpvs carrying the fipv e1 gene ( rrpv - fipv e1 ) were isolated and purified from psc11 - fipv e1 - transfected vero cells using the methods described for rrpv - fipv n , with some modifications . in this case , thymidine kinase deficient ( tk -) rrpvs from the initial virus - cell lysates were selected on tk - rat - 2 cells ( atcc crl 1764 ). this was performed by inoculating 1 ml of the initial virus - cell lysate onto a monolayer of rat - 2 cells in a 75 cm 2 flask ( approximately 5 × 10 6 cells ) in the presence of 5 - bromodeoxyuridine ( brdu ) at 30 μg / ml in mem . the infected monolayer was incubated at 37 ° c . ( 5 % co 2 ) for 3 - 4 days until greater than 70 % cpe was observed . the tk - virus - cell lysates were subjected to two cycles of freeze - thawing before storage at - 70 ° c . two individual rrpv - fipv e1 clones were selected and subjected to six cycles of plaque purification as described above for rrpv - fipv n . 5 . confirmation of fipv n and e1 genes in rrpv by polymerase chain reaction the presence of the fipv n and e1 genes in the rrpvs was confirmed using the polymerase chain reaction ( pcr ). 90 μl of a virus - cell lysate were incubated with 10 μl of tenfold concentrated pcr lysis buffer ( 100 mm tris - hcl buffer , ph 8 . 5 ; 500 mm kcl ; 25 mm mgcl 2 ; 5 % tween 20 ( polyoxyethylenesorbitan monolaurate ); 3 mg / ml proteinase k ) for 16 hours at 50 ° c ., then boiled for 10 min . 10 μl of this lysate was used in the pcr . pcr was performed in 100 μl of 10 mm tris - hcl buffer , ph 8 . 3 ; 50 mm kcl ; 200 um of each deoxyribonucleotide triphosphate , 1 . 5 mm mgcl 2 ; 30 pmoles of each oligonucleotide primer ; and 2 . 5 units of amplitaq dna polymerase ( taq dna polymerase ) ( perkin - elmer cetus , norwalk , conn .). the primers used in the pcr for fipv n were : corresponding to nucleotides 68 - 91 and 721 - 744 of the fipv n open reading frame ( seq id no : 2 ) ( primers 1 and 2 , respectively ). the primers used in the pcr for fipv e1 were : corresponding to nucleotides 334 - 355 and 721 - 742 of the fipv e1 open reading frame seq . id . no . 1 ( primers 3 and 4 , respectively ). the pcr amplifications were performed in a dna thermal cycler perkin - elmer cetus ) by first heating the reaction mixes to 94 ° c . for denaturation , and then performing 35 cycles of amplification , each consisting of 1 min at 95 ° c ., 1 min at 55 ° c ., 2 min at 72 ° c ., and , on the last cycle , a final incubation of 8 min at 72 ° c . 10 μl of the pcr products were resolved by electrophoresis in a horizontal - submarine 4 % nusieve ( agarose ) gel ( fmc bioproducts , rockland , me .) in tae buffer ( 40 mm tris base , 20 mm sodium acetate , 1 mm edta , ph 7 . 2 ) by applying 5 v / cm for 1 - 2 hours . the dna products were visualized by staining the gels with ethidium bromide . pcr amplifications with the fipv n and e1 primers gave dna fragments of 676 and 408 nucleotides , respectively ( fig7 ). pcr amplifications using the psc11 fipv n and e1 transfer plasmids served as positive controls , and showed products of the predicted sizes . pcr amplifications using wild - type raccoon pox virus - vero cell lysates served as a negative control , and no pcr products were observed in those samples . confirmation of rrpv fipv n and e1 protein expression by immunoblot analysis confluent monolayers of vero cells in a 25 cm 2 flask ( 1 - 2 × 10 6 cells ) were infected with clones of either rrpv - fipv n or rrpv - fipv e1 at an moi of 0 . 1 . the infected cell were incubated at 37 ° c . ( 5 % co 2 ) for 2 - 3 days until approximately 80 % of the cells showed cytopathic effects . a virus - cell lysate was prepared , and 20 μl of the sample were added to 5 μl of 5 x laemmli sample buffer ( 0 . 3m tris - hci buffer , ph 6 . 8 , containing 5 % sds , 50 % glycerol , 0 . 4 % bromophenol blue , and 3 % 2 - β - mercaptoethanol ) and heated at 95 ° c . for 5 min . the denatured protein samples were separated by sds / polyacrylamide electrophoresis using a 4 - 15 % gradient polyacrylamide gel as described previously . maniatis et al ., molecular cloning : a laboratory manual , 1982 , cold spring harbor press . after electrophoresis , the proteins were transferred to nitrocellulose ( bio - rad laboratories , hercules , calif .) by electrotransfer using a bio - rad transfer apparatus per manufacturer &# 39 ; s instructions . the transfer was performed in 25 mm tris - hci buffer , containing 0 . 2m glycine and 20 % methanol , for 45 minutes at 50 v with constant current . fipv n and e1 proteins were visualized on the nitrocellulose filter using specific antibodies . davis et al ., basic methods in molecular biology , 1986 , elsevier science publishing company , new york , n . y . the filter was rinsed in phosphate buffered saline ph 7 . 4 containing 0 . 1 % tween - 20 ( polyoxyethylenesorbitan monolaurate ) (&# 34 ; pbs - tw &# 34 ;), after which non - specific sites were blocked by overnight incubation at 4 ° c . in pbs containing 1 % bovine serum albumin ( pbs - bsa ) followed by a 15 min wash in pbs - tw . the filter was then incubated for 30 min at room temperature with anti - fipv antibodies , which consisted of ascites fluid from a fipv ( strain 79 - 1146 )- infected cat , diluted 1 : 100 in pbs - tw containing 1 % bsa (&# 34 ; pbs - tw - bsa &# 34 ;). after four 5 min washes in pbs - tw , the filter was incubated for 30 min at room temperature with a secondary antibody consisting of biotin - labeled mouse anti - cat igg antibody ( kirkegaard & amp ; perry laboratories inc ., gaithersburg , md .) that had been diluted 1 : 2000 in pbs - tw - bsa , followed by four 5 min washes in pbs - tw . the filter was then incubated for 30 min at room temperature with horseradish peroxidase - conjugated streptavidin ( kirkegaard a perry laboratories inc .) that had been diluted 1 : 1000 in pbs - tw . after the filter was washed four times ( 5 min each ) in pbs - tw , the antigen - antibody complexes were visualized with peroxidase chromogenic substrate ( kirkegaard & amp ; perry laboratories inc .). sucrose - gradient purified fipv and wild - type raccoon pox virus - vero cell lysates were used as the positive and negative controls , respectively . a typical immunoblot is shown in fig8 . serial tenfold dilutions of virus are prepared in mem and inoculated in replicates of five onto vero cells ( 1 × 10 4 cells per well ) in a 96 well plate . virus preparations may be pretreated by dilution into an equal volume of 0 . 5 % trypsin and incubation at 37 ° c . for 30 min in order to release virus from inclusions . plates are incubated for 3 - 5 days at 37 ° c . ( 5 % co 2 ) and observed for cytopathology typical of raccoon poxvirus . titers are calculated as 50 % endpoints based on cytopathology using the methods of reed and muench , the american journal of hygiene 27 ( 3 ): 493 - 497 ) ( 1938 ). a single clone of each recombinant virus was selected for large - scale expansion to serve as a master seed virus . the criteria for selection were : 1 ) demonstration of purity . polymerase chain reaction was utilized to insure that the clone was uncontaminated with wild type virus . 2 ) demonstration of adequate recombinant proten expression by western blot or other antigen detection methods . all recombinant virus expansions and titrations were done on vero cells in mem containing 2 . 5 % fbs . each plaque purified virus clone was expanded by inoculating a confluent monolayer of vero cells in a 150 cm 2 flask ( 1 × 10 7 cells ) with 1 ml of viral - cell lysate ( approximately 10 7 infectious virus particles ), and incubating at 37 ° c . ( 5 % co 2 ) until 100 % cytopathic effect was observed ( 2 - 3 days ). this virus - cell lysate was titrated on vero cells as described in example 1 , and served as a premaster seed virus stock to obtain the master seed virus . the moi to be used to produce the highest titer master seed virus was determined by inoculating a confluent monolayer of vero cells in a roller bottle ( 1 × 10 8 cells ) with various mois of recombinant virus ( e . g . 0 . 1 , 0 . 05 , 0 . 01 , 0 . 005 , and 0 . 001 tcid 50 / cell .) the infected cells were incubated at 37 ° c . until greater than 80 % cpe was observed ( approximately 3 days ), and the titers of each infected roller bottle was determined . the master seed viruses were aliquoted into 1 . 5 ml ampules , which were sealed and stored in a liquid nitrogen freezer . 3 × 10 7 vero cells were seeded into 850 cm 2 roller bottles in 200 ml of growth media ( mem containing 0 . 5 % lactalbumin hydrolysate and 5 % fbs ) and incubated for 18 hours at 37 ° c . the next day , the medium was removed from the cells and replaced with 50ml of rrpv - fipv n virus diluted to an moi of 0 . 01 in infection media ( mem containing 0 . 5 % lactalbumin hydrolysate and 2 . 5 % fbs ). the virus used was at the second passage beyond the master seed preparation . virus was allowed to absorb to the cells for 30 min at 37 ° c ., after which the volume of medium was adjusted to 150 ml per roller bottle . roller bottles were incubated at 37 ° c . until 100 % cytopathology was evident ( 3 days ). the virus - cell lysate was harvested and stored frozen (- 70 ° c .). the virus titer was determined to be 10 6 . 97 tcid . sub . / ml . rrpv - fipv e1 stocks were prepared in the same manner , except that an moi of 0 . 1 was used . the final virus preparation was titered and found to contain 10 6 . 5 tcid 50 / ml . wild type raccoon poxvirus was grown using the same methods as described above , and contained 10 6 . 44 tcid 50 / ml . a group of twenty - four 9 - month - old cats ( specific pathogen - free , harlan sprague dawley , madison , wis . ), comprising seven males and seventeen females , was used to demonstrate the efficacy of the rrpv - fipv n vaccine . cats were divided into five groups and vaccinated twice , 21 days apart , as indicated below : ______________________________________ # volume viral dose vaccinatongroup cats vaccine ( ml ) ( tcid . sub . 50 ) route * ______________________________________1 5 rrpv - fipv n 3 10 . sup . 7 . 44 sc2 5 rrpv - fipv n 1 10 . sup . 6 . 97 im3 5 rrpv - fipv n 3 10 . sup . 7 . 44 oral4 4 rrpv - fipv n 3 10 . sup . 6 . 44 sc ( 1 : 10 dilution ) 5 5 wild type rev 3 10 . sup . 6 . 44 sc______________________________________ * sc = subcutaneous im = intramuscular oral = oral two weeks following the second vaccination , cats were orally inoculated with 10 3 . 4 tcid 50 of feline enteric coronavirus ( strain 79 - 1683 , atcc vr - 989 ). this virus induces a subclinical infection which can enhance subsequent fipv infection . three weeks later , cats were orally challenged with 10 3 . 4 tcid 50 of fipv ( strain 79 - 1146 , atcc vr - 990 ). cats were monitored weekly for a total of 64 days after challenge for signs of clinical disease including : fever , icterus , leukopenia , anemia , weight loss , anorexia , depression , dehydration , and peritoneal swelling . cats deemed moribund were euthanized by the attending veterinarian and post - mortem pathological examination was performed . clinical disease signs were scored as follows : ______________________________________sign score______________________________________fever103 . 0 - 10 . 39 ° f . 1 point / day * 104 . 0 - 104 . 9 ° f . 2 points / day ≧ 105 . 0 ° f . 3 points / daydehydration 1 point / daydepression 1 point / dayanorexia 1 point / dayperitoneal swelling 1 point / dayicterus 1 point / dayweight loss & gt ; 20 % 1 point per observation & gt ; 30 % 2 points per observation & gt ; 50 % 5 points per observationleukopeniadecrease of 50 % 3 points per observationcounts & lt ; 6000 2 points per observationhematocrit 3 points per observation & lt ; 25 % pcvdeath 25 points______________________________________ * for cats with baseline temperatures averaging 103 ° f ., no points will be scored until temperatures are in excess of 1 ° f . above baseline . inoculation with virulent fipv induced a fatal infection in 4 / 5 ( 80 %) of the control cats , which were vaccinated with wild type raccoon poxvirus ( table 1 ). both effusive and non - effusive forms of the disease were noted in the control cats . on the other hand , clinical disease was essentially absent after challenge of the subcutaneous vaccinates . the sporadic fever in these cats could be attributed to excitability and the slight anemia on one day in cat 1264 is not a significant finding . the subcutaneous vaccinates showed a statistically significant reduction in clinical signs ( p & lt ; 0 . 05 , by anova ) and death ( p & lt ; 0 . 01 , by chi square analysis ) when compared to the control cats . the intramuscular route of vaccination was less effective in that 2 / 5 ( 40 %) of the cats succumbed to fipv - induced disease . however , the onset of disease in these cats was delayed when compared to the controls . the decreased efficacy may be related to the lower titer of virus inoculated into these cats because only a 1 ml dose could be administered by this route . there was also decreased efficacy when cats were inoculated by the oral route ( 60 % mortality ) which may indicate the need for a higher virus dose when vaccinated by this route . the protection conferred against fipv - caused disease by the subcutaneously administered vaccine was shown to be dose - dependent , confirming the benefit of a high - titer rrpv - fipv vaccine in inducing protection against clinical disease induced by fipv virus . a suitable vaccine dose contains viral antigen in the range of 10 4 - 10 8 tcid 50 / ml , preferably 10 7 - 10 8 tcid 50 / ml . a typical dose for administration to cats is 1 - 3 ml , and delivery by the subcutaneous route is preferred . table 1__________________________________________________________________________total clinical scores following challenge with fipvcat id fever weight loss leukopenia anemia chemical signs death * total score__________________________________________________________________________subcutaneous vaccinates1260 0 0 0 0 0 0 01262 1 0 0 0 0 0 11264 1 0 0 0 0 0 41266 0 0 0 0 0 0 01268 2 0 0 0 0 0 2intramusculuar vaccinates1270 13 6 0 0 33 25 771272 2 0 0 0 0 0 21297 23 0 0 0 34 25 911301 3 0 0 0 0 0 3oral vaccinates1303 1 0 4 0 0 0 51305 0 1 0 0 5 0 61307 3 1 0 0 4 25 331309 2 1 6 0 6 25 401311 4 3 0 0 18 25 501 / 10 dose subcutaneous vaccinates1313 29 5 9 3 36 25 1071315 17 0 0 0 20 25 621317 3 0 3 0 9 25 401319 1 0 0 0 6 0 7controls1321 39 3 18 3 63 25 1511323 5 0 2 0 14 25 461327 0 1 2 0 15 25 431329 5 1 6 0 11 25 481337 1 0 0 0 0 0 1__________________________________________________________________________ __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 10 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 789 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna ( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : fipv e1 ( xi ) sequence description : seq id no : 1 : atgaagtacattttgctaatactcgcgtgcataattgcatgcgtttatggtgaacgctac60tgtgccatgcaagacagtggcttgcagtgtattaatggcacaaattcaagatgtcaaacc120tgctttgaacgtggtgatcttatttggcatcttgctaactggaacttcagctggtctgta180atattgattgtttttataacagtgttacaatatggcagaccacaatttagctggctcgtt240tatggcattaaaatgctgatcatgtggctattatggcctattgttctagcgcttacgatt300tttaatgcatactctgagtaccaagtttccagatatgtaatgttcggctttagtgttgca360ggtgcagttgtaacgtttgcactttggatgatgtattttgtgagatctgttcagctatat420agaagaaccaaatcatggtggtcttttaatcctgagactaatgcaattctttgtgttaat480gcattgggtagaagttatgtgcttcccttagatggtactcctacaggtgttacccttact540ctactttcaggaaatctatatgctgaaggtttcaaaatggctggtggtttaaccatcgag600catttgcctaaatacgtcatgattgctacacctagtagaaccatcgtttatacattagtt660ggaaaacaattaaaagcaactactgccacaggatgggcttactacgtaaaatctaaagct720ggtgattactcaacagaagcacgtactgacaatttgagtgaacatgaaaaattattacat780atggtgtaa789 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1134 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna ( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : fipv n ( xi ) sequence description : seq id no : 2 : atggccacacagggacaacgcgtcaactggggagatgaaccttccaaaagacgtggtcgt60tctaactctcgtggtcggaagaataatgatatacctttgtcattctacaaccccattacc120ctcgaacaaggatctaaattttggaatttatgtccgagagaccttgttcccaaaggaata180ggtaataaggatcaacaaattggttattggaatagacagattcgttatcgtattgtaaaa240ggccagcgtaaggaactcgctgagaggtggttcttttacttcttaggtacaggacctcat300gctgatgctaaattcaaagacaagattgatggagtcttctgggttgcaagggatggtgcc360atgaacaagcccacaacgcttggcactcgtggaaccaataacgaatccaaaccactgaga420tttgatggtaagataccgccacagtttcagcttgaagtgaaccgttctaggaacaattca480aggtctggttctcagtctagatctgtttcaagaaacagatctcaatctagaggaagacac540cattccaataaccagaataataatgttgaggatacaattgtagccgtgcttgaaaaatta600ggtgttactgacaaacaaaggtcacgttctaaacctagagaacgtagtgattccaaacct660agggacacaacacctaagaatgccaacaaacacacctggaagaaaactgcaggcaaggga720gatgtgacaactttctatggtgctagaagtagttcagctaactttggtgatagtgatctc780gttgccaatggtaacgctgccaaatgctaccctcagatagctgaatgtgttccatcagtg840tctagcataatctttggcagtcaatggtctgctgaagaagctggtgatcaagtgaaagtc900acgctcactcacacctactacctgccaaaggatgatgccaaaactagtcaattcctagaa960cagattgacgcttacaagcgaccttctgaagtggctaaggatcagaggcaaagaagatcc1020cgttctaagtctgctgataagaagcctgaggagttgtctgtaactcttgtggaggcatac1080acagatgtgtttgatgacacacaggttgagatgattgatgaggttacgaactaa1134 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 8710 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : circular ( ii ) molecule type : dna ( genomic )( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : psc11f1 ( xi ) sequence description : seq id no : 3 : cgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttct60tagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttc120taaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataa180tattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt240gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgct300gaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc360cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta420tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacac480tattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggc540atgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaac600ttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggg660gatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgac720gagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggc780gaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt840gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctgga900gccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcc960cgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacag1020atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactca1080tatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc1140ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca1200gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc1260tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagcta1320ccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcctt1380ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc1440gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccggg1500ttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg1560tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgag1620cattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc1680agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat1740agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg1800gggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgc1860tggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtatt1920accgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtca1980gtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccg2040attcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac2100gcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccg2160gctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgac2220catgattacgccaagcttttgcgatcaataaatggatcacaaccagtatctcttaacgat2280gttcttcgcagatgatgattcattttttaagtatttggctagtcaagatgatgaaatctt2340cattatctgatatattgcaaatcactcaatatctagactttctgttattattattgatcc2400aatcaaaaaataaattagaagccgtgggtcattgttatgaatctctttcagaggaataca2460gacaattgacaaaattcacagactttcaagattttaaaaaactgtttaacaaggtcccta2520ttgttacagatggaagggtcaaacttaataaaggatatttgttcgactttgtgattagtt2580tgatgcgattcaaaaaagaatcctctctagctaccaccgcaatagatcctgttagataca2640tagatcctcgtcgcaatatcgcattttctaacgtgatggatatattaaagtcgaataaag2700tgaacaataattaattctttattgtcatcatgaacggcggacatattcagttgataatcg2760gccccatgttttcaggtaaaagtacagaattaattagacgagttagacgttatcaaatag2820ctcaatataaatgcgtgactataaaatattctaacgataatagatacggaacgggactat2880ggacgcatgataagaataattttgaagcattggaagcaactaaactatgtgatctcttgg2940aatcaattacagatttctccgtgataggtatcgatgaaggacagttctttccagacattg3000ttgaattccgagcttggctgcaggtcggggatcccccctgcccggttattattatttttg3060acaccagaccaactggtaatggtagcgaacggcgctcagctgaattccgccgatactgac3120gggctccaggagtcgtcgccaccaatccccatatggaaaccgtcgatattcagccatgtg3180ccttcttccgcgtgcagcagatggcgatggctggtttccatcagttgctgttgactgtag3240cggctgatgttgaactggaagtcgccgcgccactggtgtgggccataattcaattcgcgc3300gtcccgcagcgcagaccgttttcgctcgggaagacgtacggggtatacatgtctgacaat3360ggcagatcccagcggtcaaaacaggcggcagtaaggcggtcgggatagttttcttgcggc3420cctaatccgagccagtttacccgctctgctacctgcgccagctggcagttcaggccaatc3480cgcgccggatgcggtgtatcgctcgccacttcaacatcaacggtaatcgccatttgacca3540ctaccatcaatccggtaggttttccggctgataaataaggttttcccctgatgctgccac3600gcgtgaccggtcgtaatcagcaccgcatcagcaagtgtatctgccgtgcactgcaacaac3660gctgcttcggcctggtaatggcccgccgccttccagcgttcgacccaggcgttagggtca3720atgcgggtcgcttcacttacgccaatgtcgttatccagcggtgcacgggtgaactgatcg3780cgcagcggcgtcagcagttgttttttatcgccaatccacatctgtgaaagaaagcctgac3840tggcggttaaattgccaacgcttattacccagctcgatgcaaaaatccatttcgctggtg3900gtcagatgcgggatggcgtgggacgcggcggggagcgtcacactgaggttttccgccaga3960cgccactgctgccaggcgctgatgtgcccggcttctgaccatgcggtcgcgttcggttgc4020actacgcgtactgtgagccagagttgcccggcgctctccggctgcggtagttcaggcagt4080tcaatcaactgtttaccttgtggagcgacatccagaggcacttcaccgcttgccagcggc4140ttaccatccagcgccaccatccagtgcaggagctcgttatcgctatgacggaacaggtat4200tcgctggtcacttcgatggtttgcccggataaacggaactggaaaaactgctgctggtgt4260tttgcttccgtcagcgctggatgcggcgtgcggtcggcaaagaccagaccgttcatacag4320aactggcgatcgttcggcgtatcgccaaaatcaccgccgtaagccgaccacgggttgccg4380ttttcatcatatttaatcagcgactgatccacccagtcccagacgaagccgccctgtaaa4440cggggatactgacgaaacgcctgccagtatttagcgaaaccgccaagactgttacccatc4500gcgtgggcgtattcgcaaaggatcagcgggcgcgtctctccaggtagcgaaagccatttt4560ttgatggaccatttcggcacagccgggaagggctggtcttcatccacgcgcgcgtacatc4620gggcaaataatatcggtggccgtggtgtcggctccgccgccttcatactgcaccgggcgg4680gaaggatcgacagatttgatccagc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( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 9019 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : circular ( ii ) molecule type : dna ( genomic )( vi ) original source :( a ) organism : feline immunodeficiency virus ( vii ) immediate source :( b ) clone : psc11e1 ( xi ) sequence description : seq id no : 4 : gaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttctt60agacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttct120aaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataat180attgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttg240cggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctg300aagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatcc360ttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctat420gtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacact480attctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggca540tgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaact600tacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggggg660atcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacg720agcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcg780aactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttg840caggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggag900ccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctccc960gtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacaga1020tcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcat1080atatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcc1140tttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcag1200accccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgct1260gcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctac1320caactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttc1380tagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcg1440ctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggt1500tggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgt1560gcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagc1620attgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggca1680gggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttata1740gtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggg1800ggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgct1860ggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtatta1920ccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcag1980tgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccga2040ttcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacg2100caattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccgg2160ctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc2220atgattacgccaagcttttgcgatcaataaatggatcacaaccagtatctcttaacgatg2280ttcttcgcagatgatgattcattttttaagtatttggctagtcaagatgatgaaatcttc2340attatctgatatattgcaaatcactcaatatctagactttctgttattattattgatcca2400atcaaaaaataaattagaagccgtgggtcattgttatgaatctctttcagaggaatacag2460acaattgacaaaattcacagactttcaagattttaaaaaactgtttaacaaggtccctat2520tgttacagatggaagggtcaaacttaataaaggatatttgttcgactttgtgattagttt2580gatgcgattcaaaaaagaatcctctctagctaccaccgcaatagatcctgttagatacat2640agatcctcgtcgcaatatcgcattttctaacgtgatggatatattaaagtcgaataaagt2700gaacaataattaattctttattgtcatcatgaacggcggacatattcagttgataatcgg2760ccccatgttttcaggtaaaagtacagaattaattagacgagttagacgttatcaaatagc2820tcaatataaatgcgtgactataaaatattctaacgataatagatacggaacgggactatg2880gacgcatgataagaataattttgaagcattggaagcaactaaactatgtgatctcttgga2940atcaattacagatttctccgtgataggtatcgatgaaggacagttctttccagacattgt3000tgaattccgagcttggctgcaggtcggggatcccccctgcccggttattattatttttga3060caccagaccaactggtaatggtagcgaacggcgctcagctgaattccgccgatactgacg3120ggctccaggagtcgtcgccaccaatccccatatggaaaccgtcgatattcagccatgtgc3180cttcttccgcgtgcagcagatggcgatggctggtttccatcagttgctgttgactgtagc3240ggctgatgttgaactggaagtcgccgcgccactggtgtgggccataattcaattcgcgcg3300tcccgcagcgcagaccgttttcgctcgggaagacgtacggggtatacatgtctgacaatg3360gcagatcccagcggtcaaaacaggcggcagtaaggcggtcgggatagttttcttgcggcc3420ctaatccgagccagtttacccgctctgctacctgcgccagctggcagttcaggccaatcc3480gcgccggatgcggtgtatcgctcgccacttcaacatcaacggtaatcgccatttgaccac3540taccatcaatccggtaggttttccggctgataaataaggttttcccctgatgctgccacg3600cgtgaccggtcgtaatcagcaccgcatcagcaagtgtatctgccgtgcactgcaacaacg3660ctgcttcggcctggtaatggcccgccgccttccagcgttcgacccaggcgttagggtcaa3720tgcgggtcgcttcacttacgccaatgtcgttatccagcggtgcacgggtgaactgatcgc3780gcagcggcgtcagcagttgttttttatcgccaatccacatctgtgaaagaaagcctgact3840ggcggttaaattgccaacgcttattacccagctcgatgcaaaaatccatttcgctggtgg3900tcagatgcgggatggcgtgggacgcggcggggagcgtcacactgaggttttccgccagac3960gccactgctgccaggcgctgatgtgcccggcttctgaccatgcggtcgcgttcggttgca4020ctacgcgtactgtgagccagagttgcccggcgctctccggctgcggtagttcaggcagtt4080caatcaactgtttaccttgtggagcgacatccagaggcacttcaccgcttgccagcggct4140taccatccagcgccaccatccagtgcaggagctcgttatcgctatgacggaacaggtatt4200cgctggtcacttcgatggtttgcccggataaacggaactggaaaaactgctgctggtgtt4260ttgcttccgtcagcgctggatgcggcgtgcggtcggcaaagaccagaccgttcatacaga4320actggcgatcgttcggcgtatcgccaaaatcaccgccgtaagccgaccacgggttgccgt4380tttcatcatatttaatcagcgactgatccacccagtcccagacgaagccgccctgtaaac4440ggggatactgacgaaacgcctgccagtatttagcgaaaccgccaagactgttacccatcg4500cgtgggcgtattcgcaaaggatcagcgggcgcgtctctccaggtagcgaaagccattttt4560tgatggaccatttcggcacagccgggaagggctggtcttcatccacgcgcgcgtacatcg4620ggcaaataatatcggtggccgtggtgtcggctccgccgccttcatactgcaccgggcggg4680aaggatcgacagatttgatccagcgatacagcgcgtcgtgattagcgccgtggcctgatt4740cattccccagcgaccagatgatcacactcgggtgattacgatcgcgctgcaccattcgcg4800ttacgcgttcgctcatcgccggtagccagcgcggatcatcggtcagacgattgattggca4860ccatgccgtgggtttcaatattggcttcatccaccacatacaggccgtagcggtcgcaca4920gcgtgtaccacagcggatggttcggataatgcgaacagcgcacggcgttaaagttgttct4980gcttcatcagcaggatatcctgcaccatcgtctgctcatccatgacctgaccatgcagag5040gatgatgctcgtgacggttaacgcctcgaatcagcaacggcttgccgttcagcagcagca5100gaccattttcaatccgcacctcgcggaaaccgacatcgcaggcttctgcttcaatcagcg5160tgccgtcggcggtgtgcagttcaaccaccgcacgatagagattcgggatttcggcgctcc5220acagtttcgggttttcgaccttgagacgtagtgtgacgcgatcggcataaccaccacgct5280catcgataatttcaccgccgaaaggcgcggtgccgctggcgacctgcgtttcaccctgcc5340ataaagaaactgttacccgtaggtagtcacgcaactcgccgcacatctgaacttcagcct5400ccagtacagcgcggctgaaatcatcattaaagcgagtggcaacatggaaatcgctgattt5460gtgtagtcggtttatgcagcaacgagacgtcacggaaaatgccgctcatccgccacatat5520cctgatcttccagataactgccgtcactccaacgcagcaccatcaccgcgaggcggtttt5580ctccggcgcgtaaaaatgcgctcaggtcaaattcagacggcaaacgactgtcctggccgt5640aaccgacccagcgcccgttgcaccacagatgaaacgccgagttaacgccatcaaaaataa5700ttcgcgtctggccttcctgtagccagctttcatcaacattaaatgtgagcgagtaacaac5760ccgtcggattctccgtgggaacaaacggcggattgaccgtaatgggataggttacgttgg5820tgtagatgggcgcatcgtaaccgtgcatctgccagtttgaggggacgacgacagtatcgg5880cctcaggaagatcgcactccagccagctttccggcaccgcttctggtgccggaaaccagg5940caaagcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcct6000cttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaa6060cgccagggttttcccagtcacgacgttgtaaaacgacgggatccctcgaggaattcattt6120atagcatagaaaaaaacaaaatgaaattctactatatttttacatacatatattctaaat6180atgaaagtggtgattgtgactagcgtagcatcgcttctagacatatactatatagtaata6240ccaatactcaagactacgaaactgatacaatctcttatcatgtgggtaatgttctcgatg6300tcgaatagccatatgccggtagttgcgatatacataaactgatcactaattccaaaccca6360cccgctttttatagtaagtttttcacccataaataataaatacaataattaatttctcgt6420aaaagtagaaaatatattctaatttattgcacggtaaggaagtagaatcataaagaacag6480tgacggatcccgggatggccacacagggacaacgcgtcaactggggagatgaaccttcca6540aaagacgtggtcgttctaactctcgtggtcggaagaataatgatatacctttgtcattct6600acaaccccattaccctcgaacaaggatctaaattttggaatttatgtccgagagaccttg6660ttcccaaaggaataggtaataaggatcaacaaattggttattggaatagacagattcgtt6720atcgtattgtaaaaggccagcgtaaggaactcgctgagaggtggttcttttacttcttag6780gtacaggacctcatgctgatgctaaattcaaagacaagattgatggagtcttctgggttg6840caagggatggtgccatgaacaagcccacaacgcttggcactcgtggaaccaataacgaat6900ccaaaccactgagatttgatggtaagataccgccacagtttcagcttgaagtgaaccgtt6960ctaggaacaattcaaggtctggttctcagtctagatctgtttcaagaaacagatctcaat7020ctagaggaagacaccattccaataaccagaataataatgttgaggatacaattgtagccg7080tgcttgaaaaattaggtgttactgacaaacaaaggtcacgttctaaacctagagaacgta7140gtgattccaaacctagggacacaacacctaagaatgccaacaaacacacctggaagaaaa7200ctgcaggcaagggagatgtgacaactttctatggtgctagaagtagttcagctaactttg7260gtgatagtgatctcgttgccaatggtaacgctgccaaatgctaccctcagatagctgaat7320gtgttccatcagtgtctagcataatctttggcagtcaatggtctgctgaagaagctggtg7380atcaagtgaaagtcacgctcactcacacctactacctgccaaaggatgatgccaaaacta7440gtcaattcctagaacagattgacgcttacaagcgaccttctgaagtggctaaggatcaga7500ggcaaagaagatcccgttctaagtctgctgataagaagcctgaggagttgtctgtaactc7560ttgtggaggcatacacagatgtgtttgatgacacacaggttgagatgattgatgaggtta7620cgaactaaacgcatgcccgggaattctgtgagcgtatggcaaacgaaggaaaaattagtt7680atagtagccgcactcgatgggacatttcaacgtaaaccgtttaataatattttgaatctt7740attccattatctgaaatggtggtaaaactaactgctgtgtgtatgaaatgctttaaggag7800gcttccttttctaaacgattgggtgaggaaaccgagatagaaataataggaggtaatgat7860atgtatcaatcggtgtgtagaaagtgttacatcgactcataatattatattttttatcta7920aaaaactaaaaataaacattgattaaattttaatataatacttaaaaatggatgttgtgt7980cgttagataaaccgtttatgtattttgaggaaattgataatgagttagattacgaaccag8040aaagtgcaaatgaggtcgcaaaaaaactgccgtatcaaggacagttaaaactattactag8100gagaattattttttcttagtaagttacagcgacacggtatattagatggtgccaccgtag8160tgtatataggatctgctcccggtacacatatacgttatttgagagatcatttctataatt8220taggagtgatcatcaaatggatgctaattgacggccgccatcatgatcctattttaaatg8280gattgcgtgatgtgactctagtgactcggttcgttgatgaggaatatctacgatccatca8340aaaaacaactgcatccttctaagattattttaatttctgatgtgagatccaaacgaggag8400gaaatgaacctagtacggcggatttactaagtaattacgctctacaaaatgtcatgatta8460gtattttaaaccccgtggcgtctagtcttaaatggagatgcccgtttccagatcaatgga8520tcaaggacttttatatcccacacggtaataaaatgttacaaccttttgctccttcatatt8580cagggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcg8640ccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcg8700cccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctctt8760tacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtaccatctgctctga8820tgccgcatagttaagccagtacactccgctatcgctacgtgactgggtcatggctgcgcc8880ccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgc8940ttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatc9000accgaaacgcgcgaggcag9019 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 262 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : n - terminal ( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : fipv e1 ( xi ) sequence description : seq id no : 5 : metlystyrileleuleuileleualacysileilealacysvaltyr151015glygluargtyrcysalametglnaspserglyleuglncysileasn202530glythrasnserargcysglnthrcysphegluargglyaspleuile354045trphisleualaasntrpasnphesertrpservalileleuileval505560pheilethrvalleuglntyrglyargproglnphesertrpleuval65707580tyrglyilelysmetleuilemettrpleuleutrpproilevalleu859095alaleuthrilepheasnalatyrserglutyrglnvalserargtyr100105110valmetpheglypheservalalaglyalavalvalthrphealaleu115120125trpmetmettyrphevalargservalglnleutyrargargthrlys130135140sertrptrpserpheasnprogluthrasnalaileleucysvalasn145150155160alaleuglyargsertyrvalleuproleuaspglythrprothrgly165170175valthrleuthrleuleuserglyasnleutyralagluglyphelys180185190metalaglyglyleuthrilegluhisleuprolystyrvalmetile195200205alathrproserargthrilevaltyrthrleuvalglylysglnleu210215220lysalathrthralathrglytrpalatyrtyrvallysserlysala225230235240glyasptyrserthrglualaargthraspasnleusergluhisglu245250255lysleuleuhismetval260 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 377 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : n - terminal ( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : fipv n ( xi ) sequence description : seq id no : 6 : metalathrglnglyglnargvalasntrpglyaspgluproserlys151015argargglyargserasnserargglyarglysasnasnaspilepro202530leuserphetyrasnproilethrleugluglnglyserlysphetrp354045asnleucysproargaspleuvalprolysglyileglyasnlysasp505560glnglnileglytyrtrpasnargglnileargtyrargilevallys65707580glyglnarglysgluleualagluargtrpphephetyrpheleugly859095thrglyprohisalaaspalalysphelysasplysileaspglyval100105110phetrpvalalaargaspglyalametasnlysprothrthrleugly115120125thrargglythrasnasngluserlysproleuargpheaspglylys130135140ileproproglnpheglnleugluvalasnargserargasnasnser145150155160argserglyserglnserargservalserargasnargserglnser165170175argglyarghishisserasnasnglnasnasnasnvalgluaspthr180185190ilevalalavalleuglulysleuglyvalthrasplysglnargser195200205argserlysproarggluargseraspserlysproargaspthrthr210215220prolysasnalaasnlyshisthrtrplyslysthralaglylysgly225230235240aspvalthrthrphetyrglyalaargserserseralaasnphegly245250255aspseraspleuvalalaasnglyasnalaalalyscystyrprogln260265270ilealaglucysvalproservalserserileilepheglysergln275280285trpseralagluglualaglyaspglnvallysvalthrleuthrhis290295300thrtyrtyrleuprolysaspaspalalysthrserglnpheleuglu305310315320glnileaspalatyrlysargprosergluvalalalysaspglnarg325330335glnargargserargserlysseralaasplyslysproglugluleu340345350servalthrleuvalglualatyrthraspvalpheaspaspthrgln355360365valglumetileaspgluvalthrasn370375 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 24 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : n primer # 1 ( xi ) sequence description : seq id no : 7 : ctcgtggtcggaagaataatgata24 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 24 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : n primer # 2 ( xi ) sequence description : seq id no : 8 : agcaccatagaaagttgtcacatc24 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 22 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : e1 primer # 1 ( xi ) sequence description : seq id no : 9 : tatgtaatgttcggctttagtg22 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 22 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : other nucleic acid ( vi ) original source :( a ) organism : feline infectious peritonitis virus ( vii ) immediate source :( b ) clone : e1 primer # 2 ( xi ) sequence description : seq id no : 10 : gtgcttctgttgagtaatcacc22__________________________________________________________________________