Paramyxovirus and uses thereof

Abstract

The present invention relates to a novel feline paramyxovirus. The paramyxovirus of the present invention is a (-)ssRNA virus and has in one aspect a genome which is complementary to the nucleic acid according to SEQ ID NO:1 or SEQ ID NO:8. The invention further relates to corresponding nucleic acids and polypeptides, antibodies and vaccines. Further, the invention relates to medical uses and diagnostic methods concerning the paramyxovirus of the invention.

Claims

1. A nucleic acid comprising a nucleotide sequence encoding a paramyxovirus wherein the nucleotide sequence comprises a polynucleotide selected from the group consisting of a DNA sequence being at least 80% identical to SEQ ID NO: 1 or SEQ ID NO:8 and the complementary strand of any of the nucleotide sequences thereof.

2. A vector comprising the nucleic acid according to claim 1.

3. A host cell comprising the vector according to claim 2.

4. A polypeptide having an amino acid sequence encoded by the nucleic acid of claim 1 [selected from the group consisting of (a) an amino acid sequence which is at least 90% identical to SEQ ID NO:2 or 9, (b) an amino acid sequence which is at least 76% identical to SEQ ID NO:3 or 10, (c) an amino acid sequence which is at least 92% identical to SEQ ID NO:4 or 11, (d) an amino acid sequence which is at least 89% identical to SEQ ID NO:5 or 12, (e) an amino acid sequence which is at least 86% identical to SEQ ID NO:6 or 13, or (f) an amino acid sequence which is at least 91% identical to SEQ ID NO:7 or 14].

5. An immunogenic composition comprising at least one member selected from the group consisting of: (a); a nucleic acid according to claim 1; (b) a polypeptide according to claim 4; and (c) a polypeptide according to claim 4, which is fused to a heterologous or autologous (poly-)peptide, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient suitable for intradermal or intramuscular application, and optionally said immunogenic composition further comprises an adjuvant.

6. A kit [for vaccinating a feline against a disease associated with and/or reducing the incidence or the severity of one or more clinical signs associated with or caused by a paramyxovirus in a subject] comprising: (a) a dispenser capable of administering a vaccine to the subject; (b) the immunogenic composition according to claim 5; and (c) optionally an instruction leaflet.

7. An immunogenic composition comprising; the paramyxovirus which is deposited under accession no. CNCM I-5123 or the paramyxovirus which is deposited under accession no. CNCM I-5123 wherein the paramyxovirus is attenuated, a pharmaceutically acceptable carrier or excipient suitable for intradermal or intramuscular application, and optionally said immunogenic composition further comprises an adjuvant.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Detection of feline paramyxovirus (FPaV-2) in cell culture supernatants.

(2) Shown is the analysis of the cell culture supernatant from passage 3 of CrFK and LLC-MK2 cells, respectively, after infection with the FPaV-2 “Gordon” strain. M: DNA size standard; 1: cell culture supernatant of CrFK cells; 2: cell culture supernatant of LLC-MK2 cells; 3: water; 4: positive control.

(3) FIG. 2: Immunofluorescence of LLC-MK2 cells infected with the FPaV-2 “Gordon” isolate.

(4) Cell nuclei stained with DAPI. Cells that are FPaV-2 infected show green fluorescence. Magnification: 200×; staining 5 days after infection.

(5) FIG. 3: Isolation of feline primary kidney cells. Magnification: 100×, 48 hours after seeding.

(6) FIG. 4: Infection of feline primary kidney cells with FPaV-2.

(7) Cell nuclei are stained with DAPI. Cells that are FPaV-2 infected show green fluorescence. Magnification: 200×; staining 5 days after infection.

(8) FIG. 5: Antibody diversity of FPaV-2-infected cats.

(9) Western-blot analysis of semi-purified whole FPaV-2 separated by SDS-PAGE and blotted onto a nitrocellulose membrane. A.=Incubation with 1:100 diluted serum sample from FPaV-2-positive cat ‘TV25’. B.=Incubation with 1:100 diluted sample from paramyxovirus-negative cat. C.=Incubation with 1:200 diluted anti-nucleocapsid antibody.

(10) Specific reactions to viral proteins are annotated at the right bottom based on their reactivity with target antibodies (nucleocapsid-protein and phospho-protein) or based on their predicted molecular weight (polymerase- and hemagglutinin-protein).

(11) FIG. 6: Development of an ELISA for the detection of FPaV-2 antibodies in cat serum samples.

(12) Cat serum samples were screened in FPaV-2-IFA for the presence of specific antibodies. IFA result was set as gold standard and compared to OD values. Samples having in OD value below 0.5 are defined as FPaV-2 negative, whereas samples with an OD value higher than 0.7 are defined as FPaV-2-positive. Grey box indicates ‘borderline’-samples which need to be checked in IFA to evaluate the ELISA result.

(13) FIG. 7: Primary immune target cells of FPaV-2.

(14) Flow cytometric analysis of PBMCs 48 hours after infection with FPaV-2 at an MOI of 0.1. Cells were stained for surface markers of T-cells (CD4) or B-cells (CD20) and for the intracellular presence of FPaV-2 using a polyclonal nucleocapsid antibody.

(15) A. and C.=Mock-infected PBMCs

(16) B. and D.=FPaV-2-infected PBMCs

(17) FIG. 8: Western-blot analysis of an FPaV-2-immunized rabbit.

(18) Semi-purified whole FPaV-2 was separated by SDS-PAGE and blotted onto a nitrocellulose membrane.

(19) A.=Incubation with serum sample (1:100 dilution) from rabbit no. 2 before immunization (pre-immune serum).

(20) B.=Incubation with serum sample (1:100 dilution) from rabbit no. 2 five weeks after immunization with heat-inactivated FPaV-2.

(21) Specific reactions to viral proteins are annotated at the right bottom based on reactivity shown in FIG. 5.

(22) In contrast to the pre-immune serum FPaV-2-specific antibodies were detected five weeks after Immunization.

EXAMPLES

Example 1: Detection of FPaV-2

(23) Collection of Sample:

(24) Urine from a 13 year old male cat with a chronic kidney disease is collected and stored on ice.

(25) RNA-Isolation:

(26) RNA is isolated from 300 μl urine using the ‘QIAamp Viral RNA Mini Kit’ (Qiagen, Hilden), eluted in 50 μl of buffer AVE and stored at −80° C.

(27) RT-PCR:

(28) RT-PCR is performed in a single step using the ‘SuperScript III One Step RT-PCR System with Platinum Taq High Fidelity’ (Life Technologies) as described by Tong et al. (2008) (J Clin Microbiol. 46(8):2652-8) with some minor modifications: Nine microliter of RNA are mixed with 12.5 μl reaction buffer (2-fold, 0.4 mM dNTPs each and 2.4 mM MgSO.sub.4), 2 μl magnesium sulphate (5 mM), 0.25 μl primer RES-MOR-HEN-R (100 μM), 0.25 μl primer RES-MOR-HEN-F1 (100 μM), 0.5 μl RNase inhibitor (40 U/μl, Life Technologies) and 0.5 μl SuperScript III/Platinum Taq High Fidelity Enzyme Mix′. Samples are then treated according to the following thermal profile: one minute at 60° C., 30 minutes at 45° C. and 2 minutes at 94° C. After this treatment samples are heated as follows: 45 cycles at 94° C. for 15 seconds, 48° C. for 30 seconds and 68° C. for 30 seconds with a final elongation step at 68° C. for 5 minutes. PCR products are visualized using agarose gel electrophoresis in Tris-acetate-EDTA-buffer (40 mM Tris-acetate, 1 mM EDTA, pH=8.3) including 0.2 μg/ml of ethidium bromide. A specific PCR fragment having a size of about 611 bp is cut out of the gel and purified using the ‘Gel/PCR DNA Fragments Extraction Kit’ (Geneaid, Taiwan).

(29) Sequencing:

(30) The PCR fragment is sequenced by applying the Sanger didesoxy method using RES-MOR-HEN-R (10 μM) and RES-MOR-HEN-FI (10 μM) as sequencing priming. Resulting chromatograms are edited using the software ‘BioEdit’ (version 7.2.4) and aligned with the ‘Basic Local Alignment Search Tool’ (BLAST) on the NCBI website.

Example 2: Isolation of FPaV-2

(31) For virus cultivation LLC-MK2 and CrFK cells are seeded in 75 cm.sup.2 cell culture flasks in DMEM (with sodium pyruvat and non-essential amino acids) with 5% of FBS in an atmosphere including 5% carbon dioxide at 37° C. and 90% humidity. At 70-80% confluence cells are infected with a mixture of one milliliter urine and 5 ml DMEM (with penicillin and streptomycin) over night at 37° C., 5% CO.sub.2 and 90% humidity.

(32) After 24 hours the infection medium was replaced by 8 ml of cultivation medium (DMEM, sodium pyruvat, non-essential amino acids, 5% FBS, penicillin and streptomycin) and cultivated for further 6 days at the indicated conditions. The cell culture supernatant from this infection is passaged for further three times. Afterwards 600 μl of the cell culture supernatant are tested as described in Example 1 for the presence of feline paramyxoviruses. FIG. 1 shows the result of such an experiment.

Example 3: Immunofluorescence Detection of FPaV-2

(33) To detect FPaV-2 infections LLC-MK2 cells are infected as described in Example 2 and stained with a FPaV-2-specific antibody using immunofluorescence techniques.

(34) For this purpose adherent cells are washed with PBS after an infection period of 5 days and subsequently fixed with 80% of acetone at −20° C. for 10 minutes. Cells are washed twice with PBS and unspecific binding is blocked by incubation with 5% BSA in PBS at 37° C. for one hour.

(35) This is followed by an incubation step with anti-FPaV-2 antibody (anti-FPaV-2 nucleocapsid, polyclonal, rabbit) at a final concentration of 1 μg/ml in 1% BSA in PBS for one hour at 37° C. Cells are washed three time with PBS followed by the application of ‘Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate’ (Thermo Fisher Scientific) at a final dilution of 1:1000 in 1% BSA in PBS. After an incubation time of one hour at 37° C. cells are washed twice with PBS and cells were screened for the presence of FPaV-2 using a fluorescence microscope. Results are shown in FIG. 2.

Example 4: Infection Spectrum of FPaV-2

(36) To analyze the in vitro susceptibility of FPaV-2 different cell lines are infected and then analyzed using immunofluorescence techniques as described in Example 3. Table 3 reflects the result of such an experiment.

(37) TABLE-US-00003 TABLE 3 in vitro spectrum of FPaV-2 infection Infektion Cell line Tissue Species with FPaV-2 CrFK Kidney, epithel Cat positive CrFK/CatSLAM Kidney, epithel, Cat positive transfected with feline CD150 FE Embryonal, epithelial Cat positive and fibroblastic Vero (CCL-81) Kidney, epithel Vervet monkey positive LLC-MK2 Kidney, epithel Rhesus monkey positive BHK-21 Kidney, fibroblast Syrian golden positive hamsters

Example 5: Infection of Feline Primary Kidney Cells with FPaV-2

(38) To investigate whether primary cells are also susceptible to FPaV-2 primary feline kidney cells are isolated. For this purpose kidneys from an euthanized cat are removed under aseptic conditions and stored on ice immediately. Then the capsule of the kidney is detached and the cortex is cut into small pieces and rinsed five times in HBSS. These tissue pieces are then treated with 0.1 percent of trypsin in HBSS for 20 minutes at 37° C. in a vertical shaker. The cell suspension is filtered through a 100 μm nylon filter and the filtrate is centrifuged at 400×g for 10 minutes. The cell pellet is re-suspended in complete kidney medium (1:1 mixture of DMEM and Hams-F12 medium supplemented with ‘Insulin-Transferrin-Selenium-Ethanolamine’ [Thermo Fisher Scientific], sodium pyruvate, non-essential amino acids, 10% FBS, penicillin and streptomycin), seeded out in tissue flasks and incubated at 37° C., 5% CO.sub.2 and 90% humidity. FIG. 3 shows a typical result of such an experiment.

(39) The previously described primary feline kidney cells are infected with FPaV-2 as described in Example 2 and stained for the presence of FPaV-2 as described in Example 3. FIG. 4 shows a result of such an experiment.

Example 6: Determination and Analysis of the Full-Length FPaV-2 Genome

(40) For obtaining the whole genome sequence of the FPaV-2-cell culture isolate ‘Gordon’ RNA is isolated from the cell culture supernatant as described in Example 1. FPaV-2-specific PCR products are then generated by using the one-step-PCR-system described in example no. 1 and a primer-walking strategy. Amplification products are isolated from the agarose gel with the help of the ‘ Gel/PCR DNA Fragments Extraction Kit’ (Geneaid) and sequenced by the sanger didesoxy method with the corresponding amplification primers. Each PCR fragment is sequenced twice. The alignment result of the obtained FPaV-2 sequence with the FmoPV-isolate M252A (Woo et al. (2012), Proc. Nat. Acad. Sci. 109(14):5435-5440); Accession number: JQ411016.1) is shown in table 4.

(41) TABLE-US-00004 TABLE 4 Analysis of whole genome sequence of FPaV-2 (strain, Gordon‘) Nucleotide Amino acid Nucleotide homology to homology to position FmoPV-M252A FmoPV M252A Genome region (cRNA) (nt's/nt's) (aa/aa) 3′ untranslated  1-107 84.1% (90/107) not applicable Region (UTR) Nucleocapsid  108-1667 81.1% (1266/1560) 89.8% (466/519) protein Intergenic sequence 1668-1780 39.8% (45/113) not applicable Phospho protein 1781-3256 80.5% (1188/1476) 75.1% (369/491) Intergenic sequence 3257-3388 44.7% (59/132) not applicable Matrix protein 3389-4402 83.3% (845/1014) 91.7% (309/337) Intergenic sequence 4403-4949 48.6% (268/551) not applicable Fusion protein 4950-6581 80.9% (1320/1632) 88.7% (482/543) Intergenic sequence 6582-6958 56% (211/377) not applicable Hemagglutinin 6959-8746 80.5% (1435/1788) 85.9% (511/595) protein Intergenic sequence 8747-8887 54.6% (77/141) not applicable Polymerase protein  8888-15496 82.5% (5453/6609)  90.9% (2001/2202) 5′ untranslated 15497-16047 52.4% (289/551) not applicable region (UTR) Whole genome   1-16047 78.2% (12546/16047) not applicable nt’s: nucleotides aa: amino acids

Example 7: Prevalence of FPaV-2 in Urine Samples

(42) To elucidate the prevalence of FPaV-2 in domestic cats, urine samples were collected by cystocentesis, stored immediately at −20° C. and analyzed for the presence of FPaV-2-RNA as described in example 1. Results are shown in table 5.

(43) TABLE-US-00005 TABLE 5 Prevalence of FPaV-2 in urine samples of domestic cats Characteristics Diseased group Healthy group Number of 325 238 tested urines Clinical and FLUTD, nephritis, anuria, No history of laboratory polyuria, urolithiasis, urotract diseases findings cystitis, hematuria, leukocyturia, lipiduria, proteinuria Mean age in years 8 (1-19 years) 10 (0.5-21 years) Male cats 72% 61% RT-PCR positive 4 (1.2%) 0 (0%)

Example 8: Heterogeneity of FPaV-2 Strains

(44) Using the procedure described in example 1, a second strain of FPaV-2 was isolated from a male cat suffering from feline urologic syndrome. The viral isolate (named ‘TV25’) was subjected to whole genome sequencing using primer walking strategy and sanger dideoxy DNA sequencing method. Results are summarized in table 6, nucleotide sequence is shown in SEQ ID NO:8.

(45) TABLE-US-00006 TABLE 6 Comparison of whole genome sequences of the two FPaV-2 isolates ‘Gordon’ and ‘TV25’ Nucleotide Amino acid similarity between similarity between Nucleotide ‘Gordon’ and ‘Gordon’ and position ‘TV25’ ‘TV25’ Genome region (cRNA) (nucleotides) (amino acids) 3′ untranslated  1-107 100% (107/107) not applicable Region (UTR) Nucleocapsid  108-1667 99.2% (1547/1560) 99.2% (515/519) protein Intergenic sequence 1668-1780 98.2% (111/113) not applicable Phospho protein 1781-3256 99.6% (1470/1476) 98.8% (485/491) Intergenic sequence 3257-3388 98.5% (130/132) not applicable Matrix protein 3389-4402 99.4% (1008/1014) 99.7% (336/337) Intergenic sequence 4403-4949 98% (540/551) not applicable Fusion protein 4950-6581 99.4% (1622/1632) 99.4% (540/543) Intergenic sequence 6582-6958 97.6% (368/377) not applicable Hemagglutinin 6959-8746 99.2% (1774/1788) 99.5% (592/595) protein Intergenic sequence 8747-8887 97.9% (138/141) not applicable Polymerase protein  8888-15496 99.2% (65 54/6609)  99.5% (2191/2202) 5′ untranslated 15497-16047 99.4% (548/551) not applicable region (UTR) Whole genome   1-16047 99.2% (15917/16047) not applicable

Example 9: Electron Microscopy of FPaV-2

(46) 15 ml of the a FPaV-2-cell culture supernatant (described in example 2) was centrifuged at 3.000×g for 10 minutes at 4° C. followed by filtration through a 0.45 μm cellulose nitrate filter. The filtrate was overlayed on a 20% (w/v) sucrose cushion and centrifuged at 100.000×g for 90 minutes at 4° C. The pellet was then suspended in 100 μl of PBS and the virus was allowed to absorb to a formvar/carbon coated 300 mesh copper grid for five minutes at room temperature. After three washing steps with distillated water viral particles were stained with 2% (w/v) uranyl acetate for 30 seconds. Analysis of this sample using a transmission electron microscope revealed typical paramyxoviral morphology: pleomorphic, enveloped viral particles having a size of 100-150 nanometers.

Example 10: Antibody Diversity of FPaV-2-Infected Cats

(47) To investigate the antibody diversity of cats being naturally infected with FPaV-2, semi-purified viral particles (as shown in example 9) were mixed with an equal volume of SDS-loading buffer (100 mM Tris-HCl, pH 6.8; 4% (w/v) sodium dodecyl sulfate; 0.2% (w/v) bromophenol blue; 20% (v/v) glycerol; 200 mM β-mercaptoethanol), heated at 95° C. for five minutes and loaded onto an 8% polyacrylamide gel. Viral proteins were separated by electrophoresis at 130 V for 90 minutes in SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) followed by blotting to a nitro cellulose membrane.

(48) After blocking with 5% (w/v) non-fat dry milk in PBS-T (0.05 tween 20) for 30 minutes at room temperature the membrane was incubated over night at 4° C. with cat serum samples diluted 1:100 in block buffer. The membrane was washed three times with PBS-T and incubated with horseradish peroxidase conjugated α-Cat-IgG antibody diluted 1:1.000 in blocking buffer for one hour at room temperature. 3,3′-Diaminobenzidine was used for signal development. As shown in FIG. 5A, FPaV-2-infected cats can develop antibodies against a broad spectrum of viral structural proteins, e.g. the polymerase-, phospho-, nucleocapsid- and hemagglutinin-protein. Specific reactions to viral proteins were annotated based on their reactivity with target antibodies as shown for the nucleocapsid-protein (FIG. 5C).

(49) The phospho-protein was proved to be heavily phosphorylated using a phospho-serine Antibody (Q5 from QIAGEN N.V.) shifting the calculated molecular weight from 53 kDa to about 75 kDa. This phenomenon of molecular weight shift is known from other morbilliviruses like Measles Virus (phospho-protein=70 kDa) and Canine Distemper Virus (phospho-protein=73 kDa). Annotations of specific reactions against the polymerase- and hemagglutinin-protein were done based on the predicted molecular weight from their amino acid sequences.

Example 11: Development of a Serum Neutralization Test for FPaV-2

(50) To detect neutralizing antibodies against FPaV-2 a serum neutralization assay (SNT) was established. Therefore, cat serum samples were treated at 56° C. for 30 minutes to inactivate complement factors. 50 μl of these heat inactivated serum samples were mixed with 50 μl DMEM containing 100 fluorescence forming units (FFU) of FPaV-2 (isolate ‘Gordon’) and were then incubated for one hour at 4° C. The mixture was used to infect LLC-MK2-cells in a 96-well cell culture plate for two hours at 37° C. Then the serum/virus-mixture was removed and replaced by DMEM containing 2% (v/v) heat inactivated FBS, sodium pyruvate, non-essential amino acids, penicillin and streptomycin. The cells were incubated for five days at 37° C., 5% CO.sub.2 and 90% humidity followed by immunofluorescence staining as described in example 3. The neutralization titer of the test serum sample is defined as the reciprocal of the highest test serum dilution for which the virus infectivity is reduced by 50% when compared to the virus control without serum incubation.

Example 12: Screening for Neutralizing Antibodies in FPaV-2-Infected Cats

(51) Serum samples of naturally FPaV-2-infected cats were screened for the presence of neutralizing antibodies using the SNT described in example 11. The results of these experiments are shown in table 7. They clearly show that an FPaV-2-infection can induce high titers of neutralizing antibodies against the virus (see FPaV-2-SNT results of cat serum samples 98450 and TV25 in table 7). In contrast, serum samples from canine distemper virus-infected cats (sample CDV in table 7) and feline paramyxovirus-negative (sample TV26 in table 7) cats show no neutralizing antibodies, highlighting that the detected antibody titers are FPaV-2 specific.

(52) TABLE-US-00007 TABLE 7 Results of FPaV-2 serum neutralization tests. Cat serum ID 98450 TV25 TV26 CDV PCR result FPaV2 positive Paramyxovirus — from urine negative Presence of viral >14 >18 — — RNA in urine weeks months IFA Pos. Pos. Neg. Neg. FPaV-2 320 320 <10 <10 SNT titer IFA: immune fluorescence assay, CDV: canine distemper virus-positive sample

Example 13: ELISA-Development for Screening of FPaV-2 Antibodies in Cat Serum Samples

(53) To elucidate the prevalence of FPaV-2 infections in German cat populations, an ELISA-system based on recombinant expressed nucleocapsid was established. Therefore, the complete open-reading frame of the FPaV-2-nucleocapsid (SEQ ID NO:2) was cloned into the expression vector ‘pGEX-4T-1’ using the restriction enzymes BamHI and XhoI. The resulting recombinant expression plasmid (′pGEX-Gordon-NC) was transformed into chemical competent E. coli BL21(DE3) by standard techniques and recombinant bacteria were selected on LB-agar with 100 μg/ml Ampicillin resulting in an E. coli-clone named ‘E. coli-Gordon-NC’. This clone was inoculated into 1.000 ml LB medium with 0.2% glucose and 100 μl/ml ampicillin and was shaken at 37° C. with 200 rpm until the culture reached an optical density of A.sub.600 nm=1.0. At that point the culture was cooled down to 22° C. and recombinant protein expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM. After incubating for 20 hours at 22° C. and 220 rpm the culture was centrifuged at 3.000×g, 4° C. for 20 minutes and the resulted pellet was sonicated to disrupt the E. coli cells. Purification of recombinant GST-fusion protein was performed using Glutathione Sepharose 4B (GE Healthcare Life Science) as described by the manufacture.

(54) 200 ng of the resulted GST-Gordon-NC-protein was coated per well in a Nunc MaxiSorp ELISA plate over night at 4° C. followed by three washing steps with PBS-T (0.05% tween 20). Free protein binding sites were blocked using 5% (w/v) non-fat dry milk in PBS-T (blocking buffer) for 30 minutes at 37° C. Serum samples were diluted 1:100 in blocking buffer and incubated for two hours at 37° C. on the primed ELISA-plate. After three washing steps with PBS-T, wells were incubated with secondary antibody (goat anti cat IgG (Fc):HRP, Bio-Rad, diluted 1:10.000 in blocking buffer) for one hour at 37° C. Unbound antibody was washed off by three PBS-T washing cycles and subtract solution (OPD Substrate Tablets, Thermo Fisher Scientific) was applied to each wells, incubated for five minutes at 22° C. before enzymatic reaction was stopped by 2.5 M sulfuric acid. Absorbance was measured at 490 nm. To define the cut-off of this ELISA-system, serum samples from cats were screened for FPaV-2-specific reactions applying the immunofluorescence test described in example 3.

(55) Results are shown in FIG. 6. IFA result was set as gold standard and compared to OD values. Samples having in OD value below 0.5 were defined as FPaV-2-negative, whereas samples with an OD value higher than 0.7 were defined to be FPaV-2-positive. ‘Borderline’-samples with an OD value between 0.5 and 0.7 need to be checked in IFA to evaluate the ELISA result. Using this approach, 13 out of 230 serum samples (=5.6%) from domestic cats were positive for FPaV-2. This result reflects the relatively high prevalence of FPaV-2 in German cat populations.

Example 14: Primary Target Cells of FPaV-2

(56) Feline PBMCs were in vitro infected with FPaV-2 to uncover whether feline immune cells are also targets of FPaV-2. For this purpose peripheral blood mononuclear cells (PBMCs) were isolated from a healthy male cat using standard Ficoll density gradient centrifugation. PBMCs were treated with 2% (v/v) phytohemagglutinin (M-from, crude extract, Thermo Fisher Scientific) in RPMI for four hours at 37° C. followed by infection with FPaV-2 at an MOI of 0.1 or were mock-infected for two hours at 37° C. PBMCs were washed once with PBS and then incubated at 37° C., 5% CO.sub.2 and 90% humidity for 48 hours. Cells were stained for CD4 and CD20 surface markers using standard flow cytometry protocols. After a fixation step with 2% (w/v) paraformaldehyde for 10 minutes at 4° C. cells were intracellularly stained with anti-FPaV-2-nucleocapsid antibody (see example 2) in facs-buffer (PBS, pH=7.4; 3% FBS; 0.1% sodium azide) with 0.5 (w/v) saponin for 30 minutes at 22° C. Stained cells were washed tree times with facs-buffer and analyzed using the LSRFortessa™ (Becton Dickinson) cell analyzer. As shown in FIG. 7 feline CD4-positive T-cells as well as feline CD20-positive B-cells are target cells of FPaV-2. These results clearly indicate that a systemic state of FPaV-2 is likely to occur.

Example 15: Immunization of Rabbits with Inactivated FPaV-2

(57) The aim of this ‘proof-of-concept’-experiment was to elucidate whether an immunization with inactivated FPaV-2 will induce neutralizing antibodies and can therefore serve as a potential vaccine candidate. A male rabbit was immunized with a vaccine mixture of 1 ml heat-inactivated (3 hours at 56° C.) FPaV-2-strain ‘Gordon’ (1*10.sup.5 FFU/ml, see example no. 2) and 2 ml an adjuvant (92.8% mineral oil; 3.48% Tween 80; 3.48% Span 80; 23% lipopolysaccharide). For the negative control animal the same volume of a cell culture supernatant from a mock-infection (no virus) mixed with the adjuvant was used instead of FPaV-2.

(58) Immunization was done according to the following scheme: 1.sup.st Immunization: Collecting of pre-immune serum, 1 ml subcutaneous and 1 ml intramuscular of vaccine mixture was than inoculated. 2.sup.nd Immunization 14 days after 1.sup.st immunization: 1 ml subcutaneous and 1 ml intramuscular of vaccine mixture was inoculated. 3.sup.rd Immunization 7 days after 2.sup.nd immunization: 1 ml subcutaneous and 1 ml intramuscular of vaccine mixture was inoculated. final bleed (rabbits were killed): 14 days after the third immunization

(59) The final serum samples were first tested in a western-blot analysis using a whole virus preparation as described in example 10. This experiment showed that five weeks after immunization specific antibodies against the polymerase-, phospho-, hemagglutinin- and nucleocapsid-protein were detected in the FPaV-2 vaccinated animal (see FIG. 8B). The specificity of the observed reactions was confirmed by analyzing the pre-immune serum of this rabbit. As shown in FIG. 8A no virus-specific antibodies were present before immunization. In addition, the staining pattern of the post-immunization serum sample (FIG. 8B) was identical to the results from naturally FPaV-2-infected cats (see FIG. 5A) proving the virus-specific nature of the observed bands in the presented western-blot analyses. Thus, the applied FPaV-2 vaccination formula is able to induce a broad spectrum of anti-FPaV-2 antibodies in animals. To elucidate whether these antibodies have neutralizing properties an FPaV-2-SNT was performed (described in example 11) using the serum samples collected five weeks after immunization with FPaV-2 or with the cell culture supernatant from a mock-infection. The results are shown in table 8.

(60) TABLE-US-00008 TABLE 8 FPaV-2-SNT result of immunized rabbit. Rabbit no. 1 2 Immunogen Heat inactivated Heat-inactivated cell culture cell culture supernatant supernatant from a mock- from a FPaV-2 infection infection (‘Gordon’ strain) SNT result: Serum dilution Virus neutralizing activity of serum dilution (samples 5 weeks after immunization) 1:10 Negative Positive 1:20 Negative Positive 1:40 Negative Positive 1:80 Negative Positive 1:160 Negative Positive 1:320 Negative Negative 1:640 Negative Negative 1:1280 Negative Negative SNT-Titer <10 160

(61) Rabbits were immunized with either heat-inactivated FPaV-2 strain ‘Gordon’ (rabbit no. 2) or with cell culture supernatant as a negative control (rabbit no. 1) as described in example 15. Five weeks after immunization serum samples of these rabbits were tested for the presence of neutralizing antibodies against FPaV-2. “Positive” in table 8 means no virus growth, i.e. there is virus neutralization activity. Virus growth was measured via immunofluorescence staining according to Example 3.

(62) These experiments clearly show that a heat inactivated FPaV-2 formula can induce a high titer of neutralizing antibodies which are able to inhibit virus infection. Although tested in rabbits it can be assumed that a similar vaccination strategy would be effective in other animals like domestic cats.