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1. (WO2018037100) NEW PARAMYXOVIRUS AND USES THEREOF
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New paramyxovirus and uses thereof

Field of the invention

[0001] The present invention relates to a novel feline paramyxovirus and vaccines and treatments against said novel paramyxovirus. The invention further relates to the detection of said paramyxovirus and the diagnosis of infections with said paramyxovirus.

Background of the invention

[0002] Paramyxoviruses are enveloped, negative-sense single-stranded R A ((-)ssR A) viruses that have been associated with a number of infectious diseases in humans and animals. There are two subfamilies of Paramoxoviruses, Paramyxovirmae and Pneumovirinae and at least five genera within the subfamily Paramyxovirmae, namely Respirovirus, Rubulavirus, Morbillivirus, Henipavirus, and Avulavirus. Examples of Paramyxoviruses include canine distemper virus, measles virus, rinderpest virus, mumps virus and human parainfluenza viruses. Paramyxoviruses have a linear genome encoding seven viral polypeptides: a nucleocapsid protein, a phospho-protein, a matrix protein, a fusion protein, a haemagglutinin protein and a polymerase. Paramyxovirus virions are enveloped and can be spherical, filamentous or pleomorphic with a diameter of around 150 nm. Fusion proteins and attachment proteins (hemagglutinin, "H") appear as spikes on the virion surface. Matrix proteins ("M") inside the envelope stabilize the structure of the virus. The nucleocapsid core is composed of the genomic RNA, nucleocapsid proteins ("N"), phosphoproteins ("P") and polymerase proteins ("L" for "large protein"). The fusion protein ("F") projects from the envelope surface as a trimer, and mediates cell entry by inducing fusion between the viral envelope and the cell membrane.

[0003] Paramyxoviruses have for example been isolated from wild-living and domestic animals including cats, rodents and bats but also humans. Paramyxovirus infections, particularly of the Paramyxovirmae subfamily, have been associated with kidney diseases due to renal tissue damage shown in various species. Kidney disease, especially chronic kidney disease (CKD) is, for instance, among the most common diseases and one of the most common causes of death in domestic cats, particularly in older individuals. Lulich et al. (Compendium on continuing education for the practicing veterinarian (1992) 14(2): 127-125) report a prevalence of chronic kidney disease among total domestic cat populations of about 1.5 % and about 7.5 % in domestic cats older than 10 years. The causes of these diseases can be very diverse. In many cases the exact etiology cannot be determined. On the other hand, it is known that chronic kidney disease most often occurs as a result of inflammation of the renal tubules and renal interstitial tissue. This is called idiopathic tubulo interstitial nephritis (TIN).

[0004] Several feline paramyxoviruses have been described in the art.

US 2013/0230529 Al and Woo et al. (Proc. Nat. Acad. Sci. (2012) 109(14):5435-5440) describe a feline morbillivirus (FmoPV) isolated in Hong Kong which is associated with TIN in domestic cats. Other research groups from Japan (Sakaguchi et al. (2014) General Virology, 95(7), 1464-1468; Furuya et al. (2014) Archives of virology, 159(2), 371-373), Italy (Lorusso et al. (2013) Vet Ital. 51(3):235-237) and the USA (Sharp et al. (2016) Emerging Infectious Diseases 22(4): 760) also detected paramyxoviruses in urine samples from cats. Sieg et al. (Virus Genes (2015) 51(2):294-297) describe the discovery of feline paramyxoviruses in domestic cats with chronic kidney disease.

[0005] However, these paramyxoviruses are genetically diverse and there is a need to identify relevant paramyxoviruses and provide vaccines and treatments against said paramyxoviruses .

Description of the invention

[0006] The present invention provides a novel feline paramyxovirus designated Feline Paramyxo virus-Type 2 (FPaV-2). 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: l or SEQ ID NO:8. These two sequences represent the complementary DNA sequences of the genome of two strains of FPaV-2 that were isolated by the present inventors, the 'Gordon' strain (SEQ ID NO: l) and strain 'TV25' (SEQ ID NO: 8). SEQ ID NO: l and SEQ ID NO: 8 are given as DNA sequences that correspond to the positive RNA-strand into which the negative RNA strand viral genome is transcribed, i.e. they comprise the ORFs in 5' to 3' direction like an mRNA. The

skilled person is aware that the viral genome of the paramyxovirus of the invention is a (-)ssR A genome. Hence, in one aspect the invention also relates to the nucleic acid according to SEQ ID NO: l or SEQ ID NO:8 or a sequence being at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to SEQ ID NO: l or SEQ ID NO:8. The nucleic acid sequence of SEQ ID NO: l or SEQ ID NO:8 comprises six open reading frames (ORFs) for the six viral polypeptides of the paramyxovirus. The sequence of elements on the nucleic acid of SEQ ID NO: l or SEQ ID NO:8 is (from 5' to 3'): 3' untranslated region (UTR) of the viral genome, nucleocapsid protein ("N") ORF, phosphoprotein ("P") ORF, matrix protein ("M") ORF, fusion protein ("F") ORF, hemagglutinin ("H") ORF, RNA-dependent RNA polymerase ("L" for "large protein") ORF, 5' UTR. The following Table 1 describes the structure of the nucleic acids of SEQ ID NOs: l and 8:

Table 1: Overview of the genome of a paramyxovirus according to the present invention

Genome region nucleotide position corresponding

(in SEQ ID NO:l) polypeptide sequences

3 ' UTR 1 - 107 —

nucleocapsid protein ORF 108 - 1667 SEQ ID NO:2,

SEQ ID NO:9

intergenic sequence 1668 - 1780 —

phosphoprotein ORF 1781 - 3256 SEQ ID NO:3,

SEQ ID NO: 10

intergenic sequence 3257 - 3388 —

matrix protein ORF 3389 - 4402 SEQ ID NO:4,

SEQ ID NO: 11

intergenic sequence 4403 - 4949 —

fusion protein ORF 4950 - 6581 SEQ ID NO:5,

SEQ ID NO: 12

intergenic sequence 6582 - 6958 —

hemagglutinin ORF 6959 - 8746 SEQ ID NO:6,

SEQ ID NO: 13

intergenic sequence 8747 - 8887 —

polymerase ORF 8888 - 15496 SEQ ID NO:7,

SEQ ID NO: 14

5 ' UTR 15497 - 16047 —

SEQ ID NOs:2 to 7 represent the polypeptide sequences of the open reading frames of SEQ ID NO: 1 (Gordon strain) and SEQ ID NOs:9 to 14 represent the polypeptide sequences of the open reading frames of SEQ ID NO: 8 (strain TV25).

[0007] The invention thus relates in one aspect to a nucleic acid comprising a polynucleotide having a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence according to SEQ ID NO: 1 or SEQ ID NO: 8;

(b) a nucleotide sequence being at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to SEQ ID NO: l or SEQ ID NO:8;

(c) a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:9 (i.e. the nucleocapsid protein), SEQ ID NO:3, SEQ ID NO:10 (i.e. the phosphoprotein), SEQ ID NO:4, SEQ ID NO: l l (i.e. the matrix protein), SEQ ID NO:5, SEQ ID NO: 12 (i.e. the fusion protein), SEQ ID NO:6, SEQ ID NO: 13 (i.e. the hemagglutinin) SEQ ID NO:7 and SEQ ID NO: 14 (i.e. the polymerase);

(d) a nucleotide sequence encoding a polypeptide having an amino acid sequence which is

(i) at least 90 %, preferably at least 95 %, more preferably at least 98 % identical to SEQ ID NO:2,

(ii) at least 76 %, preferably at least 80 %, more preferably at least 90 %, more preferably at least 95 % and most preferably at least 98 % identical to SEQ ID NO:3,

(iii) at least 92 %, preferably at least 95 %, more preferably at least 98 % identical to SEQ ID NO:4,

(iv) at least 89 %, preferably at least 90 %, more preferably at least 95 %, more preferably at least 98 % identical to SEQ ID NO:5,

(v) at least 86 %, preferably at least 90 %, more preferably at least 95 %, most preferably at least 98 % identical to SEQ ID NO:6 and/or

(vi) at least 91 %, preferably at least 95 %, more preferably at least 98 % identical to SEQ ID NO:7; and

(e) the complementary strand of any of the nucleotide sequences of (a) to (d).

[0008] The nucleic acids of the present invention code for the genome of a feline paramyxovirus. Said paramyxovirus is in one embodiment able to induce an infection, particularly an infection of the urogenital system, more in particular an infection of the urinary system, or renal disease, particularly chronic renal disease in a human or non- human mammal, preferably a felid, canid, rodent or human, most preferably a domestic cat (Felis silvestris catus sometimes also referred to as Felis catus). SEQ ID NOs: 2 to 7 relate to the polypeptide gene products of the six open reading frames (ORFs) in SEQ ID NO: l . SEQ ID NOs: 9 to 14 relate to the polypeptide gene products of the six open reading frames (ORFs) in SEQ ID NO:8. Hence, the polypeptide encoded by the nucleic acid of item (d)(i) is a viral nucleocapsid protein, the polypeptide encoded by the nucleic acid of item (d)(ii) is a viral phosphoprotein, the polypeptide encoded by the nucleic acid of item (d)(iii) is a viral matrix protein, the polypeptide encoded by the nucleic acid of item (d)(iv) is a viral fusion protein, the polypeptide encoded by the nucleic acid of item (d)(v) is a viral hemagglutinin and the polypeptide encoded by the nucleic acid of item (d)(vi) is a viral polymerase.

Preferably herein, the nucleic acid of the invention comprises

(i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which is at least 90 % identical to SEQ ID NO:2,

(ii) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which is at least 76 % identical to SEQ ID NO:3,

(iii) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which is at least 92 % identical to SEQ ID NO:4,

(vi) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which is at least 89 % identical to SEQ ID NO:5,

(v) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which is at least 86 % identical to SEQ ID NO:6, and/or

(vi) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which is at least 91 % identical to SEQ ID NO:7.

[0009] Also part of the invention is a polypeptide having an amino acid sequence

(a) selected from the group consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,

SEQ ID NO:6, SEQ ID NO:7, SEQ ID N0:9, SEQ ID NO: 10, SEQ ID N0: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; or

(b) an amino acid sequence which is at least 90 %, preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:2,

(c) an amino acid sequence which is at least 76 %, preferably at least 80 %, more preferably at least 85 %, more preferably at least 90 %, more preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:3,

(d) an amino acid sequence which is at least 92 %, preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:4,

(e) an amino acid sequence which is at least 89 %, preferably at least 90 %, more preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:5,

(f) an amino acid sequence which is at least 86 %, preferably at least 90 %, more preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:6, or

(g) an amino acid sequence which is at least 91 %, preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:7.

The polypeptide may in some embodiments comprise one or more post-translational modifications such as a glycosylation.

The invention also pertains to a feline paramyxovirus the genome of which comprises a ribonucleic acid complementary to the nucleic acid of the invention as set out herein above. In particular, the invention relates to the paramyxovirus which has been deposited on 16 August 2016 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France under accession no. CNCM 1-5123. This strain is herein designated the 'Gordon' strain. The invention further pertains to any descendant of the paramyxovirus deposited as CNCM 1-5123, whereby said descendant may be attenuated or non-attenuated. Hence, the paramyxovirus of the present invention may be selected from the group consisting of: a paramyxovirus deposited as CNCM 1-5123, a descendant of the paramyxovirus deposited as CNCM 1-5123, an attenuated

descendant of the paramyxovirus deposited as CNCM 1-5123, and a paramyxovirus strain having the same characteristics as the paramyxovirus deposited as CNCM I-5123.

The paramyxovirus of the present invention preferably comprises one or more polypeptide(s) having an amino acid sequence

(a) selected from the group consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l , SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; or

(b) an amino acid sequence which is at least 90 %, preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:2,

(c) an amino acid sequence which is at least 76 %, preferably at least 80 %, more preferably at least 85 %, more preferably at least 90 %, more preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:3,

(d) an amino acid sequence which is at least 92 %, preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:4,

(e) an amino acid sequence which is at least 89 %, preferably at least 90 %, more preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:5,

(f) an amino acid sequence which is at least 86 %, preferably at least 90 %, more preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:6, and/or

(g) an amino acid sequence which is at least 91 %, preferably at least 95 %, more preferably at least 97 %, most preferably at least 98 % identical to SEQ ID NO:7.

Preferably, the paramyxovirus of the present invention comprises the polypeptides of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 or the polypeptides of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l , SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 or all the polypeptides of items (b) to (g).

12] In accordance with the present invention, the term "at least % identical to" in connection with nucleic acid sequences or amino acid sequences describes the number of matches ("hits") of identical nucleic acids or amino acids of two or more aligned sequences as compared to the number of residues making up the overall length of the sequences (or the overall compared part thereof). In other terms, using an alignment, for two or more sequences or subsequences, the percentage of residues that are the same (e.g. at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) may be determined, when the (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected.

It is well known in the art how to determine percent sequence identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 1988, 85; 2444). Although the FASTA algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % sequence identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, Nucl. Acids Res., 25 (1977), 3389). The BLASTN program for nucleic acid sequences uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff, Proc. Natl. Acad. Sci., 89 (1989), 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. All those programs may be used for the purposes of the present invention. However, preferably the BLAST program is used. Accordingly, all the nucleic acid molecules or amino acid sequences having the prescribed function and further having a sequence identity of at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%o, 97%), 98%o or 99%> as determined with any of the above recited or further programs available to the skilled person and preferably with the BLAST program fall under the scope of the invention.

[0013] In one aspect said paramyxovirus is able to induce an infection, particularly an infection of the urogenital system, more in particular an infection of the urinary system, or renal/kidney disease, particularly chronic renal disease in a human or non- human mammal, most preferably tubulo-interstitial nephritis (TIN) in a subject, preferably a mammal, more preferably in a feline, even more preferably in a domestic cat (Felis silvestris catus).

[0014] However, the paramyxovirus of the present invention may also be attenuated relative to the paramyxovirus which has been deposited on 16 August 2016 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France under accession no. CNCM 1-5123, e.g. for use in an immunogenic composition or vaccine.

[0015] Methods for attenuating the paramyxovirus of the present invention are known to the skilled person. Traditional methods of attenuating viruses may, for example, comprise the continuous passaging of the clinically isolated virus in embryonic hen eggs and chicken embryos and/or chicken embryo cell cultures (e.g. see Buynak et al. (1966), Experimental Biology and Medicine, 123(3), 768-775) or the adaption of wild-type virus to cell lines of foreign species and serial passaging to reduce virulence and pathogenicity (e.g. see Enders et al. (1960), New England Journal of Medicine, 263(4), 153-159). More recent approaches are, for instance, based on restricting the virulence through engineering the codon pair bias of the wild-type virus (e.g. see Coleman et al. (2008), Science, 320(5884), 1784-1787) or the genetic modification of the clinically isolated virus strains by deletion of some viral proteins which are responsible for virulence and pathogenicity (e.g. see Xu et al. (2014) Journal of virology, 88(5), 2600- 2610).

[0016] Hence, the invention further relates to the use of the nucleic acid molecule according to the invention (particularly the nucleic acid according to SEQ ID NO: l or SEQ ID NO: 8 or the nucleic acid which is at least 80 % identical to SEQ ID NO: l or SEQ ID NO: 8) for producing an attenuated feline paramyxovirus, wherein one or more mutations are introduced into the nucleic acid molecule. Consequently, the invention also provides a method of producing an attenuated feline paramyxovirus comprising the step of introducing one or more mutations into the nucleic acid molecule according to the invention, particularly to the nucleic acid according to SEQ ID NO: l or SEQ ID NO: 8 or the nucleic acid which is at least 80 % identical to SEQ ID NO: l or SEQ ID NO:8.

[0017] The term "attenuated" paramyxovirus, as described herein, is in particular directed to a paramyxovirus which is attenuated in vitro and/or in vivo, more particular in susceptible cell lines and/or the host.

As mentioned herein, "attenuated" particularly relates to a reduced virulence of a pathogen, in particular of the paramyxovirus, wherein "virulence" is understood to be the degree of pathogenicity, and wherein "pathogenicity" is directed to the ability of the pathogen to induce clinical signs in the host or the offspring of the host. Possible clinical signs of the infection with the paramyxovirus of the present invention comprise, for example, an increased thirst, increased urination, weight loss, decreased appetite, lethargy, and vomiting in the subject. Possible laboratory findings associated with an infection with the paramyxovirus of the present invention in a subject comprise, for example, increased levels of creatinine and symmetric dimethyl arginine (SDMA). Possible histological findings associated with an infection with the paramyxovirus of the present invention in a subject comprise, for example, cortical and medullary scarring, tubular degeneration, interstitial inflammation due to infiltration of primarily lymphocytes, plasma cells, macrophages and granulocytes.

[0018] The term "host", as used herein, is in particular directed to mammals infectable with the feline paramyxovirus of the present invention, in particular the subjects as defined below, preferably a feline, more preferably a domestic cat.

[0019] In the context of the present invention, the term "subject" (e.g. the subject which is susceptible to an infection with the paramyxovirus of the invention or which is treated with the vaccine of the present invention or which is diagnosed in the context of the present invention) is an animal, preferably a mammal, particularly a mammal selected from the group consisting of a member of the orders of Carnivora (preferred), Rodentia, Chiroptera and Primates, more preferably of the family of Felidae, Canidae, Cricetidae, Muridae, or Hominidae. Preferably, the subject is a member of the family of Felidae, particularly of the genera of Felis (which is preferred herein), Lynx, Panthera, Neofelis, Caracal, Leopardus, Puma, Acinonyx, Prionailurus, and Otocolobus. Canidae include for example the genera Canis and Vulpes, e.g. Canis

lupus, preferably Canis lupus familiaris (the domestic dog). However, the invention is not limited to these species, orders and families.

The Felis genus includes for example the species of Felis silvestris, e.g. Felis silvestris silvestris (European wildcat), feral cat, preferably Felis silvestris catus (also known as Felis catus, i.e. the domestic cat), Felis chaus, Felis nigripes, Felis margarita, and Felis bieti.

The genus Panthera e.g. includes Tiger (Panthera tigris), Lion (Panthera leo), Jaguar (Panthera onca), Leopard (Panthera pardus), Snow leopard (Panthera uncial), and Liger.

Other Felidae include but are not limited to Lynx lynx, Lynx rufus, Acinonyx jubatus (Cheetah), Puma concolor (Cougar), Leopardus pardalis (Ocelot).

Mammals of the family of Hominidae include Bornean orangutan (Pongo pygmaeus), Sumatran orangutan (Pongo abelii), Gorilla gorilla, Chimpanzee (Pan troglodytes), Bonobo (Pan paniscus), and Homo sapiens sapiens (human).

Chiroptera (bats) include Megachiroptera (megabats) and Microchiroptera (microbats). The mammal may also be a typical pet species, e.g. guinea pig (Cavia porcellus), domestic rabbit (Oryctolagus cuniculus), fancy mouse (Mus musculus), fancy rat (Rattus norvegicus), ferret (Mustela putorius furo), or Syrian hamster (Mesocricetus auratus). Mammalian pet and zoo animals are preferred herein. The mammal is preferably a felid, canid, rodent, human or bat, more preferably a felid ("feline"), most preferably a domestic cat.

The present invention further provides a DNA construct comprising the nucleic acid molecule according to the invention, wherein said DNA construct is in particular a DNA vector such as a plasmid. DNA vectors or plasmids into which the nucleotide molecule of the present invention can be inserted will be recognized by those of ordinary skill in the art. The DNA construct, as described herein, is preferably an isolated DNA construct. As used herein, the term "comprising the nucleic acid molecule" or "comprising a DNA molecule", respectively, is in particular understood to be equivalent to the term "comprising the sequence of the nucleic acid molecule" or "comprising the sequence of a DNA molecule", respectively. Thus, the invention also relates to a vector comprising said nucleic acid of the invention and a host cell comprising said vector.

[0021] Further, the present invention provides a R A transcript of the DNA construct described herein, wherein said RNA transcript is preferably an isolated RNA transcript. The present invention also provides a cell transfected with the DNA construct described herein, wherein said cell is preferably an isolated cell. Thus, the present invention also provides a feline paramyxovirus produced by the aforementioned cell, wherein said feline paramyxovirus is preferably an isolated feline paramyxovirus. Further, the present invention provides a cell transfected with the RNA transcript mentioned herein, wherein said cell is preferably an isolated cell. Hence, the present invention also provides a feline paramyxovirus produced by the aforementioned cell, wherein said feline paramyxovirus is preferably an isolated feline paramyxovirus. Host cells in which the paramyxovirus of the present invention or the polypeptides of the invention can be produced include but are not limited to cells selected from HEK293, HEK293T, Vera, CrFK, LLC-MK2, BHK21, CHO, BSR- T7/5, MA- 104 and HELA cells.

[0022] The present invention further provides a feline paramyxovirus whose genome comprises the nucleic acid molecule of the present invention or whose genome comprises an RNA molecule encoded by a nucleic acid molecule of the present invention, wherein said feline paramyxovirus is preferably an isolated feline paramyxovirus. In another aspect, the present invention provides a method for producing a feline paramyxovirus, said method comprising transfecting a cell with the DNA construct described herein or the nucleic acid of the invention.

[0023] The paramyxovirus according to the current invention may also be a chimeric virus, i.e. a paramyxovirus of the present invention the genome of which comprises a heterologous nucleic acid sequence. The chimeric virus may for example be encoded by a vector which further comprises a heterologous nucleotide sequence. In accordance with the invention a chimeric virus may, thus, be encoded by a viral vector to which heterologous nucleotide sequences have been added, inserted or substituted for native or non-native sequences. A chimeric virus may for instance be used for the generation of recombinant vaccines protecting against two or more viruses, e.g. two or more strains of paramyxoviruses. Attenuated and replication-defective viruses may be of used for vaccination purposes with live vaccines. The heterologous sequence may encode an antigen of any infectious pathogen or an antigen associated with any disease that is capable of eliciting an immune response.

The invention further relates to an antibody that is specific for the paramyxovirus of the invention and/or the polypeptide of the invention. The term "antibody" as used herein, unless indicated otherwise, is used broadly to refer to both, antibody molecules and a variety of antibody-derived molecules. Such antibody derived molecules comprise at least one variable region (either a heavy chain or a light chain variable region), as well as individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, and the like. Functional immunoglobulin fragments according to the present invention may be Fv, scFv, disulfide- linked Fv, Fab, and F(ab')2. The antibodies may for example be IgMs, IgDs, IgEs, IgAs or IgGs (e.g. IgGl, IgG2, IgG2b, IgG3 or IgG4 or the respective IgG subclasses in the respective species). Also encompassed by the term "antibody" are polyclonal antibodies, monoclonal antibodies ("mAbs"), chimeric monoclonal antibodies; humanized antibodies, genetically engineered monoclonal antibodies. For example, such antibodies may be obtained by administering a non-human mammal such as a mouse or a rabbit with the (full) paramyxovirus of the invention or a part thereof (e.g. a polypeptide of the invention or antigenic fragment thereof) and subsequently isolating the antibodies or antibody-producing cells. Alternatively, antibody libraries can be screened for such antibodies. Methods for producing human (or feline or canine or any other desired species), humanized (or caninized, felinized or any other species) or chimeric antibodies are well-known in the art. For example, phage display and xenomouse-based technologies can be used for generating human, feline, canine or other species-based monoclonal antibodies. Antibodies generated or identified in accordance with the methods described herein above may be tested for specificity for antigens of the feline paramyxovirus of the invention and/or the ability to neutralize the feline paramyxovirus of the present invention using biological assays known in the art including. Preferred herein are antibodies that are specific against a surface epitope of the feline paramyxovirus of the present invention, particularly antibodies against the hemagglutinin protein (polypeptide according to SEQ ID NO: 6 or SEQ ID NO: 13) or the fusion protein (polypeptide according to SEQ ID NO:5 or SEQ ID NO: 12). In the context of the antibodies of the present invention the terms "specific for" and "specific binding" refer to antibodies raised against the molecule of interest or a fragment thereof. An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least 10- fold higher, preferably 50-fold higher, more preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. The antibodies of the invention may be used to detect the feline paramyxovirus of the invention in a sample and/or for diagnostic purposes, e.g. to monitor the efficacy of a therapy and/or disease progression.

[0025] In a further aspect, the present invention relates to immunogenic compositions and vaccines against the paramyxovirus of the present invention. Said vaccines may e.g. comprise

(a) a paramyxovirus according to the present invention;

(b) a nucleic acid according the present invention;

(c) a polypeptide according to the present invention; or

(d) an antibody according to the present invention;

and a pharmaceutically acceptable carrier or excipient, preferably said carrier is suitable for intradermal or intramuscular application, optionally said vaccine further comprises an adjuvant.

Said immunogenic composition may e.g. comprise

(a) the paramyxovirus of any one of claims 5, 6 or 7;

(b) a nucleic acid according to any one of claims 1 or 2;

(c) a polypeptide according to claim 10;

(d) an antibody according to claim 11 ;

(e) a polypeptide according to claim 10, which is fused to a heterologous or autologous (poly-)peptide

and optionally a pharmaceutically acceptable carrier or excipient, preferably said carrier is suitable for intradermal or intramuscular application, optionally said vaccine further comprises an adjuvant.

[0026] An "immunogenic composition" as used herein refers to a composition of matter that comprises at least one paramyxovirus, nucleic acid, polypeptide or antibody according to the present invention, or an immunogenic portion thereof, that elicits an immunological response (cellular or antibody-mediated immune response) in the host to the composition. In a specific aspect, an immunogenic composition induces an immune response and, more preferably, confers protective immunity against one or more of the clinical signs of a paramyxovirus infection.

The term "vaccine" as used in specific aspects of the present invention refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in an animal and possibly but not necessarily one or more additional components that enhance the immunological activity of the active component. A vaccine may additionally comprise further components typical to pharmaceutical compositions. By way of distinction the immunologically active component of a vaccine may comprise complete virus particles in either their original form or as attenuated particles in a so called modified live vaccine (MLV) or particles inactivated by appropriate methods in a so called killed vaccine (KV). In another form the immunologically active component of a vaccine may comprise appropriate elements of the organisms (subunit vaccines) whereby these elements are generated either by destroying the whole particle or the growth cultures containing such particles and optionally subsequent purification steps yielding the desired structure(s), or by synthetic processes including an appropriate manipulation by use of a suitable system based on, for example, bacteria, insects, mammalian, or other species plus optionally subsequent isolation and purification procedures, or by induction of the synthetic processes in the animal needing a vaccine by direct incorporation of genetic material using suitable pharmaceutical compositions (polynucleotide vaccination). A vaccine may comprise one or simultaneously more than one of the elements described above. The term "vaccine" as used in specific aspects of the present invention describes a modified live, attenuated vaccine for veterinary use comprising antigenic substances and is administered for the purpose of inducing a specific and active immunity against a disease provoked by a paramyxovirus infection, preferably by a feline paramyxovirus infection. In further specific aspects of the present invention the vaccine may inter alia be a live vaccine, a live-attenuated vaccine, an inactivated vaccine, or a conjugate vaccine.

Various physical and chemical methods of inactivation are known in the art. The term "inactivated" refers to a previously virulent or non-virulent virus that has been irradiated (ultraviolet (UV), X-ray, electron beam or gamma radiation), heated (for instance for 30 min to several hours at a temperature between 55°C and 65°C, e.g. 3 h at 56°C), or chemically treated to inactivate, kill, such virus while retaining its immunogenicity. In one aspect, the inactivated paramyxoviruses disclosed herein may be inactivated by treatment with an inactivating agent. Suitable inactivating agents include beta-propio lactone, binary or beta- or acetyl-ethyleneimine, gluteraldehyde, ozone, and formalin (formaldehyde).

For inactivation by formalin or formaldehyde, formaldehyde is typically mixed with water and methyl alcohol to create formalin. The addition of methyl alcohol prevents degradation or cross reaction during the in activation process. One embodiment uses about 0.1 to 1% of a 37% solution of formaldehyde to inactivate the virus. It is critical to adjust the amount of formalin to ensure that the material is inactivated but not so much that side effects from a high dosage occur.

[0028] More particularly, the term "inactivated" in the context of a virus means that the virus is incapable of replication in vivo or in vitro. For example, the term "inactivated" may refer to a virus that has been propagated in vitro, and has then been deactivated using chemical or physical means so that it is no longer capable of replicating. In another example, the term "inactivated" may refer to a virus that has been propagated, and then deactivated using chemical or physical means resulting in a suspension of the virus, fragments or components of the virus, which may be used as a component of a vaccine. As used herein, the terms "inactivated", "killed" or "KV" are used interchangeably.

[0029] The term "live vaccine" refers to a vaccine comprising a living, in particular, a living viral active component.

[0030] The optionally one or more pharmaceutically acceptable carriers or excipients, as mentioned herein include any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. In some aspects, and especially those that include lyophilized immunogenic compositions, stabilizing agents include stabilizers for lyophilization or freeze-drying.

[0031] In a preferred aspect, the immunogenic composition of the invention comprises an amount of 101 to 107 viral particles of the attenuated paramyxovirus virus described herein per dose, preferably 103 to 106 particles per dose, more preferably 104 to 106 particles per dose.

In another preferred aspect, the immunogenic composition of the invention comprises an amount of the paramyxovirus according to the invention which is equivalent to a virus titre of at least about 103 TCID50/mL per dose, preferably between 103 to 105 TCID50/ml per dose.

[0032] As used herein, the terms "vaccine" and "vaccine composition" are used interchangeably and in particular refer to a composition that will elicit a protective immune response in a subject that has been exposed to the composition. An immune response may include induction of antibodies and/or induction of a T-cell response.

[0033] The vaccine of the present invention may also be a marker vaccine, i.e. a vaccine that allows for the immunological differentiation or segregation of infected from vaccinated animals. For example, the marker vaccine may lack an immunogenic antigen present in the pathogen being vaccinated against (i.e. the paramyxovirus of the invention), thus creating a negative marker of vaccination. Such marker vaccines are also referred to as DIVA or SIVA vaccines ("Differentiation/Segregation of infected from vaccinated animals") in veterinary medicine and are particularly useful for productive livestock such as farm animals.

[0034] Usually, an "immune response" includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or a protective immunological (memory) response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in number or severity of, or lack of one or more of the clinical signs associated with the infection of the pathogen, in the delay of onset of viremia, in a reduced viral persistence, in a reduction of the overall viral load and/or in a reduction of viral excretion.

Thus, an "immune response" in particular means but is not limited to the development in a subset of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest.

[0035] The vaccine may in one aspect comprise a fusion protein of the polypeptide of the invention with another peptide or polypeptide. The fusion partner may be a heterologous polypeptide/peptide or an autologous polypeptide/peptide. Heterologous polypeptides that can be fused to the polypeptide of the present invention include but are not limited to a polypeptide of a canarypox virus (in particular for a vaccine in cats), of a myxoma virus (particularly for a vaccine in rabbits) or of a herpes virus.

[0036] In another aspect, the present invention relates to a method of detecting the paramyxovirus of the invention in a sample from a subject. The sample in the context of the present invention is a biological sample, particularly a sample of bodily fluid or tissue obtained for the purpose of diagnosis, prognosis, or evaluation of a subject. "Subject" for the purposes of the present invention includes both humans and other mammals. Thus, the methods are applicable to both human diagnostics and veterinary applications. Preferred test samples include whole blood, serum, plasma, urine, saliva, sputum, aspirate, punctate and mucosal swabs. In addition, one of skill in the art would realize that some test samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components. Thus, in a preferred embodiment of the invention the sample is selected from the group comprising a blood sample, a serum sample, a plasma sample, a saliva sample, an aspirate sample, a punctuate sample, a mucosal swab and a urine sample or an extract of any of the aforementioned samples. Also paraffin tissue section samples may be used. Preferably, the sample is a blood or a urine sample, most preferably a serum sample or a plasma sample. Where appropriate, the sample may need to be homogenized, or extracted with a solvent prior to use in the present invention in order to obtain a liquid sample. A liquid sample hereby may be a solution or suspension. Liquid samples may be subjected to one or more pre-treatments prior to use in the present invention. Such pre-treatments include, but are not limited to dilution, filtration, centrifugation, concentration, sedimentation, precipitation, dialysis. Pre-treatments may also include the addition of chemical or biochemical substances to the solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifiers, chelators. "Plasma" in the context of the present invention is the virtually cell-free supernatant of blood containing anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium ion binding compounds such as EDTA or citrate and thrombin inhibitors such as heparinates or hirudin. Cell- free plasma can be obtained by centrifugation of the anticoagulated blood (e.g. citrated, EDTA or heparinized blood) for at least 15 minutes at 2000 to 3000 g. The detection can either be at the nucleic acid level, i.e. the genomic RNA of the virus is detected in a sample, or on the level of the viral proteins, e.g. using an immunoassay. Any immunoassay system known in the art may be used for this purpose including, but not limited to, competitive and noncompetitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assays), "sandwich" immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and

immuno electrophoresis assays. Also Western Blot assays or lateral flow immunoassays can be used. The viral nucleic acid can be detected e.g. with a reverse transcription and polymerase chain reaction (RT-PCR) using specific primers and/or probes. For example the detection of the nucleic acid can be performed directly in a urine sample of the subject. Also other nucleic acid based samples such as NASBA may be used. PCR assays may for example comprise formats such as nested PCR, semi-nested PCR and quantitative PCR.

[0038] In particular, the present invention relates to a method for detecting the paramyxovirus according to the invention in a sample, comprising the steps of

(i) contacting the sample with one or more oligonucleotide primers and/or probes which are specific for a nucleic acid according to the invention (i.e. which preferably hybridize under stringent conditions to said nucleic acid), and

(ii) detecting binding between said paramyxovirus and said one or more oligonucleotide primers and/or probes.

In another particular aspect, the present invention relates to a method for detecting the paramyxovirus according to the invention in a sample, comprising the steps of

(i) contacting the sample with an antibody according to the invention, and

(ii) detecting binding between said paramyxovirus and said antibody.

[0039] The method for detecting the paramyxovirus of the present invention can thus be based on the direct detection of the paramyxovirus in a sample. However, it can also be based on a more indirect approach, i.e. by detecting the presence of antibodies against said paramyxovirus in the sample. The presence of antibodies against said paramyxovirus indicates that the subject's immune system has already been in contact with the paramyxovirus such that a corresponding immune response was initiated. In this context, a true infection with the paramyxovirus can be distinguished from a subject vaccinated with a marker vaccine (DIVA, see above) by detecting antigens (i.e. the polypeptide of the invention or an antigenic fragment thereof) in the sample which were deleted from the marker vaccine.

[0040] Hence, the invention also pertains to a method for detecting the paramyxovirus according to the invention in a sample, comprising detecting the presence or absence of an antibody against said paramyxovirus in said sample. Suitable approaches for detecting the antibodies in the sample include immunoassays as described above (e.g. ELISA, particularly sandwich ELISA and lateral-flow assays). Hence, the invention

also relates to methods (and corresponding kits) for detecting the paramyxovirus of the present invention in a sample, comprising contacting the sample with an antigen of the paramyxovirus and detecting binding of antibodies (if present) in the sample against said paramyxovirus to said antigen. Said antigen is preferably immobilized on a surface. The antibodies bound to said antigen can, for example, be detected by using a secondary detection antibody, e.g. in the case of a feline sample an antibody against feline immunoglobulin (Ig) such as a mouse-anti-cat Ig antibody. Exemplary antigenic epitopes of the polypeptides of the present invention are listed in the following Table 2:


These epitopes (or fragments of the polypeptides of the invention containing one or more of said epitopes) can, for example, be used to raise antibodies against the paramyxovirus of the present invention.

In another aspect, the present invention relates to a method of diagnosing an infection of a subject with the paramyxovirus of the invention. In particular, the invention also pertains to a method for diagnosing an infection with the paramyxovirus according to the invention in a sample from a subject, preferably a feline, more preferably a domestic cat, comprising a method for detecting the paramyxovirus as described herein above, wherein the presence of said paramyxovirus is indicative for an infection. In another aspect, the present invention pertains to a method for diagnosing an infection with the paramyxovirus according to the invention in a sample from a subject, preferably a domestic cat, comprising a method for detecting an antibody against said paramyxovirus of the invention, wherein the presence of said antibody is indicative for an infection with said paramyxovirus.

[0042] In the context of the methods of detecting and methods of diagnosing of the present invention, the detection of the fusion protein or the hemagglutinin of the paramyxovirus is preferred. It is also preferred in one aspect that the viral polymerase is not detected or at least epitopes or regions corresponding to nucleotide residues 10055 to 10560 of the viral genome (SEQ ID NO: l or 8) are not detected.

[0043] In another aspect, the present invention relates to a kit for the detection of the paramyxovirus of the invention. The kit may in one embodiment comprise one or more antibodies specific for the paramyxovirus of the present invention. In another embodiment, the kit may comprise oligonucleotide primers and/or probes for the detection of the paramyxovirus of the invention.

[0044] Finally, the invention relates to medical uses of the vaccines, nucleic acids, polypeptide, antibodies and immunogenic compositions of the invention and to methods of treating and/or preventing an infection with the paramyxovirus in a subject. For example, the vaccine or immunogenic composition of the present invention may be used to reduce the clinical signs of an infection with the paramyxovirus, preferably the clinical signs of an infection of the urogenital system, more in particular an infection of the urinary system, or renal/kidney disease, particularly chronic renal disease in a human or non-human mammal, most preferably tubulo -interstitial nephritis (TIN). In particular, the present invention relates to the vaccine or immunogenic composition of the present invention for use in treating or preventing an infection with a paramyxovirus, particularly the feline paramyxovirus of the present invention.

[0045] The invention further pertains to the vaccine or immunogenic composition of the invention for use in treating or preventing an infection, particularly an infection of the urogenital system, more in particular an infection of the urinary system, or renal/kidney disease, particularly chronic renal disease in a human or non-human mammal, most preferably tubulo-interstitial nephritis (TIN) in a mammal, preferably a feline, more preferably a domestic cat.

[0046] The invention also pertains to a method for treating or preventing an infection with a paramyxovirus, in particular, the feline paramyxovirus according to the invention in a subject, preferably a domestic cat, comprising the step of administering the vaccine or immunogenic composition to said subject.

The invention also relates to a method for treating or preventing a kidney disease, preferably chronic kidney disease, most preferably tubulo-interstitial nephritis (TIN), in a subject, preferably a feline, more preferably a domestic cat, comprising the step of administering the vaccine or immunogenic composition of the present invention to said subject.

[0047] In a preferred aspect, the vaccine or immunogenic composition of the present invention is used for preventing an infection with the paramyxovirus of the invention in a subject.

[0048] The present invention also relates to the immunogenic composition or the vaccine of the present invention for use in a method of reducing or preventing the clinical signs or disease caused by an infection with a pathogenic paramyxovirus, preferably the paramyxovirus of the invention, in a subject, or for use in a method of treating or preventing an infection with a pathogenic paramyxovirus, preferably the paramyxovirus of the invention, in a subject, wherein preferably said subject is a feline. The present invention further relates to the immunogenic composition or the vaccine of the present invention for use in a method of protecting a subject, preferably a feline, from an infection with a paramyxovirus, preferably the paramyxovirus according to the invention or from clinical signs or disease caused by an infection with a pathogenic paramyxovirus, preferably the paramyxovirus of the invention.

The invention further pertains to a method of immunizing a subject, preferably a feline, against a clinical disease or against clinical signs caused by a paramyxovirus, preferably the paramyxovirus of the invention, in a subject, said method comprising the step of administering to the subject, preferably a feline, an immunogenic composition or vaccine according to the invention, whereby said virus fails to cause clinical signs of paramyxovirus infection but is capable of inducing an immune response that immunizes the subject, preferably the feline, against pathogenic forms of paramyxovirus, preferably the paramyxovirus of the invention.

The invention thus also relates to a method of protecting or reducing the clinical signs of a paramyxovirus infection (preferably with the paramyxovirus of the invention) in a subject, preferably a feline, against a clinical disease or against clinical signs caused by a paramyxovirus, preferably the paramyxovirus of the invention, in a subject, said method comprising the step of administering to the subject, preferably a feline, an immunogenic composition or vaccine according to the invention, whereby said virus fails to cause clinical signs of paramyxovirus infection but is capable of inducing an immune response that immunizes the subject, preferably the feline, against pathogenic forms of paramyxovirus, preferably the paramyxovirus of the invention.

[0049] The invention further pertains to a kit for vaccinating a subject, preferably a feline, against diseases associated with and/or reducing the incidence or the severity of one or more clinical signs associated with or caused by a paramyxovirus, preferably the paramyxovirus according to the invention, in a subject comprising:

(a) a dispenser capable of administering a vaccine to the subject, preferably a feline; and

(b) the immunogenic composition or the vaccine according to the invention, and

(c) optionally an instruction leaflet.

[0050] All cited patent and non-patent documents are herewith incorporated by reference in their entirety.

[0051] Description of the drawings

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

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.

Figure 2: Immunofluorescence of LLC-MK2 cells infected with the FPaV-2 „Gordon" isolate.

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

Figure 3: Isolation of feline primary kidney cells. Magnification: 100 x, 48 hours after seeding.

Figure 4: Infection of feline primary kidney cells with FPaV-2.

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

Figure 5: Antibody diversity of FPaV-2-infected cats.

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 V25 ' .

B. = Incubation with 1 : 100 diluted sample from paramyxo virus-negative cat.

C. = Incubation with 1 :200 diluted anti-nucleocapsid antibody.

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).

Figure 6: Development of an ELISA for the detection of FPaV-2 antibodies in cat serum samples.

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.

Figure 7: Primary immune target cells of FPaV-2.

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.

A. and C. = Mock-infected PBMCs

B. and D. = FPaV-2-infected PBMCs

Figure 8: Western-blot analysis of an FPaV-2-immunized rabbit.

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

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

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

Specific reactions to viral proteins are annotated at the right bottom based on reactivity shown in figure 5.

In contrast to the pre-immune serum FPaV-2-specific antibodies were detected five

weeks after Immunization.

Examples

Example 1 : Detection ofFPaV-2

Collection of sample:

Urine from a 13 year old male cat with a chronic kidney disease is collected and stored on ice. RNA-Isolation:

RNA is isolated from 300 μΐ urine using the 'QIAamp Viral R A Mini Kit' (Qiagen, Hilden), eluted in 50 μΐ of buffer AVE and stored at -80°C.

RT-PCR:

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 μΐ reaction buffer (2-fold, 0.4 mM dNTPs each and 2.4 mM MgS04), 2 μΐ magnesium sulphate (5 mM), 0.25 μΐ primer RES-MOR-HEN-R (100 μΜ), 0.25 μΐ primer RES-MOR-HEN-Fl (100 μΜ), 0.5 μΐ RNase inhibitor (40 U/μΙ, Life Technologies) and 0.5 μΐ 'Superscript Ill/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).

Sequencing:

The PCR fragment is sequenced by applying the Sanger didesoxy method using RES-MOR-HEN-R (10 μΜ) and RES-MOR-HEN-Fl (10μΜ) as sequencing priming. Resulting chromatograms are edited using the software "BioEdif (version 7.2.4) and aligned with the 'Basic Local Alignment Search Tool' (BLAST) on the NCBI website.

Example 2: Isolation of FPaV-2

For virus cultivation LLC-MK2 and CrFK cells are seeded in 75 cm2 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% C02 and 90% humidity.

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 μΐ of the cell culture supernatant are tested as described in Example 1 for the presence of feline paramyxoviruses. Figure 1 shows the result of such an experiment.

Example 3: Immunofluorescence detection of FPaV-2

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.

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.

This is followed by an incubation step with anti-FPaV-2 antibody (anti-FPaV-2 nucleocapsid, polyclonal, rabbit) at a final concentration of ^g/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 Figure 2.

Example 4: Infection spectrum of FPaV-2

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.


Tab. 3: in vitro spectrum of FPaV-2 infection

Example 5 : Infection of feline primary kidney cells with FPaV-2

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 μιη nylon filter and the filtrate is centrifuged at 400 x 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% C02 and 90% humidity. Figure 3 shows a typical result of such an experiment.

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.

Figure 4 shows a result of such an experiment.

Example 6: Determination and analysis of the full-length FPaV-2 genome

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.


Tab. 4: Analysis of whole genome sequence of FPaV-2 (strain , Gordon')

nt's: nucleotides aa: amino acids

Example 7: Prevalence of FPaV-2 in urine samples

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.

Characteristics Diseased group Healthy group

Number of tested urines 325 238

FLUTD, nephritis, anuria,

Clinical and polyuria, urolithiasis, cystitis, No history of uro tract laboratory findings hematuria, leukocyturia, diseases

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 %)

Tab. 5: Prevalence of FPaV-2 in urine samples of domestic cats

Example 8: Heterogeneity of FPaV-2 strains

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.

Nucleotide Amino acid similarity between similarity between

Nucleotide position

Genome region 'Gordon' and 'Gordon' and

(cRNA)

'TV25' 'TV25' (nucleotides) (amino acids)

3' untranslated

1 - 107 100 %(107/107) not applicable Region (UTR)

Nucleocapsid protein 108 - 1667 99.2 %(1547/1560) 99.2 % (515/519)

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 %(6554/6609) 99.5 %(2191/2202)

5' untranslated

15497 - 16047 99.4 %(548/551) not applicable region (UTR)

99.2 %

Whole genome 1 - 16047 not applicable

(15917/16047)

Tab. 6: Comparison of whole genome sequences of the two FPaV-2 isolates 'Gordon' and 'TV25'

Example 9: Electron microscopy of FPaV-2

15 ml of the a FPaV-2-cell culture supernatant (described in example 2) was centrifuged at 3.000 x g for 10 minutes at 4 °C followed by filtration through a 0.45 μιη cellulose nitrate filter. The filtrate was overlayed on a 20 % (w/v) sucrose cushion and centrifuged at 100.000 x g for 90 minutes at 4°C. The pellet was then suspended in 100 μΐ 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

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.

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 a-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 figure 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).

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

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 μΐ of these heat inactivated serum samples were mixed with 50 μΐ 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, nonessential amio acids, penicillin and streptomycin. The cells were incubated for five days at 37 °C, 5 % CO2 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

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.

Cat serum ID 98450 TV25 TV26 CDV

PCR result Paramyxovirus

FPaV2 positive - from urine negative

Presence of viral RNA in > 14 > 18

- - urine weeks months

IFA Pos. Pos. Neg. Neg.

FPaV-2

320 320 < 10 < 10 SNT titer

Tab. 7: Results of FPaV-2 serum neutralization tests.

IFA: immune fluorescence assay, CDV: canine distemper virus-positive sample

Example 13: ELISA-development for screening of FPaV-2 antibodies in cat serum samples

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-l ' using the restriction enzymes BamHI and Xhol. 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. co/z-clone named Έ. coli-Gordon-NC. This clone was inoculated into 1.000 ml LB medium with 0.2 % glucose and 100 μΐ/ml ampicillin and was shaken at 37 °C with 200 rpm until the culture reached an optical density of
= 1.0. At that point the culture was cooled down to 22 °C and recombinant protein expression was induced with Isopropyl β-D-l-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 x 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.

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. Results are shown in figure 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

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 % C02 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 figure 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 ofFPaV-2 is likely to occur.

Example 15: Immunization of rabbits with inactivated FPaV-2

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 * 105 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.

Immunization was done according to the following scheme:

- 1st Immunization: Collecting of pre-immune serum, 1 ml subcutaneous and 1 ml intramuscular of vaccine mixture was than inoculated.

- 2nd Immunization 14 days after 1st immunization: 1 ml subcutaneous and 1 ml intramuscular of vaccine mixture was inoculated.

- 3rd Immunization 7 days after 2nd immunization: 1 ml subcutaneous and 1 ml intramuscular of vaccine mixture was inoculated.

- final bleed (rabbits were killed): 14 days after the third immunization

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 figure 8B). The specificity of the observed reactions was confirmed by analyzing the pre-immune serum of this rabbit. As shown in figure 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. 5 A) 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.


Tab. 8: FPaV-2-SNT result of immunized rabbit.

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.

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.