Vaccine Comprising An Attenuated Pestivirus

Abstract

Attenuated pestiviruses, in particular attenuated BVDV, wherein at least one mutation is in the coding sequence for glycoprotein E.sup.ms and at least another mutation in the coding sequence for N.sup.pro which preferably leads to combined inactivation of the RNase activity residing in glycoprotein E.sup.ms in addition to the inactivation of the (hypothesized) immunomodulating activity residing in N.sup.pro. Methods for attenuating pestiviruses such as BVDV, nucleic acids encoding the pestiviruses, in particular BVDV, compositions and vaccines comprising the attenuated pestiviruses, in particular BVDV, of the invention.

Description

RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Ser. No. 60/589,361, filed Jul. 20, 2004, and claims priority to German Application No. 10 2004 025 452.4, filed May 19, 2004, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of animal health and in particular to attenuated pestiviruses such as bovine viral diarrhea virus (BVDV).

BACKGROUND OF THE INVENTION

[0003] Pestiviruses are causative agents of economically important diseases of animals in many countries worldwide. Presently known virus isolates have been grouped into four different species which together form one genus within the family Flaviviridae. [0004] I./II. Bovine viral diarrhea virus (BVDV) type 1 (BVDV-1) and type 2 (BVDV-2) cause bovine viral diarrhea (BVD) and mucosal disease (MD) in cattle (Baker, 1987; Moennig and Plagemann, 1992; Thiel et al., 1996). The division of BVDV into 2 species is based on significant differences at the level of genomic sequences (summarized in Heinz et al., 2000) which are also obvious from limited cross neutralizing antibody reactions (Ridpath et al. 1994). [0005] III. Classical swine fever virus (CSFV), formerly named hog cholera virus, is responsible for classical swine fever (CSF) or hog cholera (HC) (Moennig and Plagemann, 1992; Thiel et al., 1996). [0006] IV. Border disease virus (BDV) is typically found in sheep and causes border disease (BD). After intrauterine infection of lambs with BDV persistently infected lambs can be born that are weak and show different abnormalities among which the "hairy shaker" syndrome is best known (Moennig and Plagemann, 1992; Thiel et al., 1996).

[0007] Pestiviruses are small enveloped viruses with a single stranded RNA genome of positive polarity lacking both 5' cap and 3' poly(A) sequences. The viral genome codes for a polyprotein of about 4000 amino acids giving rise to final cleavage products by co- and post-translational processing involving cellular and viral proteases. The viral proteins are arranged in the polyprotein in the order NH.sub.2-N.sup.pro-C-E.sup.ms-E1-E2-p7-NS2--NS3-NS4A-NS4B-NS5A-NS5B-COOH (Lindenbach and Rice, 2001). Protein C (=core- or capsidprotein) and the glycoproteins E.sup.ms, E1, and E2 represent structural components of the pestivirus virion as demonstrated for CSFV (Thiel et al., 1991). This also holds true for BVDV. E2 and, to a lesser extent, E.sup.ms were found to be targets for antibody neutralization (Donis et al., 1988; Paton et al., 1992; van Rijn et al., 1993; Weiland et al., 1990, 1992). E.sup.ms lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells; this protein has been reported to exhibit RNase activity (Hulst et al., 1994; Schneider et al., 1993; Windisch et al., 1996). The function of this enzymatic activity for the viral life cycle is presently unknown. The enzymatic activity depends on the presence of two stretches of amino acids conserved between the pestivirus E.sup.ms and different known RNases of plant and fungal origin. Both of these conserved sequences contain a histidine residue (Schneider et al., 1993). Exchange of each of these residues against lysine in the E.sup.ms protein of a CSFV vaccine strain resulted in the destruction of RNase activity (Hulst et al., 1998). Introduction of these mutations into the genome of the CSFV vaccine strain did not influence viral viability or growth properties but led to a virus exhibiting a cytopathogenic phenotype (Hulst et al., 1998). Similarly, Meyers et al. showed that an RNase negative variant of the virulent CSFV strain Alfort/Tubingen was fully viable. However, the respective virus mutant showed no cytopathogenic phenotype (Meyers et al., 1999).

[0008] N.sup.pro represents the first protein encoded by the long open reading frame in the pestivirus RNA. N.sup.pro represents a nonstructural protein that has protease activity and cleaves itself of the nascent polyprotein (Stark et al., 1993; Wiskerchen et al., 1991) presumably already during translation. N.sup.pro is a cysteine protease (Rumenapf et al., 1998) that is not essential for virus replication (Tratschin et al., 1998). Recently, it was shown that N.sup.pro somehow interferes with the cellular antiviral defense so that it can be hypothesized to modulate the immune system within an infected host (Ruggli et al., 2003). Mayer and coworkers presented indications for an attenuation of CSFV in consequence of a deletion of the N.sup.pro gene (Mayer et al., 2004).

[0009] Present BVDV vaccines for the prevention and treatment of BVDV infections still have drawbacks (Oirschot et al., 1999). Vaccines against the classical BVDV-1 provide only partial protection from BVDV-2 infection, and vaccinated dams may produce calves that are persistently infected with virulent BVDV-2 (Bolin et al., 1991; Ridpath et al., 1994). This problem is probably due to the great antigenic diversity between type 1 and type 2 strains which is most pronounced in the glycoprotein E2, the major antigen for virus neutralization (Tijssen et al., 1996). Most monoclonal antibodies against type 1 strains fail to bind to type 2 viruses (Ridpath et al., 1994).

[0010] Vaccines comprising attenuated or killed viruses or viral proteins expressed in heterologous expression systems have been generated for CSFV and BVDV and are presently used. Killed vaccines (inactivated whole virus) or subunit vaccines (conventionally purified or heterologously expressed viral proteins) are most often inferior to live vaccines in their efficacy to produce a full protective immune response even in the presence of adjuvants.

[0011] The structural basis of the attenuation of BVDV used as live vaccines is not known. These vaccines, although attenuated, are most often associated with safety problems. The vaccine viruses may cross the placenta of pregnant animals, e.g., cows, and lead to clinical manifestations in the fetus and/or the induction of persistently infected calves. Therefore, they cannot be applied to breeding herds that contain pregnant cows. Pregnant cows have to be kept separate from vaccinated cattle to protect fetuses and must not be vaccinated themselves. Furthermore, revertants of attenuated live BVDV pose a serious threat to animals. For conventionally derived attenuated viruses wherein the attenuation is achieved by conventional multiple passaging, the molecular origin as well as the genetic stability of the attenuation remains unknown and reversion to the virulent wild-type is unpredictable.

[0012] Because of the importance of an effective and safe as well as detectable prophylaxis and treatment of pestiviral infections, there is a strong need for improved attenuated pestiviruses, such as BVDV, with a high potential for induction of immunity as well as a defined basis of attenuation which can also be distinguished from pathogenic pestiviruses, such as BVDV, as well as compositions and vaccines comprising the attenuated pestiviruses, such as BVDV.

[0013] Therefore, the technical problem underlying the present invention is to provide improved attenuated pestivirus, preferably an attenuated BVDV for use as live attenuated vaccines. Such improved attenuated pestivirus, preferably BVDV, should especially (i) not cross the placenta themselves, and (ii) induce an immunity that prevents viral transmission across the placenta and thereby prevents pregnancy problems like abortion of the fetus or birth of persistently infected host such calves in the case of BVDV infection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows the serum neutralization against NY93/C (BVDV type II);

[0015] FIG. 2 shows the serum neutralization assay against KE9 (BVDV type I); and

[0016] FIG. 3 shows the serum neutralization assay against NY93/C (BVDV type II).

[0017] All subsequent sequences show the deleted regions indicated with dashes (-), which are also numbered, whereas the sequences in the sequence listing attached hereto are continuously numbered without the deleted regions or amino acid codons.

SEQ ID NO:1 XIKE-A-cDNA sequence SEQ ID NO:2 XIKE-A-NdN-cDNA sequence SEQ ID NO:3 XIKE-B-cDNA sequence

SEQ ID NO:4 XIKE-B-NdN-cDNA

[0018] SEQ ID NO:5 XIKE-A amino acid sequence SEQ ID NO:6 XIKE-A-NdN amino acid sequence SEQ ID NO:7 XIKE-B amino acid sequence SEQ ID NO:8 XIKE-B-NdN amino acid sequence SEQ. ID NO:9 XIKE-C-NdN amino acid sequence SEQ ID NO:10 XIKE-C-NdN-cDNA sequence SEQ ID NO:11 XIKE-C-cDNA sequence SEQ ID NO:12 XIKE-C amino acid sequence

SUMMARY OF THE INVENTION

[0019] The present invention relates to attenuated pestivirus, preferably to attenuated BVDV, wherein at least one mutation is in the coding sequence for glycoprotein E.sup.ms and at least another mutation in the coding sequence for N.sup.pro which preferably leads to combined inactivation of the RNase activity residing in glycoprotein E.sup.ms in addition to the inactivation of the (hypothesized) immunomodulating activity residing in N.sup.pro. The invention also relates to methods for attenuating pestivirus in such that the attenuation results in an attenuated pestivirus, preferably in an attenuated BVDV, as described above. The present invention furthermore relates to nucleic acid molecules encoding the attenuated pestiviruses, preferably encoding attenuated BVDV, compositions and vaccines comprising the attenuated pestivirus, preferably BVDV as disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions of Terms Used in the Description

[0020] Before the embodiments of the present invention it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a BVDV" includes a plurality of such BVDV, reference to the "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies as reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0021] The term "pestivirus" as used herein refers to all members of the genus Pestivirus, including BVDV, CSFV, and BDV, within the family Flaviviridae.

[0022] The term "CSFV" as used herein refers to all viruses belonging to species of classical swine fever virus (CSFV) in the genus Pestivirus within the family Flaviviridae.

[0023] The term "BVDV" as used herein refers to all viruses belonging to species bovine viral diarrhea virus (BVDV) type 1 (BVDV-1) and BVDV type 2 (BVDV-2) in the genus Pestivirus within the family Flaviviridae (Heinz et al., 2000). The more classical BVDV type 1 strains and the more recently recognized BVDV type 2 strains display some limited but distinctive differences in nucleotide and amino acid sequences.

[0024] The term "N.sup.pro" as understood herein relates to the first protein encoded by the viral open reading frame and cleaves itself from the rest of the synthesized polyprotein (Stark et al., J. Virol. 67:7088-7093 (1993); Wiskerchen et al., Virol. 65:4508-4514 (1991)). The term, depending on the context, may also relate to the remaining "N.sup.pro" amino acids after mutation of the encoding nucleotide sequence or to the coding nucleotide sequence for the protein itself "Protease activity residing in N.sup.pro" relates to the polypeptide cleavage activity of the "N.sup.pro".

[0025] "E.sup.ms" as used herein relates to the glycoprotein E.sup.ms which represents a structural component of the pestivirus virion (Thiel et al., 1991). E.sup.ms lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells; this protein has been reported to exhibit RNase activity (Hulst et al., 1994; Schneider et al., 1993; Windisch et al., 1996). It should be noted that the term glycoprotein E0 is often used synonymously to glycoprotein E.sup.ms in publications. The term, depending on the context, may also relate to the mutated "E.sup.ms" protein after mutation of the encoding nucleotide sequence or to the coding nucleotide sequence for the protein itself. "RNase activity residing in glycoprotein E.sup.ms" relates to the RNA cleavage activity of the glycoprotein, i.e., the ability of the glycoprotein E.sup.ms to hydrolyze RNA. The term "inactivation of the RNase activity residing in the glycoprotein" refers to the inability or reduced capability of a modified glycoprotein E.sup.ms to hydrolyze RNA as compared to the unmodified wild-type of the glycoprotein E.sup.ms.

[0026] "An attenuated pestivirus or BVDV particle" as used herein means that there is a statistically significant difference between the virulence of attenuated pestivirus or BVDV particles of the present invention, wherein the attenuated viral particles being attenuated by a method described herein, and wild-type pestivirus or BVDV isolates from which the attenuated pestivirus or BVDV particles have been derived, for the predominant clinical parameters, in case of BVDV for diarrhea, pyrexia, and lethality in animals infected with the same dose, preferably 6.times.10.sup.6 TCID.sub.50. Thus, the attenuated BVDV particles do not cause diarrhea, pyrexia, and lethality and thus may be used in a vaccine.

[0027] "Inactivation of E.sup.ms" as used herein means RNase activity not significantly above the level measured for noninfected control cells in an RNase assay as described in Meyers et al., 1999. "Not significantly above the level measured for noninfected control cells in an RNase assay as described in Meyers et al., 1999," means for example, that the RNase activity is less than 150% compared to the noninfected control cells.

[0028] "Inactivation of N.sup.pro" as used herein means the prevention or considerable reduction of the probable immunomodulating activity of N.sup.pro by mutation. In a preferred embodiment, this mutation prevents or considerably reduces the interference of N.sup.pro with the induction of an interferon response by the infected cells as described by Ruggli et al., 2003. In this case, the inactivation of N.sup.pro would allow the cell to mount a normal interferon response.

[0029] "Processing signal" as used herein relates to a substance that ensures the generation of a functional N-terminal of the C protein of the pestivirus, preferably of BVDV, in particular a substance selected from the group of ubiquitin, LC3, SUMO-1, NEDD8, GATE-16, and GABA(A)RAP. Also proteases selected from the group of intein, picornavirus 3C, caridovirus 2A, and p15 of rabbit hemorrhagic disease virus are understood as "processing signals" as used herein. Any other similar processing signal known to the skilled person that ensures the generation of a functional N-terminal of the C protein shall also be comprised in the term "processing signal".

[0030] "Protein C" or "C protein" or "C-protein" as used herein relates to a structural component of the pestivirus virion (Thiel et al., 1991). "Protein C" is the capsid or core protein of pestiviruses. The term, depending on the context, may also relate to the "Protein C" with one or several amino acids exchanges resulting from mutation of the encoding nucleotide sequence.

[0031] A "fragment" according to the invention is any subunit of a polynucleotide molecule according to the invention, i.e., any subset. For DNA, the fragment is characterized in that it is shorter than the DNA covering the full length viral genome.

[0032] A "functional variant" of the nucleotide molecule according to the invention is a nucleotide molecule which possesses a biological activity (either functional or structural) that is substantially similar to the nucleotide molecule according to the invention. The term "functional variant" also includes "a fragment", "a functional variant", "variant based on the degenerative nucleic acid code", or "chemical derivative". Such a "functional variant", e.g., may carry one or several nucleotide. exchanges, deletions, or insertions. The functional variant at least partially retains its biological activity, e.g., function as an infectious clone or a vaccine strain, or even exhibits improved biological activity. "Possess a biological activity that is substantially similar" means with respect to the pestiviruses provided herewith, for example, that the pestivirus is attenuated in a manner described herein and result in an non-pathogenic virus suitable for the production of live attenuated virus, which loss ability to pass the placenta but mediates an immune response after vaccination.

[0033] A "variant based on the degenerative nature of the genetic code" is a variant resulting from the fact that a certain amino acid may be encoded by several different nucleotide triplets. The variant at least partially retains its biological activity, or even exhibits improved biological activity.

[0034] A molecule is "substantially similar" to another molecule if both molecules have substantially similar nucleotide sequences or biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein if the nucleotide sequence is not identical, and two molecules which have a similar nucleotide sequence are considered variants as that term is used herein even if their biological activity is not identical.

[0035] A mutation as used herein relates to modifications in the nucleic acid molecules encoding the proteins/amino acids according to the invention. The mutations relate to, but are not limited to, substitutions (replacement of one or several nucleotides/base pairs), deletions (removal of one or several nucleotides/base pairs), and/or insertions (addition of one or several nucleotides/base pairs). As used herein, mutation may be a single mutation or several mutations, therefore, often the term "mutation(s)" is used and relates to both a single mutation and several mutations. The mutations include, but are not limited to point mutations (single nucleotide mutations) or larger mutations wherein, e.g., parts of the encoding nucleic acid molecules are deleted, substituted, and/or additional coding nucleic acid is inserted. The mutations may result in a modified expressed polypeptide due to the change in the coding sequence. Such modified polypeptides are desired, as set out in the disclosure of the invention as set out below.

[0036] The term "vaccine" as used herein 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. 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 understood herein is a 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 pestivirus infection, preferably by a BVDV infection. The attenuated pestivirus, in particular the attenuated BVDV as described herein, confer active immunity that may be transferred passively via maternal antibodies against the immunogens it contains and sometimes also against antigenically related organisms. A vaccine of the invention refers to a vaccine as defined above, wherein one immunologically active component is a BVDV or of pestiviral origin or derived from a nucleotide sequence that is more than 70% homologous to any known pestivirus sequence (sense or antisense).

[0037] The term "live vaccine" refers to a vaccine comprising a living, in particular, a living viral active component.

[0038] Additional components to enhance the immune response are constituents commonly referred to as "adjuvants", e.g., aluminum hydroxide, mineral or other oils, or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins, or growth factors.

[0039] A "pharmaceutical composition" essentially consists of one or more ingredients capable of modifying physiological, e.g., immunological functions, of the organism it is administered to, or of organisms living in or on the organism. The term includes, but is not restricted to, antibiotics or antiparasitics, as well as other constituents commonly used to achieve certain other objectives such as, but not limited to, processing traits, sterility, stability, feasibility to administer the composition via enteral or parenteral routes such as oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal, or other suitable route, tolerance after administration, or controlled release properties. One non-limiting example of such a pharmaceutical composition, solely given for demonstration purposes, could be prepared as follows: cell culture supernatant of an infected cell culture is mixed with a stabilizer (e.g., spermidine and/or bovine serum albumin (BSA)) and the mixture is subsequently lyophilized or dehydrated by other methods. Prior to vaccination, the mixture is then rehydrated in aqueous (e.g., saline, phosphate buffered saline (PBS)) or non-aqueous solutions (e.g., oil emulsion, aluminum-based adjuvant).

[0040] The solution to the above technical problem is achieved by the description and the embodiments characterized in the claims.

[0041] It has surprisingly been found that pestiviruses, in particular BVDV, can be more effectively attenuated by introducing at least one mutation in the coding sequence for glycoprotein E.sup.ms and at least another mutation in the coding sequence for N.sup.pro which preferably leads to combined inactivation of the RNase activity residing in glycoprotein E.sup.ms in addition to the inactivation of the immunomodulating activity residing in N.sup.pro. An immunomodulating effect in one aspect is indicated but not limited to the indicated function for one pestivirus in an exemplary manner by Ruggli et al., 2003.

[0042] A pestivirus, in particular BVDV, attenuated in accordance with the present invention may be advantageously used in vaccines. The attenuated pestivirus, in particular the attenuated BVDV, now provide live vaccines of high immunogenicity. Surprisingly, the pestivirus, in particular the BVDV, according to the invention furthermore are safe for use in pregnant animals as they do not cross the placenta. This is exemplified in a non-limiting manner for BVDV in Example 3.

[0043] Furthermore, live vaccines with defined mutations as a basis for attenuation will allow to avoid the disadvantages of the present generation of vaccines, e.g., the risk of reversion to an more pathogenic strain. A further advantage of the attenuating mutations lies in their molecular uniqueness which allows to use them as distinctive labels for an attenuated pestivirus, in particular BVDV, and to distinguish them from pestivirus, in particular BVDV, from the field. Therefore, in one aspect the present invention provides an attenuated pestivirus, in particular an attenuated BVDV, having at least one mutation in the coding sequence for glycoprotein E.sup.ms and at least another mutation in the coding sequence for N.sup.pro. Preferably, in such attenuated pestivirus, preferably in such attenuated BVDV, the mutation in the coding sequence for glycoprotein E.sup.ms leads to inactivation of the RNase activity residing in E.sup.ms and/or the mutation in the coding sequence for N.sup.pro leads to inactivation of the N.sup.pro. The inactivation may take place by any mutation known to the person skilled in the art of the E.sup.ms--and the N.sup.pro-coding sequence, wherein the mutations are any mutation as defined in the Definitions of Terms Used in the Description section above, such as deletions, insertion mutations, and/or substitution mutations. Most preferably, the mutation(s) are deletions, as the likelihood for revertation to the wild-type is the lowest for deletions.

[0044] It has been shown that the glycoprotein E.sup.ms forms a disulfide-bonded homodimer of about 97 kD, wherein each monomer consists of 227 amino acids corresponding to the amino acids 268 to 494 of the CSFV polyprotein as described by Rumenapf et al., 1993. The genome sequence of the Alfort/Tubingen strain of CSFV is available in the GenBank/EMBL data library under accession number J04358; alternatively, the amino acid sequence for the BVDV strain CP7 can be accessed in the GenBank/EMBL data library (accession number U63479); in the BVDV CP7 polyprotein, the E.sup.ms protein corresponds to residues 271 to 497. Two regions of amino acids are highly conserved in glycoprotein E.sup.ms as well as in some plant and fungal RNase-active proteins (Schneider et al., 1993). These two regions are of particular importance to the RNase enzymatic activity. The first region consists of the region at the amino acids at position 295 to 307 (298 to 310 for BVDV strain CP7) and the second region consists of the amino acids at position 338 to 357 (341 to 360 for BVDV strain CP7) of the viral polyprotein as exemplified for the Alfort strain of CSFV in Meyers et al., 1999 (numbering according to the published deduced amino acid sequence of CSFV strain Alfort/Tubingen (Meyers et al., 1989). The amino acids of particular importance to the RNase activity as mentioned above are by no means limited to the exact position as defined for the Alfort/Tubingen strain of CSFV but are simply used in an exemplary manner to point out the preferred amino acids being at that position or corresponding to that position in other strains such as found in BVDV, BDV, and pestiviruses in general since they are highly conserved. For pestiviruses other than the CSFV Alfort/Tubingen strain, the numbering of the positions of the preferred amino acids can be different but an expert in the field of the molecular biology of pestiviruses will easily identify these preferred amino acids by the high degree of conservation of this amino acid sequence and the position of these motifs in the sequence context. In one particular non-limiting example, the position of CSFV Alfort/Tubingen 346 is identical to position 349 of BVDV strain CP7.

[0045] As a consequence, the present invention preferably relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for glycoprotein E.sup.ms are located in the encoding nucleotide sequence corresponding to amino acids at position 298 to 310 and/or position 341 to 360. Preferably, such mutations are (where amino acids are given in the one letter symbols; the amino acid before the position number indicates the amino acid to be substituted, the amino acid after the position number the substituting amino acid (del indicates deletion): for example, H300L means histidine 300 was substituted by leucine):

[0046] Suitable modification of the glycoprotein E.sup.ms are for example, the single substitutions/deletions: S298G, H300K, H300L, H300R, H300del, W303G, P304del, E305A, C308G, R343G, E345del, W346G, K348A, H349K, H349L, H349del, H349Q, H349SV (mutation H349S and insertion of V), K348R, W351P, W351G, W351L, W351K, W351H; the double substitutions/deletions: H300L/H349L, K348del/H349del, H349det/G350del, E345del/H349del, W303G/E305A, H300K/H349K, H300K/H349L and the triple deletions: L299del/H300del/G300del, K348del/H349del/G350del. Numbering is according to the published amino acid sequence of BVDV CP7 for all the mutants listed above (the given numbers minus 3 would correspond to the equivalent residues of the CSFV Alfort/Tubingen amino acid sequence). All the above-listed mutants were at least tested as respective CSFV or BVDV mutants without mutations in the N.sup.pro region. Suitable mutants of the pestiviral glycoprotein E.sup.ms are provided, for example, by WO 99/64604, which is incorporated herein in its entirety. It should be noted, however, that according to the present invention, at least one additional mutation in the N.sup.pro region, as disclosed in further detail below, must be present.

[0047] It was particularly found that deletion or substitution of the histidine residue at position 346 (CSFV) or 349 (BVDV) leads to effective inactivation of E.sup.ms and therefore leads to particularly useful pestiviral live vaccines. The present invention demonstrates that pestiviruses are viable and code for an E.sup.ms protein without RNase activity when the histidine residue at position 346 of the viral polyprotein (numbering according to the published sequence of CSFV Alfort/Tubingen (Meyers et al., 1989)), or at position 349 (numbering according to the published sequence of BVDV CP7 (Meyers et al., 1996b)) if the pestivirus is BVDV, which represents one of the conserved putative active site residues of the E.sup.ms RNase, is deleted. Thus, preferably, the invention also relates to a BVDV according to the invention, wherein the mutation in the coding sequence for glycoprotein E.sup.ms is a deletion or substitution of the histidine residue at position 349. Even more specifically, the putative active site of the RNase is represented by the conserved E.sup.ms sequences SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW (sequence of the BVDV-2 NewYork93 protein given here in an exemplary manner; minor changes can possibly be found in other pestivirus sequences but the identity of the motif will always be obvious for an expert in the field. As an example, the corresponding amino acid sequences of BVDV-1 CP7 would be SLHGIWPEKICTG and/or LQRHEWNKHGWCNWYNIEPW and that of CSFV Alfort/Tubingen SLHGIWPEKICKG and/or LQRHEWNKHGWCNWYNIDPW). Thus, preferably, the invention further relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for glycoprotein E.sup.ms are located in the nucleotide sequence coding for the conserved E.sup.ms sequence SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW. These sequences are representing the putative active site of the RNase. The sequences SLHGIWPEKIC and RHEWNKHGWCNW of the putative E.sup.ms active site are even more conserved across pestiviruses. Thus, preferably, the invention also relates to a pestivirus, in particular to BVDV, having at least one mutation in the coding sequence of the N.sup.pro protein and the glycoprotein E.sup.ms, wherein the mutation(s) in the coding sequence for glycoprotein E.sup.ms are located in the nucleotide sequence coding for the conserved E.sup.ms sequence SLHGIWPEKIC and/or RHEWNKHGWCNR. Preferably, the mutation is located in only one of the sequences. Thus the invention also relates to a pestivirus, in particular to BVDV, having at least one mutation in the coding sequence of the N.sup.pro protein and the glycoprotein E.sup.ms, wherein the mutation(s) in the coding sequence for glycoprotein E.sup.ms are located in the nucleotide sequence coding for the conserved E.sup.ms sequence SLHGIWPEKIC or RHEWNKHGWCNR. Preferably, such mutations concern two different amino acids, i.e., are double mutations. Thus, the mutations may be 1 to 3 nucleotide mutations in two different triplets encoding two amino acids. Thus, the invention also relates to a pestivirus, in particular to BVDV having at least one mutation in the coding sequence of the N.sup.pro protein and the glycoprotein E.sup.ms, wherein the mutation(s) in the coding sequence for glycoprotein E.sup.ms are two mutations located in the nucleotide sequence coding for the conserved E.sup.ms sequence SLHGIWPEKIC and/or RHEWNKHGWCNR. Preferably, such mutations concern a single amino acid. Thus, the mutation may be 1 to 3 nucleotide mutations in one triplet encoding one amino acid. Thus, the invention also relates to a pestivirus, in particular to BVDV, having at least one mutation in the coding sequence of the N.sup.pro protein and the glycoprotein E.sup.ms, wherein a single mutation is located in the conserved E.sup.ms sequence SLHGIWPEKIC or RHEWNKHGWCNR.

[0048] As mentioned above, the attenuated pestiviruses provided by the present invention, having at least on mutation in the coding sequence of the glycoprotein E.sup.ms and in the coding sequence of the N.sup.pro protein, wherein the mutation preferably result in inactivation of the RNase activity residing in the glycoprotein E.sup.ms and of the immunomodulating activity residing in N.sup.pro. Inactivation of the N.sup.pro is achieved in pestiviruses, in particular BVDV, of the specified formula described more in detail below, wherein between 0 and all amino acids of N.sup.pro are present; ubiquitin or LC3 or another sequence serving as processing signal (e.g., SUMO-1, NEDD8, GATE-16, GABA(A)RAP, or proteases, e.g., intein, picornavirus 3C, caridovirus 2A, or p15 of rabbit hemorrhagic disease virus) is present or absent. In case a processing signal is present, the coding sequence of the processing signal is inserted at or close to the C-terminal end of the (remaining part of the) N.sup.pro-protein. Only in the case that a processing signal is present, any number of amino acids coding for N.sup.pro (=N.sup.pro amino acids) may be present. In case no processing signal sequence is inserted, a maximum of 12 amino acids, preferably aminoterminal amino acids, of N.sup.pro may be present, the remaining amino acids have to be deleted. Furthermore, other than the E.sup.ms mutations as disclosed above (at least one of which has to be present in the pestivirus, in particular in BVDV according to the invention), the remaining sequences of the pestivirus, in particular BVDV may remain unchanged, i.e., are not mutated, or may also have mutations close to the N-terminal end of the C-protein. A number of more specific embodiments as disclosed below exemplify this.

[0049] Thus, the invention relates to a pestivirus, in particular to BVDV according to the invention,

[0050] wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

[N.sup.pro].sub.x-[PS].sub.y[C-term],

wherein: [0051] [N.sup.pro] relates to the N.sup.pro portion of the polyprotein, wherein x represents the number of amino acids of the N.sup.pro present in the polyprotein; [0052] [PS] relates to a processing signal selected from: ubiquitin, LC3, SUMO-1, NEDD8, GATE-16 or GABA(A)RAP) or proteases, e.g., intein, picornavirus 3C, caridovirus 2A, or p15 of rabbit hemorrhagic disease virus, or the like, or any processing signal known to the skilled person that ensures the generation of a functional N-terminal of the C-protein and y may be 0, which means that no processing signal is present (i.e., that PS is absent), or y may be 1, which means that a processing signal is present (i.e., that PS is present); [0053] [C-term] relates to the complete pestivirus, in particular the complete BVDV polyprotein except for N.sup.pro, but including the capsid (C)-protein and any other protein present in the pestivirus polyprotein, in particular in the BVDV polyprotein including the carboxyterminal NS5B. Preferably, the glycoprotein E.sup.ms in the [C-term] is mutated, in such that the RNase activity residing in the glycoprotein E.sup.ms is inactivated. The term "any other protein present in the pestivirus polyprotein/BVDV polyprotein" relates to E.sup.ms, E1, E2, p7, NS2, NS3, NS4A, NS4B, and NS5A, wherein glycoprotein E.sup.ms is mutated, preferably as disclosed herein (see above), in such that the RNase activity residing in the glycoprotein E.sup.ms is inactivated. Preferably, the pestivirus, in particular the BVDV, according to the invention has a C-protein which is not mutated except for the amino acid at position 2 which is changed from D to N. Therefore, [C-term*] is the same as [C-term] but with a mutation at position 2 of the C-protein (N instead of D); [0054] if y is 0 (which means that no [PS] is present) then x is 0 to 12, (which means no N.sup.pro specific amino acid or 1 to 12 amino acids of N.sup.pro, preferably of the N-terminus of N.sup.pro, are present); and [0055] if y is 1 (which means that [PS] is present) then x is 0 to 168; (which means no N.sup.pro specific amino acid or 1 to all 168 amino acids of N.sup.pro, preferably of the N-terminus of N.sup.pro, are present).

[0056] Also more preferably, the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

[N.sup.pro].sub.1-[PS].sub.0-[C-term],

wherein the definitions are as defined above.

[0057] A specific example thereof is disclosed below, wherein the N-terminal methionine is followed by the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B. Hence, most preferably, the invention relates to a pestivirus, in particular BVDV, according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

M[C-term],

wherein the definitions are as defined above.

[0058] Also more preferably, the invention relates to a pestivirus, in particular to BVDV, according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

[N.sup.pro].sub.3-[PS].sub.0-[C-term],

wherein the definitions are as defined above.

[0059] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the N.sup.pro sequence E.sup.ms and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B. Hence, most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

MEL-[C-term],

wherein the definitions are as defined above.

[0060] Also more preferably, the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

[N.sup.pro].sub.4-[PS].sub.0-[C-term],

wherein the definitions are as defined above.

[0061] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the N.sup.pro sequence ELF and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B. Hence, most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

MELF-[C-term],

wherein the definitions are as defined above.

[0062] Also more preferably, the invention relates to a pestivirus, in particular to BVDV, according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

[N.sup.pro].sub.6-[PS].sub.0-[C-term],

wherein the definitions are as defined above.

[0063] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the N.sup.pro sequence ELFSN and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B. Hence, most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

MELFSN-[C-term],

wherein the definitions are as defined above.

[0064] Also more preferably, the invention relates to a pestivirus, in particular to BVDV, according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

[NP''].sub.4-[PS].sub.0-[C-term*],

wherein the definitions are as defined above except for the fact that the aminoterminal part of the C-protein is changed.

[0065] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the N.sup.pro sequence ELF and in the C-protein sequence, the amino acid at position 2 is changed from D to N. Therefore, the aminoterminal C-protein sequence is SNEGSK . . . instead of SDEGSK. Hence, most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

MELF-[C-term*],

wherein in the C-protein the amino acid at position 2 is changed from D to N, and the definitions are as defined above.

[0066] Also more preferably, the invention relates to a pestivirus, in particular BVDV, according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

[N.sup.pro].sub.x-[PS].sub.1-[C-term],

wherein the definitions are as defined as above, and PS is any of the PS disclosed above, preferably selected from the group of ubiquitin or LC3.

[0067] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by any 21 or 28 N.sup.pro amino acids, ubiquitin, or LC3 and the C-protein. Hence most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula:

[N.sup.pro].sub.22-[PS].sub.1-[C-term], wherein preferably the PS is ubiquitin or LC3, or [N.sup.pro].sub.29-[PS].sub.1-[C-term], wherein preferably the PS is ubiquitin or LC3.

[0068] Ubiquitin is a well known highly conserved cellular protein of 76 amino acids. Among other functions, ubiquitin is a key player in protein catabolism since conjugation with ubiquitin can mark a protein for degradation via the proteasome. Ubiquitin conjugated with or fused to other proteins via the carboxyterminal glycine can be cleaved off by cellular ubiquitin-specific proteases. Thus, fusion of a protein to the carboxyterminus of ubiquitin will usually result in defined proteolytic cleavage of the fusion protein into its components when expressed within a cell.

[0069] LC3 (light chain 3 of microtubule associated proteins) represents a cellular protein of 125 amino acids that serves a variety of functions (length given for bovine LC3). Recently, a fundamental role of the protein in autophagy has been defined. During this process, LC3 is activated by carboxyterminal cleavage. Thereby, a new carboxyterminus is generated that consists of glycine. LC3 is then conjugated via the carboxyterminal glycine to phosphatidylethanolamine present in the membranes of autophagic vesicles. Because of this process, a protein fused to the carboxyterminus of LC3 will be cleaved off by a cellular protease at a defined position.

[0070] Also more preferably, the invention relates to a pestivirus, preferably to BVDV according to the invention, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula selected from the group of:

[0071] [N.sup.pro].sub.2-[PS].sub.y-[C-term] and preferably ME-[PS].sub.y-[C-term];

[0072] [N.sup.pro].sub.5-[PS].sub.y-[C-term] and preferably MELFS-[PS].sub.y-[C-term];

[0073] [N.sup.pro].sub.7-[PS].sub.y-[C-term] and preferably MELFSNE-[PS].sub.y-[C-term];

[0074] [N.sup.pro].sub.8-[PS].sub.y-[C-term] and preferably MELFSNEL-[PS].sub.y-[C-term];

[0075] [N.sup.pro].sub.9-[PS].sub.y-[C-term] and preferably MELFSNELL-[PS].sub.y-[C-term];

[0076] [N.sup.pro].sub.10-[PS].sub.y-[C-term] and preferably MELFSNELLY-[PS].sub.y-[C-term];

[0077] [N.sup.pro].sub.11-[PS].sub.y-[C-term] and preferably MELFSNELLYK-[PS].sub.y-[C-term]; and

[0078] [N.sup.pro].sub.12-[PS].sub.y-[C-term] and preferably MELFSNELLYKT-[PS].sub.y-[C-term],

wherein the definitions are as defined as above. The preferably disclosed embodiments refers to BVDV. Most preferably, y is 0 (i.e., no PS is present).

[0079] Also more preferably, the BVDV according to the invention as described supra is a BVDV type 1 BVDV. Most preferably, the BVDV according to the invention as described supra is a BVDV type 2 BVDV. BVDV-1 and BVDV-2 are differentiated according to features of their genomic sequences (Heinz et al., 2000 and references therein). BVDV-1 as disclosed herein may be used in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 1 infections in breeding stocks of cattle, in pregnant cows and in the induction of fetal protection against BVDV type 1 infection is pregnant cows. Surprisingly, a BVDV-2 as disclosed herein may be used in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 1 infections in breeding stocks of cattle. In particular, the invention relates to the use of a BVDV type 2 according to the invention in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 1 infections in pregnant cows. Preferably, the BVDV type 2 according to the invention may be used in the manufacture of a composition for use in the induction of fetal protection against BVDV type 1 infections in pregnant cows. Surprisingly also, a BVDV-1 as disclosed herein may be used in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 2 infections in breeding stocks of cattle. In particular, the invention relates to the use of a BVDV type 1 according to the invention in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 2 infections in pregnant cows. Preferably, the BVDV type 1 according to the invention may be used in the manufacture of a composition for use in the induction of fetal protection against BVDV type 2 infections in pregnant cows. Most preferred is the use of BVDV type 1 and type 2 in combination for the manufacture of a composition for use in the prevention and/or treatment of BVDV type 1 and or type 2 infections in breeding stocks of cattle, in pregnant cows and in the induction of fetal protection against BVDV type 1 and/or type 2 infections is pregnant cows.

[0080] Most preferably, the wild-type BVDV according to the invention which is to be mutated as disclosed herein corresponds to amino acid sequence SEQ ID NO:5 (termed XIKE-A) or is a functional variant thereof. Most preferably also, the BVDV according to the invention has a N.sup.pro mutation according to the invention and corresponds to amino acid sequence SEQ ID NO:6 (termed XIKE-A-NdN) or is a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the amino acid sequence disclosed herein. On the amino acid level, homologies are very roughly: BVDV-1/-BVDV-1: 93%; BVDV-1/-BVDV-2: 84%; BVDV-2/-BVDV-2: 98%. Therefore, more preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the amino acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the amino acid sequence disclosed herein.

[0081] Most preferably also, the BVDV according to the invention has a E.sup.ms mutation according to the invention which has a deletion of the codon coding for histidine 349, and corresponds to amino acid sequence SEQ ID NO:7 (termed XIKE-B) or is a functional variant thereof. Most preferably also, the BVDV according to the invention has both a E.sup.ms mutation and a N.sup.pro mutation according to the invention, wherein the codon coding for histidine 349 of E.sup.ms is deleted and also the complete N.sup.pro coding region is deleted, except for codons 1 to 4, thus amino acids MELF of N.sup.pro remain. The mutant corresponds to amino acid sequence SEQ ID NO:8 (termed XIKE-B-NdN) or is a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the amino acid sequence disclosed herein. More preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the amino acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the amino acid sequence disclosed herein.

[0082] Most preferably also, the BVDV according to the invention has a E.sup.ms mutation according to the invention which has a substitution of the codon coding for histidine 300 by the codon coding for leucine and corresponds to amino acid sequence SEQ ID NO:9 (termed XIKE-C) or is a functional variant thereof. Most preferably also, the BVDV according to the invention has both a E.sup.ms mutation and a N.sup.pro mutation according to the invention, wherein the codon coding for histidine 300 is substituted by the codon coding for leucine and also the complete N.sup.pro coding region is deleted, except for codons 1 to 4, thus amino acids MELF of N.sup.pro remain. The mutant corresponds to amino acid sequence SEQ ID NO:10 (termed XLKE-C NdN) or is a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the amino acid sequence disclosed herein. More preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the amino acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the amino acid sequence disclosed herein.

[0083] Another important embodiment of the invention described herein is a composition comprising a pestivirus, in particular a BVDV according to the invention and a solution. The skilled person knows additional components which may be comprised in the composition (see also Remington's Pharmaceutical Sciences, 18th ed. Mack Publ., Easton (1990)). The expert may use known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as, e.g., saline or corresponding plasma protein solutions, are readily available. The pharmaceutical compositions may be present as lyophylisates or dry preparations, which can be reconstituted with a known injectable solution directly before use under sterile conditions, e.g., as a kit of parts.

[0084] The final preparation of the compositions of the present invention are prepared for, e.g., injection by mixing the pestivirus, preferably BVDV according to the invention with a sterile physiologically acceptable solution, that may be supplemented with known carrier substances or/and additives (e.g., serum albumin, dextrose, sodium bisulfite, EDTA). The solution may be based on a physiologically acceptable solvent, e.g., an aqueous solution between pH 7 and 8. The pH may be stabilized by a pharmaceutically acceptable buffer. The solution may also contain further stabilizing agents like a detergent like Tween 20, serum albumin such as bovine serum albumin (BSA), ascorbic acid, and/or spermidine. The composition may also comprise adjuvants, e.g., aluminum hydroxide, mineral or other oils or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins, or growth factors.

[0085] For example, in a composition according to the invention, the pestivirus, in particular BVDV may be solved in:

TABLE-US-00001 Pestivirus (preferably BVDV) 10.sup.2-10.sup.8 TCID.sub.50 SGS* 25% v/v Cell culture medium qsp 1 dose *SGS: Composition per 2 mL Sucrose 75 mg Gelatin 20 mg Potassium hydroxide 0.274 mg L- glutamic acid 0.72 mg Potassium dihydrogen phosphate 0.516 mg Dipotassium phosphate 1.254 mg Water for injection qsp 2 mL

[0086] If the composition is first lyophilized or dehydrated by other methods, then, prior to vaccination, the composition is rehydrated in aqueous (e.g., saline, phosphate buffered saline (PBS)) or non-aqueous solutions (e.g., oil emulsion (mineral oil, or vegetable/metabolizable oil based/single or double emulsion based), aluminum-based, carbomer based adjuvant).

[0087] Preferably, the composition according to the invention induces an immunological response in an animal. More preferred, the composition according to the invention is a vaccine. A vaccine as understood herein comprises a pestivirus, in particular BVDV according to the invention and is defined above in the Definitions of Terms Used in the Description section.

[0088] Most preferred, the composition according to the invention further comprises a pharmaceutically acceptable carrier or excipient. Several carriers or excipients are disclosed above. The composition may comprise, if aimed at injections or infusion, substances for preparing isotonic solutions, preservatives such as p-hydroxybenzoates, stabilizers such as alkali salts of ethylendiamintetracetic acid, possibly also containing emulsifying and/or dispersing agents.

[0089] The composition according to the invention may be applied intradermally, intratracheally, or intravaginally. The composition preferably may be applied intramuscularly or intranasally. In an animal body, it can prove advantageous to apply the pharmaceutical compositions as described above via an intravenous or by direct injection into target tissues. For systemic application, the intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred. A more local application can be effected subcutaneously, intradermally, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily, or directly in or near the tissue to be treated (connective-, bone-, muscle-, nerve-, or epithelial tissue). Depending on the desired duration and effectiveness of the treatment, the compositions according to the invention may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks, or months and in different dosages.

[0090] The invention also relates to the use of a pestivirus, in particular BVDV, according to the invention in the manufacture of a vaccine for the prophylaxis and treatment of pestiviral infections, in particular of BVDV infections.

[0091] Another important part of the invention is a polynucleotide molecule comprising the nucleic acid coding for a pestivirus, in particular for a BVDV, according to the invention, or a fragment, functional variant, variant based on the degenerative nucleic acid code, fusion molecule, or a chemical derivative thereof. Preferably, the polynucleotide molecule is DNA. Also preferably, the polynucleotide molecule is RNA. In a more preferred embodiment, the polynucleotide molecule also comprises the nucleotide sequence of a functional 5'- and/or 3'-non-translated region of a pestivirus, in particular of BVDV.

[0092] There are several nucleotide sequences known in the art, which represents the basis for the production of a polynucleotide molecule coding for a pestivirus attenuated according to the present invention, having at least one mutation in the coding sequence of N.sup.pro and at least one in the coding sequence of glycoprotein E.sup.ms, wherein the mutations result in an combined inactivation of the RNase activity residing in glycoprotein E.sup.ms and in the inactivation of the immunomodulating activity residing in N.sup.pro. Examples of nucleic acid sequences of wild-type sequences of several members of pestiviruses are listed below:

TABLE-US-00002 Border disease virus Strain BD31 NCBI GenBank Accession No. [U70263] Strain X818 NCBI GenBank Accession No. [AF037405] Bovine viral diarrhea virus 1 Strain NADL NCBI GenBank Accession No. [M31182] Strain Osloss NCBI GenBank Accession No. [M96687] Strain SD-1 NCBI GenBank Accession No. [M96751] Strain CP7 NCBI GenBank Accession No. [U63479] Bovine viral diarrhea virus 2 Strain 890 NCBI GenBank Accession No. [U18059] Strain C413 NCBI GenBank Accession No. [AF002227] Classical swine fever virus Strain Alfort/187 NCBI GenBank Accession No. [X87939] Strain Alfort-Tubingen NCBI GenBank Accession No. [J04358] Strain Brescia NCBI GenBank Accession No. [M31768] Strain C strain NCBI GenBank Accession No. [Z46258]

[0093] The mutations/modifications according to the invention relating to the coding sequence of N.sup.pro and E.sup.ms are described above more in detail. Having this information, a person skilled in the art is able to realize the manufacture of any polynucleotide/polynucleic acid coding for a pestivirus according to the present invention. Furthermore, this person is able to manufacture an attenuated pestivirus according to the invention. Molecular method for introducing a mutation into a polynucleotide sequence, cloning, and amplification of the mutated polynucleotide are for example provided by Sambrook et al., 1989 or Ausubel et al., 1994.

[0094] Most preferably, the wild-type BVDV according to the invention which is to be mutated as disclosed herein is encoded by the nucleic acid sequence SEQ ID NO:1 (termed XIKE-A) or a functional variant thereof. Most preferably also, the BVDV according to the invention has a N.sup.pro mutation according to the invention and is encoded by nucleic acid sequence SEQ ID NO:2 (termed XIKE-A-NdN) or a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the nucleic acid sequence disclosed herein. On the nucleic acid level, homologies are very roughly: BVDV-1/-BVDV-1: 80%; BVDV-1/-BVDV-2: 70%; BVDV-2/-BVDV-2: 96%. Therefore, more preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the nucleic acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the nucleic acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the nucleic acid sequence disclosed herein.

[0095] Most preferably also, the BVDV according to the invention has a E.sup.ms mutation according to the invention which has a deletion of codon H349 and is encoded by nucleic acid sequence SEQ ID NO:7 (termed XIKE-B) or by a functional variant thereof. Most preferably also, the BVDV according to the invention has both a E.sup.ms mutation and a N.sup.pro mutation according to the invention, wherein the codon coding for histidine 349 of E.sup.ms is deleted and also the complete N.sup.pro coding region is deleted, except for codons 1 to 4, thus amino acids MELF of N.sup.pro remain. The mutant is encoded by nucleic acid sequence SEQ ID NO:8 (termed XIKE-B-NdN) or by a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the nucleic acid sequence disclosed herein. More preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the nucleic acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the nucleic acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the nucleic acid sequence disclosed herein.

[0096] Most preferably also, the BVDV according to the invention has a E.sup.ms mutation according to the invention which is a substitution of codon "H300" by a leucine codon, and is encoded by nucleic acid sequence SEQ ID NO:11 (termed XIKE-C) or a functional variant thereof. Most preferably also, the BVDV according to the invention has both a E.sup.ms mutation and a N.sup.pro mutation according to the invention, wherein the codon coding for histidine 300 is substituted by the codon coding for leucine and also the complete N.sup.pro coding region is deleted, except for codons 1 to 4, thus amino acids MELF of N.sup.pro remain. The mutant is encoded by nucleic acid sequence SEQ ID NO:12 (termed XIKE-C-NdN) or by a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the nucleic acid sequence disclosed herein. More preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the nucleic acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the nucleic acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the nucleic acid sequence disclosed herein.

[0097] Another important aspect of the invention is a method for attenuating a pestivirus, characterized in that at least one mutation in the coding sequence for glycoprotein E.sup.ms and at least another mutation in the coding sequence for N.sup.pro is generated in a pestivirus genome. According to a preferred embodiment, the pestivirus is BVDV.

[0098] According to a more preferred embodiment, the method comprises the steps: [0099] a) reverse transcription of a wild-type pestivirus nucleotide sequence into a cDNA; [0100] b) cloning the cDNA; [0101] c) introducing mutations selected from the group of deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein E.sup.ms and the protease N.sup.pro; and [0102] d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of pestivirus cDNA into RNA in vitro or upon infection of suitable cells.

[0103] Regarding the method for attenuating a BVDV according to the invention, the preferred methods comprises the steps: [0104] a) reverse transcription of a wild-type BVDV nucleotide sequence into a cDNA; [0105] b) cloning the cDNA; [0106] c) introducing mutations selected from the group of deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein E.sup.ms and the protease N.sup.pro; and [0107] d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of pestivirus cDNA into RNA in vitro or upon infection of suitable cells.

[0108] Yet another important embodiment of the invention is a method of treatment of disease caused by a pestivirus, wherein a pestivirus according to the invention or a composition according to the invention, wherein the pestivirus or the composition is administered to an animal in need thereof at a suitable doses as known to the skilled person and the reduction of symptoms of the pestivirus infection.

[0109] Yet another important embodiment of the invention is a method of treatment of disease caused by BVDV, wherein a BVDV according to the invention or a composition according to the invention, wherein the BVDV or the composition is administered to an animal in need thereof at a suitable doses as known to the skilled person and the reduction of symptoms of BVDV infection such as viremia and leukopenia and/or pyrexia and/or diarrhea is monitored.

EXAMPLES

[0110] The following examples serve to further illustrate the present invention; but the same should not be construed as limiting the scope of the invention disclosed herein.

Example 1

BVDV XIKE-B: Fetopathogenicity Assessment in Pregnant Heifers

[0111] BVDV XIKE-B, an RNase negative mutant of the highly pathogenic BVDV type 2 isolate NewYork93/C was recovered from the infectious cDNA clone pKANE40B and showed wild-type-like (wt-like) growth characteristics in tissue culture. In animal experiments the mutant virus was found to be considerably attenuated so that it represented a promising candidate for development of a live attenuated vaccine virus (Meyer et al., 2002). To test whether this attenuated virus is still able to cross the placenta and infect the fetus, pregnant heifers were infected with XIKE-B. As a control, wild-type BVDV recovered from cDNA clone pKANE40A was used. The respective virus named XIKE-A expresses an active E.sup.ms RNase in the infected cell. The study aimed to assess the safety of XIKE-A and XIKE-B in pregnant animals.

Experimental Design

[0112] Ten pregnant heifers were selected from a BVDV negative herd. The following groups of 5 heifers were included in the trial:

TABLE-US-00003 No. Inoculation Virus Group 1: 5 One i.n. administration, XIKE-A 3 mL in each nostril Group 2: 5 One i.n. administration, XIKE-B 3 mL in each nostril

[0113] Heifers were moved to the experimental facilities 8 days before inoculations. Pregnancy status was confirmed after transport into the experimental facility. Heifers were between days 60 and 90 of gestation on the day of inoculation. Inoculation took place for all animals at one point of time.

[0114] Heifers were monitored for the presence of clinical signs of BVDV infection including abortions during the observation period. Blood samples were collected from the animals for serology, antigen detection, and white blood cells were counted. The experiment was terminated 9 weeks after infection. Non-aborted cows were slaughtered, the uterus examined, and collected. Fetal organ samples were collected during routine necropsy and examined for BVDV infection.

[0115] The presence of fetal infection was the main evaluation parameter, composed from the number of BVDV-related cow mortality, the number of BVDV-related abortions, and the number of BVDV positive fetuses at termination. In addition to the main parameter, clinical signs characteristic for BVDV infection, viremia, and white blood cell counts in cows and rectal temperature after challenge were evaluated.

Animals

[0116] Heifers were purchased from a farm free of BVDV. Only animals which met the following inclusion criteria were used.

Inclusion Criteria

[0117] Free of BVD antibodies; each individual was tested in the serum antibody test prior to transport and at the initiation of the study (at the animal test facility). [0118] Free of BVDV; plasma and/or buffy-coat preparation from each individual was tested by a suitable test. [0119] Clinically healthy at the initiation of the study judged upon physical examination. The health examination of the animals was accomplished in accordance with the current, generally accepted veterinary practice. [0120] Pregnancy confirmed by physical examination before inoculation. Pregnancy was between 60-90 days at the time of inoculation, proven by insemination records.

TABLE-US-00004 [0120] Test Strain A Description: XIKE-A, live virus BVDV strain Composition: Experimental material comprising of cell culture supernatant of low passaged XIKE-A BVD components: BVDV type II strain: XIKE-A Supplied by: Dr. Gregor Meyers, "Bundesforschungsanstalt fur Viruskrankheiten der Tiere" (BFAV), Paul- Ehrlich-Stra.beta.e 28, 72076 Tubingen, Germany Applied BVD Type 1 strain: 10.sup.5 TCID.sub.50/6 mL (TCID = Tissue virus dose: Culture Infective Dose) Applied 3 mL per nostril vaccine volume: Application Intranasal route: Preparation The inoculum was sent in a pre-diluted frozen form of dosage in a 50 mL vial on dry ice and was to be stored form: at -70.degree. C. before inoculation. Immediately before inoculation of Group 1 heifers, the material was thawed avoiding local temperatures above 37.degree. C. After no ice was visible in the fluid, material was gently stirred and immediately used for inoculation of the animals. Unused inoculum: The volume of the unused material was be measured and split on two aliquots before immediate freezing in dry ice or liquid nitrogen and stored for re-titration purposes. Virus and contaminated plastic or glassware were incubated with an appropriate volume of an 8-10% formaldehyde solution for at least 24 hours at room temperature before discarding in order to inactivate viruses.

TABLE-US-00005 Test Strain B Description: XIKE-B, live virus BVDV strain Composition: Experimental material comprising of cell culture supernatant of low passaged XIKE-B BVD components: BVDV type II strain: XIKE-B Supplied by: Dr. Gregor Meyers, "Bundesforschungsanstalt fur Viruskrankheiten der Tiere" (BFAV), Paul- Ehrlich-Stra.beta.e 28, 72076 Tubingen, Germany Applied BVD Type 1 strain: 10.sup.5 TCID.sub.50/6 mL (TCID = Tissue virus dose: Culture Infective Dose) Applied 3 mL per nostril vaccine volume: Application Intranasal route: Preparation The inoculum was sent in a pre-diluted frozen form of dosage in a 50 mL vial on dry ice and was stored at -70.degree. C. form: before inoculation. Immediately before inoculation of Group 2 heifers, the material was thawed avoiding local temperatures above 37.degree. C. After no ice was visible in the fluid, material was gently stirred and immediately used for inoculation of the animals. Unused vaccine: The volume of the unused material was measured and split on two aliquots before immediate freezing in dry ice or liquid nitrogen and stored for re- titration purposes. Virus and contaminated plastic or glassware was incubated with an appropriate volume of an 8-10% formaldehyde solution for at least 24 hours at room temperature before discarding in order to inactivate viruses.

Pregnancy Control

[0121] Pregnancy was confirmed immediately before inoculation.

Inoculation of Heifers

[0122] The inoculation is Day 0 of the experiment.

[0123] In each nostril, 3 mL of the test material was administered intranasally by syringe without needle. Each time a new sterile syringe was taken. Administration was performed during the aspiration phase in order to minimize loss of fluid via expiration of material.

Post-Inoculation Observations

[0124] Collection and Examination of Blood Samples Blood was collected following standard, aseptic procedures (disinfecting the bleeding site). A new sterile syringe and needle was used for each animal.

Blood Collection to Prepare Serum

[0125] At least 10 mL blood was collected from the heifers immediately before inoculation, then weekly after infection and at the termination of the study. Serum was stored at -20.degree. C. until required.

Blood Collection for Leukocyte Counts and Buffy Coat Preparations

[0126] For leukocyte counting, 3 mL blood was transferred immediately after collection to suitable sterile vessels (Venoject, Terumo Europe N.V., Leuven, Belgium), pre-filled with 0.06 mL EDTA (0.235 MOL/L).

[0127] For buffy coat preparations, at least 15 mL blood was transferred immediately after collection to suitable sterile vessels, pre-filled with 0.1 mL Heparin solution (Na-heparin for inj., 5000 IU/mL lot A7B 163A, exp. date: November 2000: Gedeon Richter R T, Budapest, Hungary) yielding at least 20 IU Heparin per mL blood in the blood sample. The content was carefully mixed thereafter.

[0128] For preparation of buffy coats and leukocyte counting, blood was collected from the heifers on every day, between Day 0 and Day 14 after infection; and on every second day, between Day 15 and Day 40, or until all animals were negative for virus isolation for three consecutive sampling time points.

Preparation of Serum

[0129] Blood was allowed to clot at room temperature, and separated by centrifugation. Each serum sample was divided into two aliquots of at least 2 mL each. One set of aliquots was assayed for BVDV specific antibodies by ELISA. The rest of the sera was frozen and stored at -20.degree. C. until required.

Leukocyte Counts

[0130] Leukocyte counts was determined with a coulter-counter semi-automated electronic device (Diatron Minicell-16, Messtechnik GmbH, Wien, Austria) with a claimed accuracy of 0.1.times.10.sup.9/1,100 .mu.L. The instrument was used (calibration and leukocyte-counts) according to the manufacturer's recommendations.

Preparation of Buffy Coats

[0131] Heparin blood samples was transported to the laboratory as soon as possible. Buffy coat preparation procedure, following a standard laboratory procedure, was performed under aseptic conditions (sterile pipettes, handling, clean bench, etc.).

[0132] The obtained buffy coats were re-suspended in a small volume (2 mL) of RPMI 1640 and frozen at -70.degree. C. in two aliquots of 0.5 mL. The residual 1 mL buffy coats was immediately used for determination of blood cell associated BVDV by co-cultivation in a permissive cell culture.

BVD Serum Antibody ELISA-Test

[0133] Each serum sample was tested for the presence of BVDV-antibodies using a suitable and validated ELISA test (Svanovir.TM. BVDV antibody test Cat# 10-2200-10). Test was validated and performed according to the manufacturer's recommendations. Positive samples were diluted according to the log.sub.2 scale to determine BVDV antibody titers.

BVD Antigen Assay(s)

[0134] Each buffy coat sample was assayed for the presence of BVDV by co-cultivation of the freshly prepared buffy-coats with susceptible cells or a cell-line. No freezing was allowed before co-cultivation. Plasma was collected and provided to Man-Gene from each sample.

Clinical Observations

Observation of Heifers

[0135] Animals were examined daily from Day 0-42 post-inoculation for the presence of clinical symptoms by a sufficiently trained veterinarian.

[0136] All clinical signs were recorded and described by its nature, consistence/touch, severity (mild, medium or severe) location, size of the area affected, and they will be scored according to agreed and standard definitions. Special attention was paid to respiratory signs (respiration, its rate; nasal or ocular discharge; conjunctivitis, sneezing, coughing, etc.) and diarrhea.

Rectal Temperatures

[0137] Rectal temperatures were measured daily in each heifer, at the same hour of the day (preferably in the morning) from 5 days prior to the inoculation till 21 days post-infection. Daily measurement of rectal temperature was continued until each animal had rectal temperatures below or equal to 39.degree. C. for at least 3 consecutive days.

Detection of Interrupted Pregnancy

[0138] Pregnancy was confirmed and suspicion for abortion or resorption of the fetus was established by rectal examination. A trained veterinarian examined all animals at inoculation, 1 and 2 months post-inoculation. The examination was carried out according to the generally accepted veterinary practice. Heifers were examined daily for any sign of abortion until termination of the study (8-12 weeks post-challenge).

Termination of the Study

[0139] The study was terminated by slaughtering the heifers and extracting the fetuses. Fetuses and fetal material were transferred into closed transport containers marked with the number of the cow and the date/time. Containers were transported to a selected necropsy room. Necropsy of the heifers was not required. Necropsy was performed on fetuses, findings recorded, and a panel of samples collected as described below.

Post-Mortem Examination

[0140] A detailed necropsy of the experimental animals was done in each case of death. Post-mortem examinations were carried out by an experienced veterinary surgeon and the data were recorded on appropriate data sheets. Further laboratory tests were performed according to the clinical signs and lesions observed. If the diagnosis of the necropsy referred to a disease caused by microbial agent the diagnosis was verified by an appropriate test, specific for the agent. Each tissue sample was collected in at least 2 separate, labeled containers and snap-frozen in liquid nitrogen. Samples were stored at -70.degree. C. until required.

Aborted Fetuses and Study Termination

[0141] At least the following tissue samples were collected from the fetuses: exudate from the peritoneal cavity or thorax, if present; mesenteric lymph nodes; spleen; thymus; cerebellum; kidney; bone marrow from the sternum; and sample from the placenta, if available.

Dead or Sacrificed Heifers

[0142] At least the following tissue samples were collected: blood for buffy coat, if available; blood for serum, if available; Peyer's patches; mesenteric lymph nodes; spleen; kidney; uterus, including a sample from the placenta, if available.

Storage and Transport of Samples

TABLE-US-00006 [0143] Samples: Storage: Serum -20.degree. C. Buffy coat -70.degree. C. Virus -70.degree. C. Tissue from heifers -70.degree. C. Tissue from fetuses -70.degree. C.

[0144] Samples were sent for laboratory analysis as required by the sponsor. The choice of samples and the timing of transport were agreed with the study monitor or the project manager. As a matter of general principle, samples coming from aborted material or from new-born calves were investigated as soon as possible.

Results

Mortality

[0145] Heifer No. 626 (Group 1) died on Day 13 PI (post-inoculation). The following table summarizes the observed clinical signs and lesions revealed during necropsy:

TABLE-US-00007 Heifer In-life observations Post-mortem findings No. 626 signs of disease from 7 DPI dehydration lachrymation, nasal discharge on hemorrhages on the 7-12 DPI serous membranes loss of appetite from 8-12 DPI hyperemia of the diarrhea on 11-12 DPI Peyer's patches elevated respiratory rate on 9-10 edema of the lung and 12 DPI coughing on 9 DPI abnormal breathing on 12 DPI

[0146] These clinical and gross-pathological findings are consistent with BVDV induced lesions, therefore it may be concluded that the reason of death was the BVDV infection.

Abortions after Infection

[0147] One heifer had clinical abortion in each group. Heifer No. 615 (Group 1) aborted on Day 38 PI, Heifer No. 469 (Group 2) aborted on Day 39 PI. Both fetuses showed the signs of autolysis, and they were estimated to die at least 3-7 days before the abortion (around 32-35 DPI). In Group 1, no fetus was found in Heifer No. 526 during the slaughter examination at termination. Gross-pathology of the uterus revealed the following: the right uterine horn was slightly enlarged, and the remains of placenta with progressed autolysis was retained in the lumen. The findings on the uterus of Heifer No. 526 is consistent with a "silent" abortion, most likely due to the BVD infection.

Clinical Observation of Heifers

[0148] A summary of the clinical observation data and duration of clinical signs in the groups are presented below.

[0149] Clinical Signs and the Days Post-Inoculation (DPI) When They Were Observed

TABLE-US-00008 Group 1 (XIKE-A) Animal ID 526 598 615 618 626* Clinical sign DPI Loss of appetite 8-13 8-18 8-18 8-16 8-12 Lachrymation 7-10 7-8, 10-12 8-10 8-10 7-8 Conjunctivitis 9-10 9-12 9-11 9-11 -- Nasal discharge 7-13 7-9, 11-12 8-13 8-12 7-12 Oral erosion -- -- -- -- -- Oral hemorrhage -- -- -- -- -- Diarrhea -- 11-16 10-15 11-15 11-12 Coughing 9 10, 15 9 8-9, 13 9 Abnormal breathing -- 11-14 12-14 12-14 12 Elevated respiratory rate -- 10-13 9-13 8-13 9-10, 12 Hoof erosion -- -- -- -- -- *Heifer No. 626 died on Day 13 PI

TABLE-US-00009 Group 2 (XIKE-B) Animal ID Clinical sign 469 588 565 608 619 Loss of appetite -- -- -- -- -- Lachrymation -- -- -- -- -- Conjunctivitis -- -- -- -- -- Nasal discharge -- -- -- -- -- Oral erosion -- -- -- -- -- Oral hemorrhage -- -- -- -- -- Diarrhea -- -- -- -- -- Coughing -- -- -- -- -- Abnormal breathing -- -- -- -- -- Elevated respiratory rate -- -- -- -- -- Hoof erosion -- -- -- -- --

[0150] All Group 1 animals infected with XIKE-A exhibited a broad spectrum of clinical signs. Respiratory signs appeared first accompanied by loss of appetite, and a few days later heifers developed diarrhea with the exception of Heifer No. 526. One heifer died and another one aborted (see before) after infection. All these signs are consistent with the symptoms expected after infection with a virulent BVDV strain.

[0151] All Group 2 animals infected with XIKE-B were free of clinical signs. At the same time, one heifer had abortion during the observation period.

Rectal Temperatures

[0152] No abnormal temperature changes were detected before the infection of the animals. In Group 2, all temperature values remained within the physiological range from Day 0 to Day 21 after infection. All Group 1 animals showed elevated rectal temperature after infection that were detected between Days 7-11 PI.

Findings at Study Termination

[0153] At study termination, fetuses were examined at slaughter. No fetus was recovered from Heifer No. 526 (see section 10.2 "Abortions after Infection"). The following findings were observed at the necropsy of the fetuses:

TABLE-US-00010 Animal No. Findings Conclusion Group 1 598 Ascites, general edema, autolysis Died at least 2 weeks earlier 618 Ascites, general edema, autolysis Died at least 3 weeks earlier Group 2 565 Ascites, general edema, liver degeneration Fetus considered non-viable 588 Normal -- 608 Normal, perirenal edema -- 619 General autolysis Died 3-6 weeks earlier

[0154] The findings suggest that 2 Group 1 animals (Heifers No. 598 and No. 618) and one Group 2 animal (Heifer No. 619) died several weeks before extraction, and so they can be considered abortions.

Abortions Modified by Post-Mortem Findings

[0155] After the post-mortem examination it was not clear why some of the heifers had not had abortions. Dead fetuses should be considered as abortions, therefore the clinical picture was modified after the termination of the study as follows:

TABLE-US-00011 Group 1 Animal No. Conclusion 526 BVD abortion (uterus with placenta post-mortem) 598 BVD abortion (fetus post-mortem) 615 Clinical BVD abortion 618 BVD abortion (fetus post-mortem) 626 Died due to BVD

TABLE-US-00012 Group 2 Animal No. Conclusion 469 Clinical BVD abortion 565 Expected BVD abortion; non-viable fetus 588 Normal 608 Normal 619 BVD abortion (fetus post-mortem)

Examination of Blood Samples

Leukocyte Count

[0156] WBC counting was interrupted on Day 26 PI, as all animals became negative for virus isolation for this time point. 0 DPI values were considered as individual baseline for comparison. In Group 2, the leukocyte counts never went to 40% or more below the baseline value until the end of the observation period (26 DPI). In Group 1, one animal (Heifer No. 598) had WBC count below the 40% baseline for one day.

Serology

[0157] None of the selected animals had BVDV specific antibody in their sera before the infection. After infection, all surviving Group I heifers developed BVDV specific antibodies detected from 3 weeks PI and lasted until the end of the observation period in all study animals. In Group 2, 4 out of the 5 heifers had BVDV specific antibodies detected from 4 weeks PI. Measurable antibody response lasted only in 3 animals until the end of the observation period. Titers were lower in Group 2 than in Group 1.

Virus Detection by Co-Cultivation

Buffy Coats

[0158] BVDV was detected in both groups. The duration of virus detection is summarized below. All samples were co-cultivated immediately after collection, i.e., without freezing.

TABLE-US-00013 Animal No. DPI when BVDV was detected Group 1 615 5-12 526 5-9 626 5-12 618 5-11, 14 598 5-11, 13 Group 2 565 7-9 588 8 608 6-9 469 8 619 5-11

Tissue Samples

[0159] The presence of BVD virus in the dead heifer and the fetuses is summarized below:

Heifer:

TABLE-US-00014 [0160] Animal No. Group 1 BVDV in tissue samples 626 Present.sup.#

Fetuses:

TABLE-US-00015 [0161] Animal No. BVDV in tissue samples Group 1 615 Not present.sup.# 526 NT 626 Present.sup.# 618 Not present 598 Present Group 2 565 Present 588 Not present 608 Present 469 Not present.sup.# 619 Not present NT = Not tested

[0162] Samples were co-cultivated immediately after collection (i.e., without freezing), except "#" marked ones, from which only frozen samples were available.

Summary of BVD Related Clinical and Laboratory Data

TABLE-US-00016 [0163] Group 1 Animal No. Conclusion BVD 526 BVD abortion (uterus with NT (no sample found) placenta post-mortem) 598 BVD abortion (fetus post-mortem) +(fetus)* 615 Clinical BVD abortion -(fetus)* 618 BVD abortion (fetus post-mortem) -(fetus)* 626 Died due to BVD +(fetus)/+(heifer) NT = not tested

TABLE-US-00017 Group 2 Animal No. Conclusion BVD 469 Clinical BVD abortion -(fetus)* 565 Expected BVD abortion; non-viable fetus +(fetus) 588 Normal -(fetus) 608 Normal +(fetus) 619 BVD abortion (fetus post-mortem) -(fetus)* *Fetuses were autolyzed at the time of sampling

Conclusion

[0164] The study aimed to assess the safety of XIKE-A and XIKE-B in pregnant animals. Ten pregnant heifers were selected from a BVDV negative herd. Two groups of 5 heifers were included in the trial: one was inoculated with XIKE-A the other with XIKE-B virus strain. Heifers were between days 60 and 90 of gestation on the day of inoculation. Heifers were monitored for the presence of clinical signs of BVDV infection including abortions during the observation period. Blood samples were collected from the animals for serology, antigen detection and white blood cells were counted. The experiment was terminated 9 weeks after infection. Non-aborted cows were slaughtered and the uterus examined and collected. Fetal organ samples were collected during routine necropsy and examined for BVDV infection.

[0165] The presence of fetal infection was the main evaluation parameter, composed from the number of BVDV-related cow mortality, the number of BVDV-related abortions and the number of BVD positive fetuses at termination. In addition to the main parameter, clinical signs characteristic for BVDV infection, viremia, and white blood cell count in cows and rectal temperature after challenge were evaluated. The XIKE-B virus proved to be less pathogenic than XIKE-A, nevertheless BVD-related abortion and infection of the fetus was observed in the XIKE-B group, too. Therefore it can be concluded that the inactivation of the E.sup.ms RNase does not prevent fetal infection.

Example 2

BVDV XIKE-A-NdN: Fetopathogenicity Assessment in Pregnant Heifers

[0166] The N.sup.pro gene has been shown to be nonessential for growth of CSFV in tissue culture (Tratschin et al., 1998). Even though a proof for BVDV attenuation in consequence of N.sup.pro deletion is still missing, a role of this protein in the interaction between virus and host seemed to be possible and was actually indicated by recent experiments for CSFV (Mayer et al., 2004; Ruggli et al., 2003). We therefore investigated whether the deletion of the major part of the N.sup.pro coding sequence leads to a virus that no longer infects the fetus in pregnant heifers. The N.sup.pro gene except for the 5' terminal 4 codons was deleted from the full length cDNA clone pKANE40A according to standard procedures. The resulting mutant full length clone was used as template for in vitro transcription and the resulting cRNA was transfected into MDBK cells as described (Meyer et al., 2002). The recovered virus was amplified in tissue culture and then used in the animal experiment described below. BVDV XIKE-B served as a control since it was shown before that it is able to cross the placenta (Example 1).

Objective(s)/Purpose of the Study

[0167] The study aims to assess the safety of a live attenuated BVDV with a genomic deletion of most of the N.sup.pro coding region in pregnant animals.

[0168] Materials and Methods applied are as described in Example 1

Study Design

[0169] Eight pregnant heifers were assigned at random to two groups. They were treated and observed according to the following schedule:

TABLE-US-00018 Group 1 Group 2 N 5 3 Treatments XIKE-A-NdN XIKE-B/control Route Intramuscular Vaccination time between days 60 and 90 of pregnancy (day 0 of the study) Observations Clinical signs Post-vaccination Serum at days 0, 14, 28, 42 and at termination (in life) WBC at day 0 and then daily for 14 days Buffy coat at day 0 and then daily for 14 days Post-mortem Gross-pathology (day 60) Organ panel for virus isolation Type of study: open controlled clinical study Experimental unit: Individual animal Method of blinding: Partial blinding. No detailed procedures for blinding and access to treatment schedule were applied. The observing veterinarian at the study location and the pathologist were not be aware of the treatment; they only received a protocol extract relevant to their tasks. Vaccination was performed by the investigator or his assignee. Samples for virus isolation were coded by the investigator until all results are available.

Results

[0170] All heifers were healthy and pregnant at study start. All animals proved to be free of BVDV and BVDV antibodies before the initiation of the study.

Preparation and Control of the Virus Used for the Infection Samples were collected throughout the dilution steps and assayed on the day of preparation, i.e., without freezing by co-cultivation on suitable tissue culture. The results of virus titration are shown in the following table.

TABLE-US-00019 Log.sub.10 Sample ID Virus strain Dilution/description titer/mL VT1a XIKE-A/NdN (S) 1:2 (at 4.degree. C.) 4.4 VT1b #2a on ice without 4.0 opening VT1c Return of #2b 2.8 VT2a XIKE-B 1:2.2 (at 4.degree. C.) 2.3 VT2b #3a on ice without 2.8 opening VT2c Return of #3b Negative

Clinical Symptoms of BVDV Infection

[0171] The table below gives a summary about the animals that had clinical signs during the observation period.

Clinical Signs and the Days Post-Inoculation (DPI) When They Were Observed

TABLE-US-00020 [0172] Group 1 (XIKE-A NdN) Animal ID Clinical sign 1583 Loss of appetite 8 Lachrymation -- Conjunctivitis -- Nasal discharge -- Oral erosion -- Oral hemorrhage -- Diarrhea -- Coughing -- Abnormal breathing -- Elevated respiratory rate -- Hoof erosion --

TABLE-US-00021 Group 2 (XIKE-B) Animal ID 1438 1585 -- 10 -- -- -- -- -- -- -- -- -- -- -- -- 12 10-13 -- -- -- -- -- --

[0173] Only mild and transient clinical signs were observed in some of the animals in each group. In Group 1, one out of the 5 heifers had loss of appetite on day 8 PI. In Group 2, two out of the 3 animals had clinical signs. Both heifers experienced coughing around day 21 PI that was accompanied with loss of appetite in one of the animals.

Rectal Temperatures

[0174] No abnormal temperature changes were detected before the inoculation of the animals. The few cases of elevated temperatures measured after the inoculation are summarized in the table below.

TABLE-US-00022 Group Animal ID Temperature (.degree. C.) PI day 1 1583 39.9 8 1621 39.0 5 2 1438 39.0 2 1585 40.8 9

[0175] One animal had slightly elevated temperature in each group, and also one animal had fever in each group. Fever was detected on day 8 or 9 PI. Temperature values always returned to normal value on the following day.

Leukocyte Counts

[0176] Some leukopenia was observed in all groups between PI days 3-8. The number of animals with at least 40% reduction in white blood cell count was the following:

TABLE-US-00023 Group Number of animals having leukopenia/total 1 3/5 (60%) 2 1/3 (33%)

Serology (BVDV Antibodies)

[0177] In compliance with the study protocol, all heifers were free of BVDV antibodies before vaccination. In Group 1 (inoculated with XIKE-A NdN) and Group 2 (inoculated with XIKE-B), complete seroconversion was detected only at study termination (2 months after inoculation).

BVD Virus Isolation from Buffy Coats

[0178] No viremia was detected

[0179] BVD Virus Isolation from Fetal Tissue Samples

TABLE-US-00024 Group 1 Group 2 N 5 3 Treatments XIKE-A-NdN XIKE-B/control Route Intramuscular Intramuscular Number of fetuses in which fetal 4 out of 5 2 out of 3 transmission was detected: fetuses infected fetuses infected Conclusion of the virus used for Fetal transmission for Fetal transmission for treatment has the potential to be XIKE-A-NdN observed XIKE-B observed transmitted over the placenta:

Conclusion

[0180] The N.sup.pro deletion resulted in a considerable attenuation of the BVDV in comparison to the parental virus XIKE-A that was shown to be highly pathogenic (Meyer et al., 2002). However, the N.sup.pro deletion alone is not preventing transmission of a NY93-based virus recombinant to the fetus after inoculation of pregnant cows.

Example 3

BVDV XIKE-B-NdN: Fetopathogenicity Assessment in Pregnant Heifers

[0181] To be able to test the potential of a combination of RNase inactivation and N.sup.pro deletion with regard to BVDV attenuation and fetal transmission, different BVDV-2 mutants with deletions within the N.sup.pro coding region were established based on the infectious cDNA clone pKANE40B, the RNase negative mutant of pKANE40A with a deletion of codon 349. The recovered viruses were analyzed with regard to presence of the desired mutations, the absence of second site mutations in the regions flanking the introduced changes, and their growth characteristics in tissue culture. XIKE-B-NdN (V-pK88C), a variant containing a deletion of the complete N.sup.pro coding region except for codons 1 to 4 in addition to the RNase inactivating deletion of codon 349 was chosen for an animal experiment since it combined the desired mutations with acceptable growth characteristics. The aim of the study was to assess the safety of a live attenuated BVDV isolate in pregnant animals.

[0182] Five BVDV-negative, pregnant heifers were inoculated intranasally with an infective dose of 10.sup.5 TCID.sub.50/animal XIKE-B-NdN (back titration data are depicted in Table 3.1). Clinical data were recorded daily. Blood samples were collected for white blood cell counting, for buffy-coat preparation and serology. After termination of the study, fetal tissues were collected for virus isolation.

Materials and Methods

[0183] As detailed for Example 1.

Results

[0184] No clinical data were observed (data not shown). Leukocyte counts remained virtually unchanged except for a significant decrease by approximately 40% below the baseline value (day 0) in heifer No. 1015 on a single day (day 6 PI) (data not shown).

Analysis of Buffy Coat Preparations

[0185] Approximately 10.sup.6 leukocytes were cultured in duplicates with MDBK-cells in 24-well tissue culture plates for 5 days. Samples were freeze-thawed twice. 100 .mu.L aliquots of thawed samples were inoculated onto freshly seeded 24-well tissue culture plates and tested for virus by indirect immunofluorescence staining (mAb Code 4, directed against a conserved epitope in nonstructural protein NS3). No BVDV could be isolated from the buffy coat preparations of animals # 921, 1013, 1015, 1055 and 1075 (Table 3.2) whereas positive controls clearly showed the correct conduction of the test.

Post-Mortem Examination of Fetal Tissues

[0186] After termination of the study the following fetal tissues were collected for virus isolation: spleen, kidney, thymus, sternum, cerebellum, placenta, intestine, and abdominal fluid. Briefly, tissue suspensions were made in a mortar using sterile sea sand and ice-cold PBS without Ca.sup.2+ and Mg.sup.2+. Mortars were rinsed with 1 mL ice-cold PBS without Ca.sup.2+ and Mg.sup.2+ and suspensions were centrifuged for 10 minutes at 2000.times.g (4.degree. C.). The supernatant was first passed through a disposable 0.45 .mu.m filter holder, followed by a second filter passage (0.2 .mu.m pore size). Virus isolation was carried out in duplicates (400 .mu.L of fetal tissue suspension or 100 .mu.L of fetal abdominal fluid) on a monolayer of MDBK-cells in a 24 wells tissue culture plate (37.degree. C., 7% CO.sub.2). Tissue samples were controlled daily for cytopathic effects or bacterial contamination, and after an incubation time of 5 days plates were frozen and thawed twice. 100 .mu.L of samples were passaged to freshly seeded MDBK-cells. Virus was detected by indirect immunofluorescence staining (mAb Code 4). No BVDV could be detected in the tissue samples or fetal abdominal fluid (Table 3.3).

Serological Findings

[0187] Serum neutralization titers were determined before inoculation, 1 month post-inoculation and at termination of the study. Sera from all animals were tested in triplicates for neutralizing antibodies against NY93/C, and the endpoint dilution was read by indirect immunofluorescence staining. Results were expressed as the endpoint dilution, which neutralized approximately 100 TCID.sub.50 and calculated by the method of Kaerber. No definite data could be obtained for day 0, and 1 and 2 weeks post-infection as the sera were toxic for MBDK-cells in dilutions up to 1:16 and no neutralization could be detected at higher dilutions. Starting with the third week post-vaccination all animals developed neutralizing antibodies against the homologous BVDV-2 virus NY93/C lasting till the end of the experiment (Table 3.4 and FIG. 1).

Conclusions

[0188] The data obtained during the animal study clearly show that BVDV XIKE-B-NdN represents a highly attenuated virus. In contrast to wild-type virus or the single mutants XIKE-B or XIKE-A-NdN that show fetal transmission in pregnant heifers at high rates, the double mutant did not cross the placenta. BVDV XIKE-B-NdN as well as similar double mutants are extremely suitable for the use in a live attenuated vaccine.

TABLE-US-00025 TABLE 3.1 Study No.: B01 BIVI020 and B01 BIVI022 Back Titration of Viruses Sample ID Virus Strain Dilution Titer 1a XIKE-B-NdN concentrated virus 10.sup.5.44 TCID.sub.50/mL 1 1:4 10.sup.4.86 TCID.sub.50/mL 6 residues of infection (#1) 10.sup.4.27 TCID.sub.50/mL

TABLE-US-00026 TABLE 3.2 Detection of Viremia Animal Days after vaccination ID No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0921 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2. isolation 1013 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2. isolation 1015 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2. isolation 1055 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2. isolation 1075 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2. isolation Date 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 - = sample negative

TABLE-US-00027 TABLE 3.3 Analysis of fetus tissue samples for the presence of BVDV Mesenteric Bone Date of Animal Abdominal Thoracic lymph marrow Cere- tissue No. fluid Fluid nodes Spleen Kidney Thymus (sternum) bellum Placenta Intestine collection Isolation 0921 -- NC NC -- -- -- -- -- -- -- 12.02.02 1. isolation -- -- -- -- -- -- -- -- 2. isolation 1013 -- NC NC -- -- -- -- -- -- -- 12 Dec. 1. isolation -- -- -- -- -- -- -- -- 2002 2. isolation 1015 -- NC NC -- -- -- -- -- -- -- 12 Dec. 1. isolation -- -- -- -- -- -- -- -- 2002 2. isolation 1055 -- NC NC -- -- -- -- -- -- -- 12 Dec. 1. isolation -- -- -- -- -- -- -- -- 2002 2. isolation 1075 -- NC NC -- -- -- -- -- -- -- 12 Dec. 1. isolation -- -- -- -- -- -- -- -- 2002 2. isolation - = sample negative NC = not collected

TABLE-US-00028 TABLE 3.4 B01 BIVI022/BVDV XIKE-B-NdN; fetal protection study Serum Neutralization Assay From the heifers Animal at during ID No. selection ACC 1 wPV 2 wPV 3 wPV 4 wPV 5 wPV 6 wPV 7 wPV 8 wPV 0921 * * * 1:40.sup.(2) 1:161.sup.(1) 1:256.sup.(1) 1:323.sup.(1) 1:128.sup.(1) 1:256.sup.(1) 1013 * * * 1:3.sup.(2) NA 1:161.sup.(1) 1:323.sup.(1) 1:406.sup.(1) 1:256.sup.(1) 1015 * * * 1:64.sup.(2) 1:161.sup.(1) 1:256.sup.(1) 1:323.sup.(1) 1:406.sup.(1) 1:323.sup.(1) 1055 * * * 1:32.sup.(2) 1:40.sup.(2) 1:256.sup.(1) 1:323.sup.(1) 1:406.sup.(1) 1:406.sup.(1) 1075 * * * NA 1:128.sup.(2) 1:102.sup.(1) 1:203.sup.(1) 1:161.sup.(1) 1:406.sup.(1) Date 06 Dec. 20 Dec. 27 Dec. 03 Jan. 10 Jan. 17 Jan. 24 Jan. 31 Jan. 07 Feb. 2001 2001 2001 2002 2002 2002 2002 2002 2002 .sup.(1)SNT against 1456 Nase (= NY93/C) 10.sup.2.03 TCID.sub.50/50 .mu.L .sup.(2)SNT against 1456 Nase (= NY93/C) 10.sup.1.57 TCID.sub.50/50 .mu.L * Serum toxic for MBDK-cells in dilutions up to 1:16 no data available NA data not available The Serum Neutralization Assay against NY93/C is illustrated in FIG. 1.

Efficacy and Crossprotection Study

[0189] Two possible problems have to be faced with regard to vaccination with attenuated virus mutants BVDV XIKE-B or BVDV XIKE-B-NdN. First, there is a general problem concerning crossprotection between BVDV-1 and BVDV-2. At least vaccination with inactivated BVDV-1 vaccines did not prevent the transmission of BVDV-2 to the fetus in pregnant animals. Since protection against fetal infection represents the major aim of anti-BVDV vaccination, such vaccines cannot be regarded to induce a protective immunity on a broad range. The question therefore was, whether vaccination with live attenuated BVDV-2 can prevent virus transmission to the fetus. Second, the reduced growth rates of BVDV XIKE-B-NdN might result in only a low level of protection not able to prevent transplacental infection of the fetus in pregnant heifers. To address these problems, an animal study was started. The animals (2 groups of 10 animals each) were vaccinated either with BVDV XIKE-B or XIKE-B-NdN (intended dosage: 1 mL of supernatant with 10.sup.5 TCID.sub.50 of virus; backtitration is shown in Table 3.5). None of the animals showed significant clinical signs after the vaccination except for one animal of the nonvaccinated control group with mild coughing for one day. Rectal temperature values were below 39.degree. C. except for one animal of the nonvaccinated control group that had 39.1.degree. C. for one day. Buffy coat samples prepared after vaccination were analyzed for the presence of virus as described above. The experiments showed that only 5 of the 20 animals contained virus in the blood for 1 or 2 days at 4 to 8 days post-infection (Table 3.6).

TABLE-US-00029 TABLE 3.5 Back Titration of Viruses used for vaccination Sample ID Virus Strain Dilution Titer 1a XIKE-B-NdN concentrated virus 10.sup.5.44 TCID.sub.50/mL 1 1:4 10.sup.4.86 TCID.sub.50/mL 6 residues of infection (#1) 10.sup.4.27 TCID.sub.50/mL 3 XIKE-B 1:11 10.sup.5.76 TCID.sub.50/mL 4 1:110 10.sup.4.92 TCID.sub.50/mL 5 residues of infection (#4) 10.sup.4.27 TCID.sub.50/mL

TABLE-US-00030 TABLE 3.6 Study No./Id.: B01 BIVI020/BVDV Tu XIKE-B/XIKE-B-NdN; fetal protection study Inoculation with white blood cell (buffy coat) preparations collected after vaccination Animal Days After Vaccination ID No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1134.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1141.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1142.sup.(1) OO OO OO OO OO OO OO +O OO OO OO OO OO OO OO 1. isolation OO OO OO OO O+ OO OO OO OO OO OO OO OO OO OO 2. isolation 1145.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1149.sup.(1) OO OO OO OO +O OO OO OO O+ OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1151.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1152.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1156.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1158.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1160.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation Date 13.12 14.12 15.12 16.12 17.12 18.12 19.12 20.12 21.12 22.12 23.12 24.12 25.12 26.12 27.12 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 1197.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1200.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1206.sup.(2) OO OO OO OO OO OO OO O+ OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO O+ OO OO OO OO OO OO OO OO 2. isolation 1210.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1212.sup.(1) OO OO OO OO OO OO O+ OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1214.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1216.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1217.sup.(1) OO OO OO OO OO OO O+ OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1218.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1225.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation Date 13.12 14.12 15.12 16.12 17.12 18.12 19.12 20.12 21.12 22.12 23.12 24.12 25.12 26.12 27.12 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 Immunofluorescence staining: Code 4 O sample negative + sample positive B bacterial contamination in well Code of animal numbers: .sup.(1)vaccination with BVDV XIKE-B (RNase mutant) .sup.(2)vaccination with BVDV XIKE-B-NdN (RNase and N.sup.pro double mutant)

[0190] Four weeks after vaccination, insemination of the animals was carried out. Challenge infections were performed 60 to 90 days later using either a BVDV-1 strain (BVDV KE-9, heterologous challenge, animals vaccinated with XIKE-B) or a heterologous BVDV-2 strain (BVDV KE-13, homologous challenge, animals vaccinated with XIKE-B-NdN) (intended dosage: 10.sup.5 TCID.sub.50 in 6 mL; backtitration is shown in Table 3.7). From each group of vaccinated animals 5 pregnant heifers were randomly selected for the challenge infection. Animals vaccinated with BVDV XIKE-B were challenged with the BVDV-1 strain KE-9, whereas heifers vaccinated with BVDV XIKE-B/NdN were challenged with BVDV-2 KE-13. In addition, two nonvaccinated control animals were infected with each of the challenge viruses.

TABLE-US-00031 TABLE 3.7 Study No./Id.: B01 BIVI020/BVDV Tu XIKE-B-NdN; fetal protection study Back titration of challenge viruses Mean Titer Virus Strain Sample ID Titer (TCID.sub.50/mL) (TCID.sub.50/mL) KE 9 1 10.sup.4.44 10.sup.4.94 10.sup.5.10 2 10.sup.4.69 * 10.sup.4.44 3 ** KE13 1 10.sup.4.69 10.sup.4.76 10.sup.4.82 2 10.sup.4.57 10.sup.4.63 10.sup.4.69 3 *** 10.sup.3.5 10.sup.3.5 Sample 1: stock of inoculate Sample 2: stock of inoculate returned from the stable Sample 3: excess inoculate * Second inoculation of KE9, sample 2 wasn't interpretable because of cell death. ** KE9, sample 3 wasn't interpretable because of cell death or bacterial contamination. *** First inoculation of KE13, sample 3 wasn't interpretable because of bacterial contamination.

[0191] The vaccinated animals did not show viremia or clinical symptoms upon challenge infection. The challenge was successful as all non-vaccinated controls were BVDV positive (Table 3.8). Only mild signs of disease were observed in the control groups. The white blood cell counts were nearly normal (not shown).

TABLE-US-00032 TABLE 3.8 Study No./Id.: B01 BIVI020/BVDV Tu XIKE-B/XIKE-B-NdN; fetal protection study Inoculation with white blood cell (buffy coat) preparations collected after challenge Animal Days After Challenge ID No. 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Isolation 1104.sup.(3) OO OO OO O+ ++ ++ O+ OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO +O ++ OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1108.sup.(3) OO OO OO OO ++ +O OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1126.sup.(3) OO OO ++ +O ++ O+ OO O+ O+ OO OO OO OO OO OO OO 1. isolation OO OO 2. isolation 1145.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO 2. isolation 1151.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO 2. isolation OO OO OO OO OO OO OO OO OO OO OO OO 3. isolation 1152.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO 2. isolation OO OO OO 3. isolation 1156.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO 2. isolation OO OO OO OO OO OO OO OO OO OO OO OO 3. isolation Date Apr. Apr. Apr. Apr. May May May May May May May May May May May May 22, 25, 27, 29, 01, 03, 05, 07, 09, 11, 13, 15, 17, 19, 21, 23, 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 1197.sup.(1) OO OO OO O+ ++ ++ O+ OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO +O ++ OO OO OO OO OO OO OO OO OO OO OO 2. isolation 3. isolation 1200.sup.(1) OO OO OO OO ++ +O OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1214.sup.(1) OO OO ++ +O ++ O+ OO O+ O+ OO OO OO OO OO OO OO 1. isolation OO OO 2. isolation 3. isolation 1216.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO 2. isolation 3. isolation 1217.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO 2. isolation OO OO OO OO OO OO OO OO OO OO OO OO 3. isolation 1218.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO 2. isolation 1249.sup.(3) OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation Date Apr. Apr. Apr. Apr. May May May May May May May May May May May May 22, 25, 27, 29, 01, 03, 05, 07, 09, 11, 13, 15, 17, 19, 21, 23, 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 Immunofluorescence staining: Code 4 O sample negative + sample positive B bacterial contamination in well Code of animal numbers: .sup.(1)vaccination with BVDV XIKE-B (RNase mutant) .sup.(2)vaccination with BVDV XIKE-B-NdN (RNase and N.sup.pro double mutant) .sup.(3)nonvaccinated controls

[0192] Serum neutralization titers were determined before inoculation, 1 month post-inoculation, before challenge, 1 month after challenge and at termination of the study. Sera from all animals were tested in triplicates for neutralizing antibodies against KE9 and NY93/C (1456 Nase), and the endpoint dilution was read by indirect immunofluorescence staining. Results were expressed as the endpoint dilution, which neutralized approximately 100 TCID.sub.50 and calculated by the method of Kaerber. At some of the higher antibody titers, the used endpoint dilution was not high enough. Against KE9, only animals vaccinated with XIKE-B developed low antibody titers starting about week 4. At challenge, all animals had antibody titers, which increased considerably starting around week 4 post-challenge. XIKE-B vaccinated animals had higher antibody titers then those vaccinated with XIKE-B-NdN vaccinated. All animals developed about the same neutralization titer against NY93/C four weeks post-vaccination, with marginally lower titers in XIKE-B-NdN vaccinated animals. After challenge all animals had high antibody titers. FIG. 2 shows the serum neutralization assay against KE9 (BVDV-1) and FIG. 3 shows the serum neutralization assay against NY93/C (BVDV-2).

[0193] Analysis of tissue samples obtained after termination of the study from the fetuses revealed that the material obtained from the vaccinated animals gave negative results whereas transmission had occurred in all 4 control animals (Table 3.9). Thus, it is clear that the established BVDV-2 mutants are well suited as efficient cross protective vaccine viruses.

TABLE-US-00033 TABLE 3.9 Study No./Id.: B01 BIVI020/BVDV Tu XIKE-B/XIKE-B-NdN; fetal protection study Analysis of fetus tissue samples for the presence of BVDV Mesenteric Bone Animal Abdominal Thoracic lymph Small marrow No. Fluid fluid nodes intestine Spleen Thymus Kidney (sternum) Cerebellum Placenta Date 1214.sup.(1) NA NA OO OO OO OO OO OO OO OO 17.06.02 1126.sup.(3) ++ *** ++ ++ ++ ++ ++ ++ ++ ++ 17.06.02 1249.sup.(3) ++ NA ++ ++ ++ ++ ++ ++ ++ O+ 17.06.02 1218*.sup.(1) NA NA NA NA NA NA NA NA NA OO** 17.06.02 1197.sup.(1) OO NA OO OO OO OO OO OO OO OO 17.06.02 1217.sup.(1) OO NA OO OO OO OO OO OO OO OO 17.06.02 1200.sup.(1) OO NA OO OO OO OO OO OO OO OO 17.06.02 1145.sup.(2) OO NA OO OO OO OO OO OO OO OO 21.06.02 1108.sup.(3) +O NA ++ ++ ++ ++ ++ ++ ++ OO 21.06.02 1156.sup.(2) OO NA OO OO OO OO OO OO OO OO 21.06.02 1104.sup.(3) ++ NA ++ ++ ++ ++ ++ ++ ++ ++ 21.06.02 1216.sup.(2) OO NA OO OO OO OO OO OO OO OO 21.06.02 1151.sup.(2) NA NA OO OO OO OO OO OO OO OO 21.06.02 1152.sup.(2) OO NA OO OO OO OO OO OO OO OO 21.06.02 NA = not available *No fetus was found in the uterus of heifer #1218 **Endometrium (also collected for histology) *** Sample was not sent to BFA Tubingen Code of animal numbers: .sup.(1)vaccination with BVDV XIKE-B (RNase mutant) .sup.(2)vaccination with BVDV XIKE-B-NdN (RNase and N.sup.pro double mutant) .sup.(3)nonvaccinated controls

Conclusion

[0194] The challenge was successful as all non-vaccinated controls were BVDV viremic and fetuses of all non-vaccinated controls were BVDV positive.

[0195] Both isolates gave full protection under the present test and assay conditions. Isolate XIKE-B, with the single genetic marker was shown to cross-protect against type 1 BVDV challenge in terms of BVD viremia and transmission to the fetus after challenge. Isolate XIKE-B-NdN with the double genetic marker was able to fully protect against a heterologue type 2 BVDV challenge strain in terms of BVD viremia and transmission to the fetus after challenge.

[0196] Isolate XIKE-B (type 2 isolate) was shown to cross-protect against type 1 BVDV challenge in terms of BVD viremia and transmission to the fetus after challenge under the present test and assay conditions (n=4).

[0197] Isolate XIKE-B-NdN (type 2 isolate) fully protected against a heterologues type 2 BVDV challenge strain in terms of BVD viremia and transmission to the fetus after challenge under the present test and assay conditions (n=5).

Example 4

Establishment of N.sup.pro Mutants

[0198] Further analyses of BVDV-2 mutants with N.sup.pro deletions. Different mutants with deletions in the N.sup.pro-coding region of the genome were established. Initially, only true deletions or a deletion accompanied by a point mutation were introduced. [0199] A: [N.sup.pro].sub.1-[C-term];

[0200] B: [N.sup.pro].sub.3-[C-term];

[0201] C: [N.sup.pro].sub.4-[C-term];

[0202] D: [N.sup.pro].sub.6-[C-term];

[0203] E: [N.sup.pro].sub.4-[C-term*]

[0204] In the formulas, [N.sup.pro], represents the number of residues of the aminoterminus of N.sup.pro that are left in the mutated polyprotein amino acids, [C-term] is the complete polyprotein except for N.sup.pro (starting with the C protein and ending with NS5B), and [C-term*] is the same as [C-term] but with a mutation at position 2 of the C protein (N instead of D).

[0205] The growth rates of the recovered viruses were considerably lower than those of wild-type XIKE-A or the RNase negative mutant XIKE-B. There are two possible explanations for this finding: (i) dependent on the virus strain, sequences of variable length of the N.sup.pro-coding region are necessary for efficient translation initiation (Myers et al., 2001; Tautz et al., 1999), and (ii) the fusion of additional sequences to the aminoterminus of the capsid protein interferes with capsid protein function.

[0206] To obtain better growing N.sup.pro deletion mutants, a second set of mutants was generated with either a bovine ubiquitin gene or a fragment of the bovine LC3-coding sequence replacing the major part of the N.sup.pro gene. These constructs allow efficient translation and generate a capsid protein with the correct amino terminus.

[N.sup.pro].sub.22[PS]-[C-term]

wherein PS is ubiquitin or LC3 and C-term is the complete polyprotein except for N.sup.pro (starting with the C protein and ending with NS5B).

[0207] The growth rates of these mutants were more similar to what was determined for XIKE-A. It even seemed that the two RNase positive viruses according to the formula [N.sup.pro].sub.22-[PS]-[C-term] named V-pK87F and V-pK87G showed no significant growth retardation at all, whereas the RNase negative counterpart V-pK88G once again was somewhat hampered in propagation but to a lesser extend than the formerly described mutants.

[0208] Further examples of N.sup.pro deletion mutants may be:

MESDEGSK . . .

MELFSSDEGSK . . .

MELFSNESDEGSK . . .

MELFSNELSDEGSK . . .

MELFSNELLSDEGSK . . .

MELFSNELLYSDEGSK . . .

MELFSNELL YKSDEGSK . . .

MELFSNELL YKTSDEGSK . . .

[0209] MELFSNELL YKT represents the aminoterminal sequence of N.sup.pro of the BVDV isolate NewYork93/C.

[0210] It may also be possible to use variants of this sequence with one or several mutations. Especially the naturally occurring variations as found in other pestiviruses can be expected to be functional. Therefore, the complete list of the tested or proposed variants with the different parts of the aminoterminal end of N.sup.pro can be enlarged by equivalent sets with amino acid exchanges. Below, typical examples of the respective sequences are given for several pestiviruses but the possible variations are not limited to these examples.

TABLE-US-00034 BVDV NewYork93/C: MELFSNELLYKT BVDV CP13: MEL SNELLYKT BVDV SD1: MEL NELLYKT.sub.-- CSFV Brescia: MEL FELLYKT BDV X818: MEL ELLYKT

[0211] Thus, these variants for example may include: MELI-[PS].sub.0-[C-term];

TABLE-US-00035 MELIS-[PS]9 .sub.0-[C-term]; MELISN-[PS].sub.0-[C-term]; MELISNE-[PS].sub.0-[C-term]; MELISNEL-[PS].sub.0-[C-term]; MELISNELL-[PS].sub.0-[C-term]; MELISNELLY-[PS].sub.0-[C-term]; MELISNELLYK-[PS].sub.0-[C-term]; MELISNELLYKT-[PS].sub.0-[C-term]; MELIT-[PS].sub.0-[C-term]; MELITN-[PS].sub.0-[C-term]; MELITNE-[PS].sub.0-[C-term]; MELITNEL-[PS].sub.0-[C-term]; MELITNELL-[PS].sub.0-[C-term]; MELITNELLY-[PS].sub.0-[C-term]; MELITNELLYK-[PS].sub.0-[C-term]; MELITNELLYKT-[PS].sub.0-[C-term];

[0212] These formulas may also have [PS].sub.1, i.e., PS may also be one of the PS as described herein. Sequences belonging to the N.sup.pro protein are in italics. Amino acid exchanges with regard to the sequence of BVDV NewYork93/C are in bold.

[0213] Further examples can be found, e.g., by using the GenBank accession numbers given in Becher et al., 2003, Virology 311, 96-104) or by standard sequence data searches.

[0214] A further possibility could be the use of a processing signal (PS) inserted between the (residual) N.sup.pro sequence and the aminoterminus of the capsid protein. The PS leads to a cleavage that generates a functional capsid protein. The configuration of such constructs could be as follows:

[N.sup.pro].sub.22--PS--[C-term]

where PS is a processing signal and can either be a target for a protease (e.g., ubiquitin, LC3 as defined herein or a protease or an unstable peptide leading to processing at its own carboxyterminus like e.g., intein (Chong et al. 1998 and references therein) or 3C of picornaviruses, 2A of cardioviruses or aphtoviruses, p15 of rabbit hemorrhagic disease virus, or the corresponding protease of other caliciviruses (Proter, 1993, and references therein; Meyers et al., 2000 and references therein).

[0215] When using a PS, a large number of different variants are possible since the PS ensures the generation of the correct amino terminus of the capsid protein C. Thus, when using a PS construct, all kinds of deletions or mutations of the N.sup.pro sequence are expected to result in viable mutants as long as the reading frame is not shifted or translation stopped by an in frame stop codon. As an example we established a viable CSFV N.sup.pro deletion mutant according to the formula

[N.sup.pro].sub.29--PS--[C-term]

[0216] Especially interesting could be N.sup.pro mutations blocking the proteolytic activity of the protein. Rumenapf et al., 1998, have published the identification of the active site residues of the protease for CSFV Alfort Tubingen. The respective amino acids (glutamic acid at position 22, histidine at position 49 and cysteine at position 69) are conserved for other pestiviruses. Thus, exchanges of any amino acid expect for serine or threonine for the cysteine at position 69 will result in destruction of the protease activity. Similarly, changing the glutamic acid at position 22 will most likely result in inactivation of the protease unless the new amino acid is aspartic acid. Similarly most if not all exchanges at position 49 will lead to an inactive protease).

REFERENCES

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Summerfield, 2003, Classical Swine Fever Virus Interferes with Cellular Antiviral Defense: Evidence for a Novel Function of N(pro), J. Virol. 77: 7645-7654. [0241] T. Rumenapf, R. Stark, M. Heimann, and H.-J. Theil, 1998, N-terminal Protease of Pestiviruses: Identification of Putative Catalytic Residues by Site Directed Mutagenesis, J. Virol. 72: 2544-2547. [0242] T. Rumenapf, G. Unger, J. H. Strauss, and H.-J. Theil, 1993, Processing of the Envelope Glycoproteins of Pestiviruses. J. Virol. 67: 3288-3294. [0243] R. Schneider, G. Unger, R. Stark, E. Schneider-Scherzer, and H.-J. Thiel, 1993, Identification of a Structural Glycoprotein of an RNA Virus as a Ribonuclease, Science 261: 1169-1171. [0244] J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 [0245] R. Stark, G. Meyers, T. Rumenapf, and H.-J. Theil, 1993, Processing of Pestivirus Polyprotein: Cleavage Site between Autoprotease and Nucleocapsid Protein of Classical Swine Fever Virus, J. Virol., 67: 7088-7095. [0246] H.-J. Theil, G. W. Plagemann, and V. Moennig, 1996, The Pestiviruses, in: Fields Virology, B. N. Fields, D. M. Knipe, and P. M. Howley (eds.), Lippincott-Raven, Philadelphia, pp. 1059-1073. [0247] H.-J. Theil, R. Stark, E. Weiland, T. Rumenapf, and G. Meyers, 1991, Hog Cholera Virus: Molecular Composition of Virions from a Pestivirus, J. Virol. 65: 4705-4712. [0248] J.-D. Tratschin, C. Moser, N. Ruggli, and M. A. Hofmann, 1998, Classical Swine Fever Virus Leader Proteinase N.sup.pro is Not Required for Viral Replication in Cell Culture, J. Virol. 72: 7681-7684. [0249] P. A. van Rijn, H. G. van Gennip, E. J. de Meijer, and R. J. Moormann, 1993, Epitope Mapping of Envelope Glycoprotein E1 of Hog Cholera Virus Strain Brescia, J. Gen. Virol. 74: 2053-2060. [0250] E. Weiland, H.-J. Theil, G. Hess, and F. 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Sequence CWU 1

1

16112332DNAWildtyp BVDV XIKE-A 1gtatacgaga ttagctaaag aactcgtata tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga gttgttttca aatgaacttt tatacaaaac 420atataaacaa aaaccagcag gcgtcgtgga acctgtttac gacgtcaacg ggcgcccact 480gtttggagag agcagtgact tgcacccgca gtcaacacta aaactaccac accaacgagg 540cagcgccaac atcctgacca atgctaggtc cctaccgcgg aaaggtgact gccggagagg 600taatgtgtat ggaccggtga gtggcatcta tatcaaacca ggaccgatct actaccagga 660ttatgtgggc cccgtctatc atagagcccc actggaacta tgtagggagg caagtatgtg 720cgaaacaact aggagagttg gcagagtgac cggtagtgat gggaaattat atcatatcta 780catctgcata gatgggtgta tcctcctgaa gagggcgact aggaaccaac cagaagtcct 840gaaatgggta tacaacagat taaattgtcc tttatgggtc accagctgct ccgatgaagg 900gagcaagggt gctacaagta agaagcagcc taagccagat aggatagaaa aaggtaagat 960gaaaatagcc ccaaaagaga cagaaaaaga ttgcaaaacc agaccccccg acgcgactat 1020agtagtagaa ggggttaagt accaggtgaa gaaaaaagga aaggtaaggg gaaaaaatac 1080tcaagatggg ttatatcaca acaagaataa gccccctgaa tcaagaaaaa aattggaaaa 1140ggcactgctg gcttgggcca tcttagcagc ggtcctgctt cagctggtaa caggagagaa 1200tatcacccag tggaacttga tggacaacgg caccgaggga atacagcaag cgatgttcct 1260aagaggggtg aacaggagtc tacatggaat ttggccagag aaaatttgca ccggagtacc 1320aactcactta gcaacagact atgagcttaa agagatagtg gggatgatgg acgcgagtga 1380gaagaccaac tacacgtgtt gcaggttgca aagacatgag tggaataaac atggttggtg 1440taactggttt catatagaac cgtggatatg gttgatgaac aaaacccaaa acaacctgac 1500agaagggcaa ccgcttaggg agtgtgctgt gacttgtagg tatgacaagg aaacagaatt 1560gaacatcgtg acacaggcta gggacagacc tacaactctg acaggttgca agaaaggcaa 1620gaatttctct ttcgcaggtg ttatactgga tgggccctgt aactttaaag tatcggttga 1680agatgtgctg ttcaaggagc acgattgcgg caacatgctg caagagaccg cgatacagct 1740actcgatggg gcaaccaaca ccattgaggg agcaagggta gggacggcca agttgacaac 1800ctggttaggg aagcaattag ggatccttgg taagaagttg gagaacaaaa gcaaagcatg 1860gtttggtgca catgcagcaa gtccatactg cggagtggag aggaagatcg gttacgtatg 1920gtatacaaaa aactgcactc cagcttgcct tccaagaaac actagaataa taggccccgg 1980gaaatttgat accaacgccg aagatggaaa aatactccat gagatggggg ggcacctctc 2040agaatttgtc ctattgtcct tggtggttct gtctgacttt gccccggaaa ccgcgagcgt 2100catctacttg gttctacatt ttgcgatccc gcaaagccac gttgatgtag acacatgcga 2160caagaaccag ctgaatttaa cggtagcaac cacagtagca gaggtcatac cagggacagt 2220gtggaaccta gggaagtatg tctgcataag accagactgg tggccatatg agacgacgac 2280agtcttcgtc atagaggaag cagggcaagt aatcaaattg atgctaaggg ccatcagaga 2340cttaactagg atatggaatg ctgccactac cacagctttc ttaatctttt tagtaaaagc 2400actgagggga caactaatcc aagggctatt gtggctgatg ctaataacag gagcacaggg 2460cttccctgaa tgcaaagagg gcttccaata tgccatatct aaagacagga aaatggggtt 2520attggggcca gagagcttaa ctacaacatg gcacctcccc accaaaaaaa tagtggattc 2580catggtgcat gtatggtgtg aaggaaaaga cttgaaaata ttaaaaatgt gcacaaagga 2640agagaggtat ctagtggctg tgcacgagag agccttatca accagtgccg agtttatgca 2700gatcagtgat gggacaatag gcccagacgt gatagatatg cctgatgact ttgagtttgg 2760actctgccct tgtgactcaa aaccagtgat aaagggcaaa tttaatgcca gcttactgaa 2820tggaccagct ttccagatgg tatgcccaca ggggtggact ggtacaatag aatgcaccct 2880agcgaaccaa gacaccttgg acacaactgt cattaggaca tatagaagaa ctaccccatt 2940tcagcggaga aaatggtgta cctatgaaaa aataataggg gaagatatct atgaatgcat 3000tctaggtgga aactggacat gcataaccgg tgaccatagc aggttgaaag acggacctat 3060caagaagtgt aagtggtgtg gccatgactt cgtcaactca gaggggctac cacactaccc 3120aataggcaag tgcatgctca tcaacgagag tgggtacagg tatgtagatg acacctcttg 3180cgataggggt ggtgtagcca tagttccatc tggcaccgta aagtgtagaa taggtaacgt 3240cacggtgcaa gttatcgcta ctaacaatga tctgggaccc atgccttgca gcccagctga 3300agtgatagca agtgaaggac cagtggaaaa gactgcatgc acattcaact attcaaggac 3360tctacctaat aagtattatg agccaaggga ccggtacttc caacaataca tgttaaaagg 3420ggagtggcaa tattggttcg acctggattc tgtagaccac cacaaagact acttctcaga 3480gttcataatc atagcagtgg tcgccttgtt gggtggtaag tacgtactgt ggctcttgat 3540aacatacaca atactgtctg agcagatggc tatgggtgct ggagtgaata ctgaagagat 3600agtcatgata ggcaatttgc tgacagacag tgatattgag gttgtggttt atttccttct 3660tctgtactta atagttaaag aggaactggc gaggaaatgg attatactgg tataccacat 3720ccttgtagcc aaccctatga aaacaattgg ggtcgtctta ctaatgctag ggggagtggt 3780gaaggccagc agaatcaatg ctgatgacca aagtgctatg gacccatgct ttcttctcgt 3840gacaggcgta gtggctgttt tgatgatcgc tagaagagaa cctgccacat taccactgat 3900tgtagcattg ctagcaataa gaacatcagg attcctactg cccgctagca ttgatgtaac 3960tgtagcagta gtattaattg tacttttgtt ggctagctac ataacagact actttagata 4020taaaaagtgg cttcaactct tatttagtct gatagctggt atctttatta taaggagctt 4080aaaacatatc aaccagatgg aggtaccaga aatatctatg ccaagttgga gacctctagc 4140tctggtcctt ttctatataa catctacagc aataaccact aattgggaca ttgacttagc 4200aggcttcctg ctgcaatggg cgccagcagt gatcatgatg gctaccatgt gggcagactt 4260tttgactctg atcatagtcc tgcccagtta cgagttatct aagctttact tcctaaagaa 4320cgtcaggaca gacgtggaaa agaactggct cggcaaagtg aaatacagac agatcagttc 4380agtttatgac atctgtgaca gtgaggaagc agtgtaccta tttccatcaa ggcataagag 4440tggaagcagg ccagatttca tattaccttt tttgaaagcc gtgttaataa gctgcatcag 4500cagccaatgg caagtggttt acatttctta cctaatactg gaaattacat actatatgca 4560caggaaaatc atagatgagg tgtcaggagg agcaaatttt ctatcaagac tcatagcagc 4620catcatagaa ttaaattggg ccatagatga tgaggaatgt aaaggactga agaaactgta 4680tctcttgtca gggagagcga agaatttgat agttaaacat aaggtaagaa atgaagccgt 4740ccacagatgg tttggtgagg aggaaatata cggggcaccc aaggtgatca ctatcataaa 4800agctagtacc ctaagtaaaa acaggcactg cataatctgc acgatctgtg aagggaaaga 4860atggaatgga gccaactgcc caaagtgtgg aagacaagga aagcccataa catgtggaat 4920gacactcgca gactttgagg agaaacatta caaaaagata tttataagag aagaatcttc 4980ttgtcctgtg ccttttgatc cttcttgcca ttgtaattat tttcgccacg atgggccttt 5040caggaaagag tataagggtt acgtccaata cacagccaga ggacaactct ttctgaggaa 5100cctaccaatt ctagcgacga agatgaagct attaatggtg ggaaacctcg gcgcagaaat 5160tggcgacctg gaacatctag gatgggtact gagagggcca gccgtgtgca aaaaaattac 5220caaccatgag aagtgccacg taaacatcat ggataagcta actgcatttt ttggaatcat 5280gcctagaggc acgaccccta gggcacctgt gaggttcccc acagcactac taaaagtgag 5340aagggggcta gagacgggat gggcttacac gcaccaagga gggatcagct cggtagacca 5400tgtcacagcc ggaaaggatt tactagtgtg tgacagtatg ggcaggacca gggttgtctg 5460tcatagtaac aataagatga ctgatgagac tgagtatggc atcaagaccg actcagggtg 5520tcccgaaggt gcgaggtgtt acgtgctaaa cccagaagct gttaacattt ctggcacaaa 5580aggagctatg gtacacctcc agaaaacggg gggggagttc acatgtgtca ctgcctcagg 5640gaccccggct ttcttcgatc tgaaaaatct aaaaggctgg tccgggctac caatttttga 5700agcatccagt ggcagggtgg ttggtagggt gaaagtcggc aagaatgagg attccaagcc 5760caccaaacta atgagcggaa tccagacagt gtctaagaac cagacagacc tagcggacat 5820cgtaaaaaaa ttgactagta tgaacagagg agagttcaaa cagataacat tagccactgg 5880ggcaggaaaa actacggaac tgccaaggtc cgtcatagag gagataggga ggcacaaaag 5940ggtcttagtc ctgataccat tgagagcagc agcagagtca gtgtatcagt atatgagagt 6000gaagtaccca agtatatctt tcaatttgag aataggagat atgaaggaag gtgacatggc 6060cactggtatc acctacgcct catatgggta cttttgtcag cttcctcagc ccaaactgag 6120agctgccatg gtagagtact catatatatt cttagatgag taccactgtg ctacacccga 6180gcaattagca ataattggaa agatacacag gtttgctgaa aatcttagag tggtagcaat 6240gacagcaacc ccagctggaa cggtcacaac gactggtcag aaacacccta tagaggagtt 6300catagcccca gaggtgatga aaggtgaaga tctaggtagt gaatacttgg atattgcagg 6360gttgaagata ccgactgaag agatgaaagg caacatgctc gtgttcgcgc caactaggaa 6420catggcagta gaaacagcta agaaattgaa ggctaaggga tacaactctg gatactatta 6480cagtggggaa aacccagaga acttgagggt ggtaacctcg caatccccgt atgtggtagt 6540agccaccaat gccatagagt caggtgtgac attaccagac ttagacacag ttgtagacac 6600tggactaaag tgtgagaaga gggtgaggat ttcttcaaaa atgcccttca ttgtaacagg 6660acttaagaga atggcagtca caatcggaga gcaagcccag cgcaggggta gagtaggaag 6720agtcaagcca ggtaggtact ataggagtca agaaacagct tcagggtcaa aagattacca 6780ttacgaccta ctgcaagccc agaggtacgg aatagaagat ggaattaatg taacaaagtc 6840attcagggag atgaactatg attggagcct ttacgaagag gacagcttga tgataactca 6900actcgaggtc cttaacaacc tccttatatc agaagacctg cctgccgcag tgaagaacat 6960catggcccgg accgatcacc cagaacccat acaactggcc tataacagtt atgaaaacca 7020aattccagtg ctgttcccaa agatcaaaaa tggtgaggtg acagacagtt atgagaatta 7080cacatatctc aatgcaagaa aattaggaga ggacgtgccg gcatatgtgt acgccacaga 7140ggatgaggat ctagcagtgg atcttctggg tatggattgg ccggacccag gcaaccaaca 7200ggtggtagag acagggaggg cattaaaaca agtaactggc ttatccacag cagaaaacgc 7260cctcttgata gccctattcg gctacgtcgg gtaccagaca ctttcaaaaa ggcacatacc 7320catgattact gacatctata cacttgaaga ccacaggctt gaggacacaa cccacctcca 7380gtttgcccca aacgctataa ggaccgacgg caaggactca gagttgaagg aattagctgt 7440gggagacctt gataaatatg tggacgcact ggtagactac tccaaacaag ggatgaaatt 7500catcaaagtc caagctgaaa aggtcagaga ctcccagtct acgaaggaag gcttgcaaac 7560cattaaggag tatgtggata agtttataca atcactaaca gagaataagg aggagatcat 7620caggtatgga ctatggggag ttcacacggc actctacaaa agcttggcag cgagactggg 7680gcatgaaaca gcttttgcaa ctttagtggt aaaatggttg gcttttgggg gcgaaacggt 7740atctgctcac atcaagcaag tagcagttga tctagtagta tattatatca tcaacaaacc 7800atcttttcct ggagatacag agacccaaca agaggggagg aagtttgtgg ctagtctttt 7860tatatctgca ctagcaacat acacatataa aacctggaat tacaacaatc tgcaacgggt 7920tgtcgaacct gccttagctt acctcccata tgctacaagt gccttgaagt tgttcacacc 7980cacaagatta gagagtgtgg tcatactcag ttctacaatt tacaagacat acctctctat 8040aaggaagggt aagagtgacg gcttgttagg tacaggcata agtgcagcca tggagatctt 8100aaaccaaaac ccaatctcag taggtatatc tgtgatgctg ggggtaggtg ccatcgccgc 8160ccataatgca atagaatcta gtgaacagaa aagaactttg ctgatgaagg tctttgtaaa 8220aaacttctta gaccaagcag caacagatga gctagtcaaa gagaaccctg aaaaaataat 8280catggctcta tttgaagcag tccagaccat aggaaacccc ctaagactca tctaccatct 8340gtacggggtg tactataagg ggtgggaagc aaaagaactc gcagagaaaa ctgctggccg 8400caacttattc acattgatca tgtttgaggc ctttgagctt ttaggtatgg actcagaagg 8460aaagataaga aacttgtcag gcaactacat actggactta atcttcaact tgcataataa 8520attaaacaag gggctcaaaa aactagtcct tgggtgggct cctgcacctt tgagctgtga 8580ttggacacca agtgatgaga gaataagcct acctcataac aactacttaa gggtagaaac 8640caggtgtcct tgtggctatg agatgaaggc aataaaaaat gttgctggta aattgacaaa 8700agttgaagaa aaggggtcct tcctatgcag gaatagatta gggagaggac ctccaaactt 8760caaagtaaca aagttctatg atgataactt gatagaagtc aagccagtag ctaggctaga 8820aggccaggtg gacctctatt acaagggagt aacagctaag ttagactaca acaatgggaa 8880agtactgtta gctaccaaca agtgggaggt ggaccacgct ttcctgacca gactagtaaa 8940gaagcacaca gggataggtt ttaaaggtgc atatttgggt gaccgaccag accatcaaga 9000tcttgtcgat agagattgtg caactataac gaagaactca gtacagttcc taaaaatgaa 9060gaagggttgc gctttcacat atgacctaac aatctctaac cttgtcaggc ttattgaact 9120agtccataag aataatttac aagaaagaga gatccctacc gtgacagtaa ctacttggct 9180tgcatattct tttgtcaatg aagacctggg gactatcaag cctgtattgg gggagaaagt 9240catcccagaa ccccccgagg agttgagtct ccaacccacc gtgagactag tcaccactga 9300aacagcaata accataacag gggaggctga agtgatgacg acagggatca caccagtggt 9360agagatgaaa gaagaacctc agctggacca ccagtcaact accctaaagg tagggttgaa 9420ggaaggggaa tatccagggc caggagttaa ccctaaccat ttagcagagg tgatagatga 9480gaaagatgac aggccttttg tcctaatcat cggtaacaaa ggttctacct cgaacagagc 9540aagaacggcc aagaatatac ggctgtacaa aggaaacaac ccaagagaga tcagggatct 9600gatgagccaa ggaagaatat tgacggttgc tctaaaagag ttggacccgg aattaaaaga 9660attagtagat tacaagggga cctttctcaa tagggaagct ttagaagccc taagcttagg 9720taagccaatc aagaggaaaa ccacaacagc aatgatcagg aggttaatag agccagaggt 9780tgaggaggaa ctaccagatt ggttccaagc ggaagaaccc ctatttttgg aagcaaaaat 9840acagaatgac ttataccacc taattggcag tgtagatagt ataaaaagca aagcaaagga 9900attaggggcc acagataaca caaagatagt gaaggaagtt ggggctagga cctatacgat 9960gaaattgagc agctggagca cacaagttac aaaaaaacag atgagtctag cccctctctt 10020tgaagagctg ttattaaagt gccctccatg tagtaaaatt tcaaagggac atatggtgtc 10080agcataccaa ctggctcaag gaaactggga acccctcggg tgtggggtct atatgggaac 10140cataccagct aggcgtctca agatccaccc ttatgaggct taccttaaac tcaaagagct 10200ggtggaagtt gaatcttcga gggccactgc aaaagaatcc atcataagag aacataacac 10260ctggatcctg cggaaggtga gacatgaagg gaacctaaga accaaatcaa tgatcaaccc 10320tgggaaaata tcagatcagc tatgcagaga tggacacaaa agaaacatat ataataagat 10380cataggctca acaatggcct ctgctggtat taggctggag aaactgccag tagtccgagc 10440ccaaactgac acaaccagtt tccaccaagc cataagagaa aaaattgata aaacagaaaa 10500caagcagacc cctgaattgc atgaagaact aatgaaggtc ttcgactgct taaagatccc 10560agagctgaag gaatcgtatg atgaagtttc atgggaacaa ttagaagccg ggataaaccg 10620taagggtgca gcaggctatc tagagagcaa gaacataggg gaagtcctag acacagagaa 10680acacatagta gagcagctga tcaaggatct gaggaagggg aagaagatta ggtactatga 10740aacagccatc cccaagaatg agaagagaga cgtcagcgac gactgggaag ccggagagtt 10800cgttgatgaa aagaaaccaa gagtaatcca gtacccggac gccaaggtga gactggccat 10860tacaaaagtg atgtacaaat gggtaaagca aaaaccagtg gtgatacccg gctatgaagg 10920taaaacacct ctatttgaca tattcaacaa agtgaagaag gaatgggatt cattccagga 10980ccccgtagca gtgagctttg acaccaaagc gtgggataca caagtcacca gtagagacct 11040aatgttgata aaggatatcc agaaatatta tttcaagaga agtatacaca aatttttaga 11100tacaataaca gaacacatgg tggaggtacc tgtcattaca gcagacggtg aagtttacat 11160aaggaatggt cagaggggta gtggccaacc cgacacaagt gctggtaata gtatgttgaa 11220tgtcctaacc atgatatatg ctttctgtaa aagtacaggc ataccttaca ggggattcag 11280cagagtggca agaatccatg tgtgtggtga tgatggcttt ttgataacag agagaggact 11340gggactgaaa ttctctgaga agggtatgca gatattacat gaggccggga agccccagaa 11400aataactgaa ggggacaaaa tgaaagtggc atacagattc gaggacatag agttttgttc 11460ccatactccc gtgccagtca gatgggcaga taacaccagt agttacatgg cagggaggag 11520cacagccact atactagcta agatggcaac caggctggat tccagcggag agaggggtag 11580cacagcttat gagaaggccg tagccttcag cttccttttg atgtactcat ggaatcccgt 11640agttagaagg atctgcttac tggtgttgtc acagtttcca gaaatatccc catccaaaaa 11700cacaatatac tactaccaag gggatcccat agctgcgtac agagaagtga tagggaaaca 11760gctgtgtgaa ctgaaaagaa caggatttga gaagctggct ggtctgaatt tgagtatgac 11820cactctaggc atctggacaa aacatactag taaaagacta atccaagcct gtgtagaaat 11880aggtaagaga gaaggtacct ggttagttaa tgctgacaga ctgattgcag gaaagactgg 11940gaagttttac atcccaagca ctggtgtcac tctgttggga aaacactatg aggaaattaa 12000cttaaagcaa aaggcggcac aaccgccgat agagggggtt gacagatata agttgggccc 12060catagttaat gttatcttga gaaggctgag ggtgatgctg atgacagttg ccagcggaag 12120ctggtgaatc cgtccggagc gtcgtgccct cactcaaggt ttttaattgt aaatattgta 12180aatagacagc taagatattt attgtagttg gatagtaatg cagtgatagt aaatacccca 12240atttaacact acctccaatg cactaagcac tttagctgtg tgaggttaac tcgacgtcca 12300cggttggact agggaagacc tctaacagcc cc 12332211840DNAArtificial SequenceMutated BVDV XIKE- A-NdN 2gtatacgaga ttagctaaag aactcgtata tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga gttgttttcc gatgaaggga gcaagggtgc 420tacaagtaag aagcagccta agccagatag gatagaaaaa ggtaagatga aaatagcccc 480aaaagagaca gaaaaagatt gcaaaaccag accccccgac gcgactatag tagtagaagg 540ggttaagtac caggtgaaga aaaaaggaaa ggtaagggga aaaaatactc aagatgggtt 600atatcacaac aagaataagc cccctgaatc aagaaaaaaa ttggaaaagg cactgctggc 660ttgggccatc ttagcagcgg tcctgcttca gctggtaaca ggagagaata tcacccagtg 720gaacttgatg gacaacggca ccgagggaat acagcaagcg atgttcctaa gaggggtgaa 780caggagtcta catggaattt ggccagagaa aatttgcacc ggagtaccaa ctcacttagc 840aacagactat gagcttaaag agatagtggg gatgatggac gcgagtgaga agaccaacta 900cacgtgttgc aggttgcaaa gacatgagtg gaataaacat ggttggtgta actggtttca 960tatagaaccg tggatatggt tgatgaacaa aacccaaaac aacctgacag aagggcaacc 1020gcttagggag tgtgctgtga cttgtaggta tgacaaggaa acagaattga acatcgtgac 1080acaggctagg gacagaccta caactctgac aggttgcaag aaaggcaaga atttctcttt 1140cgcaggtgtt atactggatg ggccctgtaa ctttaaagta tcggttgaag atgtgctgtt 1200caaggagcac gattgcggca acatgctgca agagaccgcg atacagctac tcgatggggc 1260aaccaacacc attgagggag caagggtagg gacggccaag ttgacaacct ggttagggaa 1320gcaattaggg atccttggta agaagttgga gaacaaaagc aaagcatggt ttggtgcaca 1380tgcagcaagt ccatactgcg gagtggagag gaagatcggt tacgtatggt atacaaaaaa 1440ctgcactcca gcttgccttc caagaaacac tagaataata ggccccggga aatttgatac 1500caacgccgaa gatggaaaaa tactccatga gatggggggg cacctctcag aatttgtcct 1560attgtccttg gtggttctgt ctgactttgc cccggaaacc gcgagcgtca tctacttggt 1620tctacatttt gcgatcccgc aaagccacgt tgatgtagac acatgcgaca agaaccagct 1680gaatttaacg gtagcaacca cagtagcaga ggtcatacca gggacagtgt ggaacctagg 1740gaagtatgtc tgcataagac cagactggtg gccatatgag acgacgacag tcttcgtcat 1800agaggaagca gggcaagtaa tcaaattgat gctaagggcc atcagagact taactaggat 1860atggaatgct gccactacca cagctttctt aatcttttta gtaaaagcac tgaggggaca 1920actaatccaa gggctattgt ggctgatgct aataacagga gcacagggct tccctgaatg 1980caaagagggc ttccaatatg ccatatctaa agacaggaaa atggggttat tggggccaga 2040gagcttaact acaacatggc acctccccac caaaaaaata gtggattcca tggtgcatgt 2100atggtgtgaa ggaaaagact tgaaaatatt aaaaatgtgc acaaaggaag agaggtatct 2160agtggctgtg cacgagagag ccttatcaac cagtgccgag tttatgcaga tcagtgatgg 2220gacaataggc ccagacgtga tagatatgcc tgatgacttt gagtttggac tctgcccttg 2280tgactcaaaa ccagtgataa agggcaaatt taatgccagc ttactgaatg gaccagcttt 2340ccagatggta tgcccacagg ggtggactgg tacaatagaa tgcaccctag cgaaccaaga 2400caccttggac acaactgtca ttaggacata tagaagaact accccatttc agcggagaaa 2460atggtgtacc tatgaaaaaa taatagggga agatatctat gaatgcattc taggtggaaa 2520ctggacatgc ataaccggtg accatagcag gttgaaagac ggacctatca agaagtgtaa 2580gtggtgtggc catgacttcg tcaactcaga

ggggctacca cactacccaa taggcaagtg 2640catgctcatc aacgagagtg ggtacaggta tgtagatgac acctcttgcg ataggggtgg 2700tgtagccata gttccatctg gcaccgtaaa gtgtagaata ggtaacgtca cggtgcaagt 2760tatcgctact aacaatgatc tgggacccat gccttgcagc ccagctgaag tgatagcaag 2820tgaaggacca gtggaaaaga ctgcatgcac attcaactat tcaaggactc tacctaataa 2880gtattatgag ccaagggacc ggtacttcca acaatacatg ttaaaagggg agtggcaata 2940ttggttcgac ctggattctg tagaccacca caaagactac ttctcagagt tcataatcat 3000agcagtggtc gccttgttgg gtggtaagta cgtactgtgg ctcttgataa catacacaat 3060actgtctgag cagatggcta tgggtgctgg agtgaatact gaagagatag tcatgatagg 3120caatttgctg acagacagtg atattgaggt tgtggtttat ttccttcttc tgtacttaat 3180agttaaagag gaactggcga ggaaatggat tatactggta taccacatcc ttgtagccaa 3240ccctatgaaa acaattgggg tcgtcttact aatgctaggg ggagtggtga aggccagcag 3300aatcaatgct gatgaccaaa gtgctatgga cccatgcttt cttctcgtga caggcgtagt 3360ggctgttttg atgatcgcta gaagagaacc tgccacatta ccactgattg tagcattgct 3420agcaataaga acatcaggat tcctactgcc cgctagcatt gatgtaactg tagcagtagt 3480attaattgta cttttgttgg ctagctacat aacagactac tttagatata aaaagtggct 3540tcaactctta tttagtctga tagctggtat ctttattata aggagcttaa aacatatcaa 3600ccagatggag gtaccagaaa tatctatgcc aagttggaga cctctagctc tggtcctttt 3660ctatataaca tctacagcaa taaccactaa ttgggacatt gacttagcag gcttcctgct 3720gcaatgggcg ccagcagtga tcatgatggc taccatgtgg gcagactttt tgactctgat 3780catagtcctg cccagttacg agttatctaa gctttacttc ctaaagaacg tcaggacaga 3840cgtggaaaag aactggctcg gcaaagtgaa atacagacag atcagttcag tttatgacat 3900ctgtgacagt gaggaagcag tgtacctatt tccatcaagg cataagagtg gaagcaggcc 3960agatttcata ttaccttttt tgaaagccgt gttaataagc tgcatcagca gccaatggca 4020agtggtttac atttcttacc taatactgga aattacatac tatatgcaca ggaaaatcat 4080agatgaggtg tcaggaggag caaattttct atcaagactc atagcagcca tcatagaatt 4140aaattgggcc atagatgatg aggaatgtaa aggactgaag aaactgtatc tcttgtcagg 4200gagagcgaag aatttgatag ttaaacataa ggtaagaaat gaagccgtcc acagatggtt 4260tggtgaggag gaaatatacg gggcacccaa ggtgatcact atcataaaag ctagtaccct 4320aagtaaaaac aggcactgca taatctgcac gatctgtgaa gggaaagaat ggaatggagc 4380caactgccca aagtgtggaa gacaaggaaa gcccataaca tgtggaatga cactcgcaga 4440ctttgaggag aaacattaca aaaagatatt tataagagaa gaatcttctt gtcctgtgcc 4500ttttgatcct tcttgccatt gtaattattt tcgccacgat gggcctttca ggaaagagta 4560taagggttac gtccaataca cagccagagg acaactcttt ctgaggaacc taccaattct 4620agcgacgaag atgaagctat taatggtggg aaacctcggc gcagaaattg gcgacctgga 4680acatctagga tgggtactga gagggccagc cgtgtgcaaa aaaattacca accatgagaa 4740gtgccacgta aacatcatgg ataagctaac tgcatttttt ggaatcatgc ctagaggcac 4800gacccctagg gcacctgtga ggttccccac agcactacta aaagtgagaa gggggctaga 4860gacgggatgg gcttacacgc accaaggagg gatcagctcg gtagaccatg tcacagccgg 4920aaaggattta ctagtgtgtg acagtatggg caggaccagg gttgtctgtc atagtaacaa 4980taagatgact gatgagactg agtatggcat caagaccgac tcagggtgtc ccgaaggtgc 5040gaggtgttac gtgctaaacc cagaagctgt taacatttct ggcacaaaag gagctatggt 5100acacctccag aaaacggggg gggagttcac atgtgtcact gcctcaggga ccccggcttt 5160cttcgatctg aaaaatctaa aaggctggtc cgggctacca atttttgaag catccagtgg 5220cagggtggtt ggtagggtga aagtcggcaa gaatgaggat tccaagccca ccaaactaat 5280gagcggaatc cagacagtgt ctaagaacca gacagaccta gcggacatcg taaaaaaatt 5340gactagtatg aacagaggag agttcaaaca gataacatta gccactgggg caggaaaaac 5400tacggaactg ccaaggtccg tcatagagga gatagggagg cacaaaaggg tcttagtcct 5460gataccattg agagcagcag cagagtcagt gtatcagtat atgagagtga agtacccaag 5520tatatctttc aatttgagaa taggagatat gaaggaaggt gacatggcca ctggtatcac 5580ctacgcctca tatgggtact tttgtcagct tcctcagccc aaactgagag ctgccatggt 5640agagtactca tatatattct tagatgagta ccactgtgct acacccgagc aattagcaat 5700aattggaaag atacacaggt ttgctgaaaa tcttagagtg gtagcaatga cagcaacccc 5760agctggaacg gtcacaacga ctggtcagaa acaccctata gaggagttca tagccccaga 5820ggtgatgaaa ggtgaagatc taggtagtga atacttggat attgcagggt tgaagatacc 5880gactgaagag atgaaaggca acatgctcgt gttcgcgcca actaggaaca tggcagtaga 5940aacagctaag aaattgaagg ctaagggata caactctgga tactattaca gtggggaaaa 6000cccagagaac ttgagggtgg taacctcgca atccccgtat gtggtagtag ccaccaatgc 6060catagagtca ggtgtgacat taccagactt agacacagtt gtagacactg gactaaagtg 6120tgagaagagg gtgaggattt cttcaaaaat gcccttcatt gtaacaggac ttaagagaat 6180ggcagtcaca atcggagagc aagcccagcg caggggtaga gtaggaagag tcaagccagg 6240taggtactat aggagtcaag aaacagcttc agggtcaaaa gattaccatt acgacctact 6300gcaagcccag aggtacggaa tagaagatgg aattaatgta acaaagtcat tcagggagat 6360gaactatgat tggagccttt acgaagagga cagcttgatg ataactcaac tcgaggtcct 6420taacaacctc cttatatcag aagacctgcc tgccgcagtg aagaacatca tggcccggac 6480cgatcaccca gaacccatac aactggccta taacagttat gaaaaccaaa ttccagtgct 6540gttcccaaag atcaaaaatg gtgaggtgac agacagttat gagaattaca catatctcaa 6600tgcaagaaaa ttaggagagg acgtgccggc atatgtgtac gccacagagg atgaggatct 6660agcagtggat cttctgggta tggattggcc ggacccaggc aaccaacagg tggtagagac 6720agggagggca ttaaaacaag taactggctt atccacagca gaaaacgccc tcttgatagc 6780cctattcggc tacgtcgggt accagacact ttcaaaaagg cacataccca tgattactga 6840catctataca cttgaagacc acaggcttga ggacacaacc cacctccagt ttgccccaaa 6900cgctataagg accgacggca aggactcaga gttgaaggaa ttagctgtgg gagaccttga 6960taaatatgtg gacgcactgg tagactactc caaacaaggg atgaaattca tcaaagtcca 7020agctgaaaag gtcagagact cccagtctac gaaggaaggc ttgcaaacca ttaaggagta 7080tgtggataag tttatacaat cactaacaga gaataaggag gagatcatca ggtatggact 7140atggggagtt cacacggcac tctacaaaag cttggcagcg agactggggc atgaaacagc 7200ttttgcaact ttagtggtaa aatggttggc ttttgggggc gaaacggtat ctgctcacat 7260caagcaagta gcagttgatc tagtagtata ttatatcatc aacaaaccat cttttcctgg 7320agatacagag acccaacaag aggggaggaa gtttgtggct agtcttttta tatctgcact 7380agcaacatac acatataaaa cctggaatta caacaatctg caacgggttg tcgaacctgc 7440cttagcttac ctcccatatg ctacaagtgc cttgaagttg ttcacaccca caagattaga 7500gagtgtggtc atactcagtt ctacaattta caagacatac ctctctataa ggaagggtaa 7560gagtgacggc ttgttaggta caggcataag tgcagccatg gagatcttaa accaaaaccc 7620aatctcagta ggtatatctg tgatgctggg ggtaggtgcc atcgccgccc ataatgcaat 7680agaatctagt gaacagaaaa gaactttgct gatgaaggtc tttgtaaaaa acttcttaga 7740ccaagcagca acagatgagc tagtcaaaga gaaccctgaa aaaataatca tggctctatt 7800tgaagcagtc cagaccatag gaaaccccct aagactcatc taccatctgt acggggtgta 7860ctataagggg tgggaagcaa aagaactcgc agagaaaact gctggccgca acttattcac 7920attgatcatg tttgaggcct ttgagctttt aggtatggac tcagaaggaa agataagaaa 7980cttgtcaggc aactacatac tggacttaat cttcaacttg cataataaat taaacaaggg 8040gctcaaaaaa ctagtccttg ggtgggctcc tgcacctttg agctgtgatt ggacaccaag 8100tgatgagaga ataagcctac ctcataacaa ctacttaagg gtagaaacca ggtgtccttg 8160tggctatgag atgaaggcaa taaaaaatgt tgctggtaaa ttgacaaaag ttgaagaaaa 8220ggggtccttc ctatgcagga atagattagg gagaggacct ccaaacttca aagtaacaaa 8280gttctatgat gataacttga tagaagtcaa gccagtagct aggctagaag gccaggtgga 8340cctctattac aagggagtaa cagctaagtt agactacaac aatgggaaag tactgttagc 8400taccaacaag tgggaggtgg accacgcttt cctgaccaga ctagtaaaga agcacacagg 8460gataggtttt aaaggtgcat atttgggtga ccgaccagac catcaagatc ttgtcgatag 8520agattgtgca actataacga agaactcagt acagttccta aaaatgaaga agggttgcgc 8580tttcacatat gacctaacaa tctctaacct tgtcaggctt attgaactag tccataagaa 8640taatttacaa gaaagagaga tccctaccgt gacagtaact acttggcttg catattcttt 8700tgtcaatgaa gacctgggga ctatcaagcc tgtattgggg gagaaagtca tcccagaacc 8760ccccgaggag ttgagtctcc aacccaccgt gagactagtc accactgaaa cagcaataac 8820cataacaggg gaggctgaag tgatgacgac agggatcaca ccagtggtag agatgaaaga 8880agaacctcag ctggaccacc agtcaactac cctaaaggta gggttgaagg aaggggaata 8940tccagggcca ggagttaacc ctaaccattt agcagaggtg atagatgaga aagatgacag 9000gccttttgtc ctaatcatcg gtaacaaagg ttctacctcg aacagagcaa gaacggccaa 9060gaatatacgg ctgtacaaag gaaacaaccc aagagagatc agggatctga tgagccaagg 9120aagaatattg acggttgctc taaaagagtt ggacccggaa ttaaaagaat tagtagatta 9180caaggggacc tttctcaata gggaagcttt agaagcccta agcttaggta agccaatcaa 9240gaggaaaacc acaacagcaa tgatcaggag gttaatagag ccagaggttg aggaggaact 9300accagattgg ttccaagcgg aagaacccct atttttggaa gcaaaaatac agaatgactt 9360ataccaccta attggcagtg tagatagtat aaaaagcaaa gcaaaggaat taggggccac 9420agataacaca aagatagtga aggaagttgg ggctaggacc tatacgatga aattgagcag 9480ctggagcaca caagttacaa aaaaacagat gagtctagcc cctctctttg aagagctgtt 9540attaaagtgc cctccatgta gtaaaatttc aaagggacat atggtgtcag cataccaact 9600ggctcaagga aactgggaac ccctcgggtg tggggtctat atgggaacca taccagctag 9660gcgtctcaag atccaccctt atgaggctta ccttaaactc aaagagctgg tggaagttga 9720atcttcgagg gccactgcaa aagaatccat cataagagaa cataacacct ggatcctgcg 9780gaaggtgaga catgaaggga acctaagaac caaatcaatg atcaaccctg ggaaaatatc 9840agatcagcta tgcagagatg gacacaaaag aaacatatat aataagatca taggctcaac 9900aatggcctct gctggtatta ggctggagaa actgccagta gtccgagccc aaactgacac 9960aaccagtttc caccaagcca taagagaaaa aattgataaa acagaaaaca agcagacccc 10020tgaattgcat gaagaactaa tgaaggtctt cgactgctta aagatcccag agctgaagga 10080atcgtatgat gaagtttcat gggaacaatt agaagccggg ataaaccgta agggtgcagc 10140aggctatcta gagagcaaga acatagggga agtcctagac acagagaaac acatagtaga 10200gcagctgatc aaggatctga ggaaggggaa gaagattagg tactatgaaa cagccatccc 10260caagaatgag aagagagacg tcagcgacga ctgggaagcc ggagagttcg ttgatgaaaa 10320gaaaccaaga gtaatccagt acccggacgc caaggtgaga ctggccatta caaaagtgat 10380gtacaaatgg gtaaagcaaa aaccagtggt gatacccggc tatgaaggta aaacacctct 10440atttgacata ttcaacaaag tgaagaagga atgggattca ttccaggacc ccgtagcagt 10500gagctttgac accaaagcgt gggatacaca agtcaccagt agagacctaa tgttgataaa 10560ggatatccag aaatattatt tcaagagaag tatacacaaa tttttagata caataacaga 10620acacatggtg gaggtacctg tcattacagc agacggtgaa gtttacataa ggaatggtca 10680gaggggtagt ggccaacccg acacaagtgc tggtaatagt atgttgaatg tcctaaccat 10740gatatatgct ttctgtaaaa gtacaggcat accttacagg ggattcagca gagtggcaag 10800aatccatgtg tgtggtgatg atggcttttt gataacagag agaggactgg gactgaaatt 10860ctctgagaag ggtatgcaga tattacatga ggccgggaag ccccagaaaa taactgaagg 10920ggacaaaatg aaagtggcat acagattcga ggacatagag ttttgttccc atactcccgt 10980gccagtcaga tgggcagata acaccagtag ttacatggca gggaggagca cagccactat 11040actagctaag atggcaacca ggctggattc cagcggagag aggggtagca cagcttatga 11100gaaggccgta gccttcagct tccttttgat gtactcatgg aatcccgtag ttagaaggat 11160ctgcttactg gtgttgtcac agtttccaga aatatcccca tccaaaaaca caatatacta 11220ctaccaaggg gatcccatag ctgcgtacag agaagtgata gggaaacagc tgtgtgaact 11280gaaaagaaca ggatttgaga agctggctgg tctgaatttg agtatgacca ctctaggcat 11340ctggacaaaa catactagta aaagactaat ccaagcctgt gtagaaatag gtaagagaga 11400aggtacctgg ttagttaatg ctgacagact gattgcagga aagactggga agttttacat 11460cccaagcact ggtgtcactc tgttgggaaa acactatgag gaaattaact taaagcaaaa 11520ggcggcacaa ccgccgatag agggggttga cagatataag ttgggcccca tagttaatgt 11580tatcttgaga aggctgaggg tgatgctgat gacagttgcc agcggaagct ggtgaatccg 11640tccggagcgt cgtgccctca ctcaaggttt ttaattgtaa atattgtaaa tagacagcta 11700agatatttat tgtagttgga tagtaatgca gtgatagtaa ataccccaat ttaacactac 11760ctccaatgca ctaagcactt tagctgtgtg aggttaactc gacgtccacg gttggactag 11820ggaagacctc taacagcccc 11840312329DNAArtificial SequenceMutated BVDV XIKE-B 3gtatacgaga ttagctaaag aactcgtata tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga gttgttttca aatgaacttt tatacaaaac 420atataaacaa aaaccagcag gcgtcgtgga acctgtttac gacgtcaacg ggcgcccact 480gtttggagag agcagtgact tgcacccgca gtcaacacta aaactaccac accaacgagg 540cagcgccaac atcctgacca atgctaggtc cctaccgcgg aaaggtgact gccggagagg 600taatgtgtat ggaccggtga gtggcatcta tatcaaacca ggaccgatct actaccagga 660ttatgtgggc cccgtctatc atagagcccc actggaacta tgtagggagg caagtatgtg 720cgaaacaact aggagagttg gcagagtgac cggtagtgat gggaaattat atcatatcta 780catctgcata gatgggtgta tcctcctgaa gagggcgact aggaaccaac cagaagtcct 840gaaatgggta tacaacagat taaattgtcc tttatgggtc accagctgct ccgatgaagg 900gagcaagggt gctacaagta agaagcagcc taagccagat aggatagaaa aaggtaagat 960gaaaatagcc ccaaaagaga cagaaaaaga ttgcaaaacc agaccccccg acgcgactat 1020agtagtagaa ggggttaagt accaggtgaa gaaaaaagga aaggtaaggg gaaaaaatac 1080tcaagatggg ttatatcaca acaagaataa gccccctgaa tcaagaaaaa aattggaaaa 1140ggcactgctg gcttgggcca tcttagcagc ggtcctgctt cagctggtaa caggagagaa 1200tatcacccag tggaacttga tggacaacgg caccgaggga atacagcaag cgatgttcct 1260aagaggggtg aacaggagtc tacatggaat ttggccagag aaaatttgca ccggagtacc 1320aactcactta gcaacagact atgagcttaa agagatagtg gggatgatgg acgcgagtga 1380gaagaccaac tacacgtgtt gcaggttgca aagacatgag tggaataaag gttggtgtaa 1440ctggtttcat atagaaccgt ggatatggtt gatgaacaaa acccaaaaca acctgacaga 1500agggcaaccg cttagggagt gtgctgtgac ttgtaggtat gacaaggaaa cagaattgaa 1560catcgtgaca caggctaggg acagacctac aactctgaca ggttgcaaga aaggcaagaa 1620tttctctttc gcaggtgtta tactggatgg gccctgtaac tttaaagtat cggttgaaga 1680tgtgctgttc aaggagcacg attgcggcaa catgctgcaa gagaccgcga tacagctact 1740cgatggggca accaacacca ttgagggagc aagggtaggg acggccaagt tgacaacctg 1800gttagggaag caattaggga tccttggtaa gaagttggag aacaaaagca aagcatggtt 1860tggtgcacat gcagcaagtc catactgcgg agtggagagg aagatcggtt acgtatggta 1920tacaaaaaac tgcactccag cttgccttcc aagaaacact agaataatag gccccgggaa 1980atttgatacc aacgccgaag atggaaaaat actccatgag atgggggggc acctctcaga 2040atttgtccta ttgtccttgg tggttctgtc tgactttgcc ccggaaaccg cgagcgtcat 2100ctacttggtt ctacattttg cgatcccgca aagccacgtt gatgtagaca catgcgacaa 2160gaaccagctg aatttaacgg tagcaaccac agtagcagag gtcataccag ggacagtgtg 2220gaacctaggg aagtatgtct gcataagacc agactggtgg ccatatgaga cgacgacagt 2280cttcgtcata gaggaagcag ggcaagtaat caaattgatg ctaagggcca tcagagactt 2340aactaggata tggaatgctg ccactaccac agctttctta atctttttag taaaagcact 2400gaggggacaa ctaatccaag ggctattgtg gctgatgcta ataacaggag cacagggctt 2460ccctgaatgc aaagagggct tccaatatgc catatctaaa gacaggaaaa tggggttatt 2520ggggccagag agcttaacta caacatggca cctccccacc aaaaaaatag tggattccat 2580ggtgcatgta tggtgtgaag gaaaagactt gaaaatatta aaaatgtgca caaaggaaga 2640gaggtatcta gtggctgtgc acgagagagc cttatcaacc agtgccgagt ttatgcagat 2700cagtgatggg acaataggcc cagacgtgat agatatgcct gatgactttg agtttggact 2760ctgcccttgt gactcaaaac cagtgataaa gggcaaattt aatgccagct tactgaatgg 2820accagctttc cagatggtat gcccacaggg gtggactggt acaatagaat gcaccctagc 2880gaaccaagac accttggaca caactgtcat taggacatat agaagaacta ccccatttca 2940gcggagaaaa tggtgtacct atgaaaaaat aataggggaa gatatctatg aatgcattct 3000aggtggaaac tggacatgca taaccggtga ccatagcagg ttgaaagacg gacctatcaa 3060gaagtgtaag tggtgtggcc atgacttcgt caactcagag gggctaccac actacccaat 3120aggcaagtgc atgctcatca acgagagtgg gtacaggtat gtagatgaca cctcttgcga 3180taggggtggt gtagccatag ttccatctgg caccgtaaag tgtagaatag gtaacgtcac 3240ggtgcaagtt atcgctacta acaatgatct gggacccatg ccttgcagcc cagctgaagt 3300gatagcaagt gaaggaccag tggaaaagac tgcatgcaca ttcaactatt caaggactct 3360acctaataag tattatgagc caagggaccg gtacttccaa caatacatgt taaaagggga 3420gtggcaatat tggttcgacc tggattctgt agaccaccac aaagactact tctcagagtt 3480cataatcata gcagtggtcg ccttgttggg tggtaagtac gtactgtggc tcttgataac 3540atacacaata ctgtctgagc agatggctat gggtgctgga gtgaatactg aagagatagt 3600catgataggc aatttgctga cagacagtga tattgaggtt gtggtttatt tccttcttct 3660gtacttaata gttaaagagg aactggcgag gaaatggatt atactggtat accacatcct 3720tgtagccaac cctatgaaaa caattggggt cgtcttacta atgctagggg gagtggtgaa 3780ggccagcaga atcaatgctg atgaccaaag tgctatggac ccatgctttc ttctcgtgac 3840aggcgtagtg gctgttttga tgatcgctag aagagaacct gccacattac cactgattgt 3900agcattgcta gcaataagaa catcaggatt cctactgccc gctagcattg atgtaactgt 3960agcagtagta ttaattgtac ttttgttggc tagctacata acagactact ttagatataa 4020aaagtggctt caactcttat ttagtctgat agctggtatc tttattataa ggagcttaaa 4080acatatcaac cagatggagg taccagaaat atctatgcca agttggagac ctctagctct 4140ggtccttttc tatataacat ctacagcaat aaccactaat tgggacattg acttagcagg 4200cttcctgctg caatgggcgc cagcagtgat catgatggct accatgtggg cagacttttt 4260gactctgatc atagtcctgc ccagttacga gttatctaag ctttacttcc taaagaacgt 4320caggacagac gtggaaaaga actggctcgg caaagtgaaa tacagacaga tcagttcagt 4380ttatgacatc tgtgacagtg aggaagcagt gtacctattt ccatcaaggc ataagagtgg 4440aagcaggcca gatttcatat tacctttttt gaaagccgtg ttaataagct gcatcagcag 4500ccaatggcaa gtggtttaca tttcttacct aatactggaa attacatact atatgcacag 4560gaaaatcata gatgaggtgt caggaggagc aaattttcta tcaagactca tagcagccat 4620catagaatta aattgggcca tagatgatga ggaatgtaaa ggactgaaga aactgtatct 4680cttgtcaggg agagcgaaga atttgatagt taaacataag gtaagaaatg aagccgtcca 4740cagatggttt ggtgaggagg aaatatacgg ggcacccaag gtgatcacta tcataaaagc 4800tagtacccta agtaaaaaca ggcactgcat aatctgcacg atctgtgaag ggaaagaatg 4860gaatggagcc aactgcccaa agtgtggaag acaaggaaag cccataacat gtggaatgac 4920actcgcagac tttgaggaga aacattacaa aaagatattt ataagagaag aatcttcttg 4980tcctgtgcct tttgatcctt cttgccattg taattatttt cgccacgatg ggcctttcag 5040gaaagagtat aagggttacg tccaatacac agccagagga caactctttc tgaggaacct 5100accaattcta gcgacgaaga tgaagctatt aatggtggga aacctcggcg cagaaattgg 5160cgacctggaa catctaggat gggtactgag agggccagcc gtgtgcaaaa aaattaccaa 5220ccatgagaag tgccacgtaa acatcatgga taagctaact gcattttttg gaatcatgcc 5280tagaggcacg acccctaggg cacctgtgag gttccccaca gcactactaa aagtgagaag 5340ggggctagag acgggatggg cttacacgca ccaaggaggg atcagctcgg tagaccatgt 5400cacagccgga aaggatttac tagtgtgtga cagtatgggc aggaccaggg ttgtctgtca 5460tagtaacaat aagatgactg atgagactga gtatggcatc aagaccgact cagggtgtcc 5520cgaaggtgcg aggtgttacg tgctaaaccc agaagctgtt aacatttctg gcacaaaagg 5580agctatggta cacctccaga aaacgggggg ggagttcaca tgtgtcactg cctcagggac 5640cccggctttc ttcgatctga aaaatctaaa aggctggtcc gggctaccaa tttttgaagc 5700atccagtggc agggtggttg gtagggtgaa agtcggcaag aatgaggatt

ccaagcccac 5760caaactaatg agcggaatcc agacagtgtc taagaaccag acagacctag cggacatcgt 5820aaaaaaattg actagtatga acagaggaga gttcaaacag ataacattag ccactggggc 5880aggaaaaact acggaactgc caaggtccgt catagaggag atagggaggc acaaaagggt 5940cttagtcctg ataccattga gagcagcagc agagtcagtg tatcagtata tgagagtgaa 6000gtacccaagt atatctttca atttgagaat aggagatatg aaggaaggtg acatggccac 6060tggtatcacc tacgcctcat atgggtactt ttgtcagctt cctcagccca aactgagagc 6120tgccatggta gagtactcat atatattctt agatgagtac cactgtgcta cacccgagca 6180attagcaata attggaaaga tacacaggtt tgctgaaaat cttagagtgg tagcaatgac 6240agcaacccca gctggaacgg tcacaacgac tggtcagaaa caccctatag aggagttcat 6300agccccagag gtgatgaaag gtgaagatct aggtagtgaa tacttggata ttgcagggtt 6360gaagataccg actgaagaga tgaaaggcaa catgctcgtg ttcgcgccaa ctaggaacat 6420ggcagtagaa acagctaaga aattgaaggc taagggatac aactctggat actattacag 6480tggggaaaac ccagagaact tgagggtggt aacctcgcaa tccccgtatg tggtagtagc 6540caccaatgcc atagagtcag gtgtgacatt accagactta gacacagttg tagacactgg 6600actaaagtgt gagaagaggg tgaggatttc ttcaaaaatg cccttcattg taacaggact 6660taagagaatg gcagtcacaa tcggagagca agcccagcgc aggggtagag taggaagagt 6720caagccaggt aggtactata ggagtcaaga aacagcttca gggtcaaaag attaccatta 6780cgacctactg caagcccaga ggtacggaat agaagatgga attaatgtaa caaagtcatt 6840cagggagatg aactatgatt ggagccttta cgaagaggac agcttgatga taactcaact 6900cgaggtcctt aacaacctcc ttatatcaga agacctgcct gccgcagtga agaacatcat 6960ggcccggacc gatcacccag aacccataca actggcctat aacagttatg aaaaccaaat 7020tccagtgctg ttcccaaaga tcaaaaatgg tgaggtgaca gacagttatg agaattacac 7080atatctcaat gcaagaaaat taggagagga cgtgccggca tatgtgtacg ccacagagga 7140tgaggatcta gcagtggatc ttctgggtat ggattggccg gacccaggca accaacaggt 7200ggtagagaca gggagggcat taaaacaagt aactggctta tccacagcag aaaacgccct 7260cttgatagcc ctattcggct acgtcgggta ccagacactt tcaaaaaggc acatacccat 7320gattactgac atctatacac ttgaagacca caggcttgag gacacaaccc acctccagtt 7380tgccccaaac gctataagga ccgacggcaa ggactcagag ttgaaggaat tagctgtggg 7440agaccttgat aaatatgtgg acgcactggt agactactcc aaacaaggga tgaaattcat 7500caaagtccaa gctgaaaagg tcagagactc ccagtctacg aaggaaggct tgcaaaccat 7560taaggagtat gtggataagt ttatacaatc actaacagag aataaggagg agatcatcag 7620gtatggacta tggggagttc acacggcact ctacaaaagc ttggcagcga gactggggca 7680tgaaacagct tttgcaactt tagtggtaaa atggttggct tttgggggcg aaacggtatc 7740tgctcacatc aagcaagtag cagttgatct agtagtatat tatatcatca acaaaccatc 7800ttttcctgga gatacagaga cccaacaaga ggggaggaag tttgtggcta gtctttttat 7860atctgcacta gcaacataca catataaaac ctggaattac aacaatctgc aacgggttgt 7920cgaacctgcc ttagcttacc tcccatatgc tacaagtgcc ttgaagttgt tcacacccac 7980aagattagag agtgtggtca tactcagttc tacaatttac aagacatacc tctctataag 8040gaagggtaag agtgacggct tgttaggtac aggcataagt gcagccatgg agatcttaaa 8100ccaaaaccca atctcagtag gtatatctgt gatgctgggg gtaggtgcca tcgccgccca 8160taatgcaata gaatctagtg aacagaaaag aactttgctg atgaaggtct ttgtaaaaaa 8220cttcttagac caagcagcaa cagatgagct agtcaaagag aaccctgaaa aaataatcat 8280ggctctattt gaagcagtcc agaccatagg aaacccccta agactcatct accatctgta 8340cggggtgtac tataaggggt gggaagcaaa agaactcgca gagaaaactg ctggccgcaa 8400cttattcaca ttgatcatgt ttgaggcctt tgagctttta ggtatggact cagaaggaaa 8460gataagaaac ttgtcaggca actacatact ggacttaatc ttcaacttgc ataataaatt 8520aaacaagggg ctcaaaaaac tagtccttgg gtgggctcct gcacctttga gctgtgattg 8580gacaccaagt gatgagagaa taagcctacc tcataacaac tacttaaggg tagaaaccag 8640gtgtccttgt ggctatgaga tgaaggcaat aaaaaatgtt gctggtaaat tgacaaaagt 8700tgaagaaaag gggtccttcc tatgcaggaa tagattaggg agaggacctc caaacttcaa 8760agtaacaaag ttctatgatg ataacttgat agaagtcaag ccagtagcta ggctagaagg 8820ccaggtggac ctctattaca agggagtaac agctaagtta gactacaaca atgggaaagt 8880actgttagct accaacaagt gggaggtgga ccacgctttc ctgaccagac tagtaaagaa 8940gcacacaggg ataggtttta aaggtgcata tttgggtgac cgaccagacc atcaagatct 9000tgtcgataga gattgtgcaa ctataacgaa gaactcagta cagttcctaa aaatgaagaa 9060gggttgcgct ttcacatatg acctaacaat ctctaacctt gtcaggctta ttgaactagt 9120ccataagaat aatttacaag aaagagagat ccctaccgtg acagtaacta cttggcttgc 9180atattctttt gtcaatgaag acctggggac tatcaagcct gtattggggg agaaagtcat 9240cccagaaccc cccgaggagt tgagtctcca acccaccgtg agactagtca ccactgaaac 9300agcaataacc ataacagggg aggctgaagt gatgacgaca gggatcacac cagtggtaga 9360gatgaaagaa gaacctcagc tggaccacca gtcaactacc ctaaaggtag ggttgaagga 9420aggggaatat ccagggccag gagttaaccc taaccattta gcagaggtga tagatgagaa 9480agatgacagg ccttttgtcc taatcatcgg taacaaaggt tctacctcga acagagcaag 9540aacggccaag aatatacggc tgtacaaagg aaacaaccca agagagatca gggatctgat 9600gagccaagga agaatattga cggttgctct aaaagagttg gacccggaat taaaagaatt 9660agtagattac aaggggacct ttctcaatag ggaagcttta gaagccctaa gcttaggtaa 9720gccaatcaag aggaaaacca caacagcaat gatcaggagg ttaatagagc cagaggttga 9780ggaggaacta ccagattggt tccaagcgga agaaccccta tttttggaag caaaaataca 9840gaatgactta taccacctaa ttggcagtgt agatagtata aaaagcaaag caaaggaatt 9900aggggccaca gataacacaa agatagtgaa ggaagttggg gctaggacct atacgatgaa 9960attgagcagc tggagcacac aagttacaaa aaaacagatg agtctagccc ctctctttga 10020agagctgtta ttaaagtgcc ctccatgtag taaaatttca aagggacata tggtgtcagc 10080ataccaactg gctcaaggaa actgggaacc cctcgggtgt ggggtctata tgggaaccat 10140accagctagg cgtctcaaga tccaccctta tgaggcttac cttaaactca aagagctggt 10200ggaagttgaa tcttcgaggg ccactgcaaa agaatccatc ataagagaac ataacacctg 10260gatcctgcgg aaggtgagac atgaagggaa cctaagaacc aaatcaatga tcaaccctgg 10320gaaaatatca gatcagctat gcagagatgg acacaaaaga aacatatata ataagatcat 10380aggctcaaca atggcctctg ctggtattag gctggagaaa ctgccagtag tccgagccca 10440aactgacaca accagtttcc accaagccat aagagaaaaa attgataaaa cagaaaacaa 10500gcagacccct gaattgcatg aagaactaat gaaggtcttc gactgcttaa agatcccaga 10560gctgaaggaa tcgtatgatg aagtttcatg ggaacaatta gaagccggga taaaccgtaa 10620gggtgcagca ggctatctag agagcaagaa cataggggaa gtcctagaca cagagaaaca 10680catagtagag cagctgatca aggatctgag gaaggggaag aagattaggt actatgaaac 10740agccatcccc aagaatgaga agagagacgt cagcgacgac tgggaagccg gagagttcgt 10800tgatgaaaag aaaccaagag taatccagta cccggacgcc aaggtgagac tggccattac 10860aaaagtgatg tacaaatggg taaagcaaaa accagtggtg atacccggct atgaaggtaa 10920aacacctcta tttgacatat tcaacaaagt gaagaaggaa tgggattcat tccaggaccc 10980cgtagcagtg agctttgaca ccaaagcgtg ggatacacaa gtcaccagta gagacctaat 11040gttgataaag gatatccaga aatattattt caagagaagt atacacaaat ttttagatac 11100aataacagaa cacatggtgg aggtacctgt cattacagca gacggtgaag tttacataag 11160gaatggtcag aggggtagtg gccaacccga cacaagtgct ggtaatagta tgttgaatgt 11220cctaaccatg atatatgctt tctgtaaaag tacaggcata ccttacaggg gattcagcag 11280agtggcaaga atccatgtgt gtggtgatga tggctttttg ataacagaga gaggactggg 11340actgaaattc tctgagaagg gtatgcagat attacatgag gccgggaagc cccagaaaat 11400aactgaaggg gacaaaatga aagtggcata cagattcgag gacatagagt tttgttccca 11460tactcccgtg ccagtcagat gggcagataa caccagtagt tacatggcag ggaggagcac 11520agccactata ctagctaaga tggcaaccag gctggattcc agcggagaga ggggtagcac 11580agcttatgag aaggccgtag ccttcagctt ccttttgatg tactcatgga atcccgtagt 11640tagaaggatc tgcttactgg tgttgtcaca gtttccagaa atatccccat ccaaaaacac 11700aatatactac taccaagggg atcccatagc tgcgtacaga gaagtgatag ggaaacagct 11760gtgtgaactg aaaagaacag gatttgagaa gctggctggt ctgaatttga gtatgaccac 11820tctaggcatc tggacaaaac atactagtaa aagactaatc caagcctgtg tagaaatagg 11880taagagagaa ggtacctggt tagttaatgc tgacagactg attgcaggaa agactgggaa 11940gttttacatc ccaagcactg gtgtcactct gttgggaaaa cactatgagg aaattaactt 12000aaagcaaaag gcggcacaac cgccgataga gggggttgac agatataagt tgggccccat 12060agttaatgtt atcttgagaa ggctgagggt gatgctgatg acagttgcca gcggaagctg 12120gtgaatccgt ccggagcgtc gtgccctcac tcaaggtttt taattgtaaa tattgtaaat 12180agacagctaa gatatttatt gtagttggat agtaatgcag tgatagtaaa taccccaatt 12240taacactacc tccaatgcac taagcacttt agctgtgtga ggttaactcg acgtccacgg 12300ttggactagg gaagacctct aacagcccc 12329411837DNAArtificial SequenceMutated BVDV XIKE-B-NdN 4gtatacgaga ttagctaaag aactcgtata tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga gttgttttcc gatgaaggga gcaagggtgc 420tacaagtaag aagcagccta agccagatag gatagaaaaa ggtaagatga aaatagcccc 480aaaagagaca gaaaaagatt gcaaaaccag accccccgac gcgactatag tagtagaagg 540ggttaagtac caggtgaaga aaaaaggaaa ggtaagggga aaaaatactc aagatgggtt 600atatcacaac aagaataagc cccctgaatc aagaaaaaaa ttggaaaagg cactgctggc 660ttgggccatc ttagcagcgg tcctgcttca gctggtaaca ggagagaata tcacccagtg 720gaacttgatg gacaacggca ccgagggaat acagcaagcg atgttcctaa gaggggtgaa 780caggagtcta catggaattt ggccagagaa aatttgcacc ggagtaccaa ctcacttagc 840aacagactat gagcttaaag agatagtggg gatgatggac gcgagtgaga agaccaacta 900cacgtgttgc aggttgcaaa gacatgagtg gaataaaggt tggtgtaact ggtttcatat 960agaaccgtgg atatggttga tgaacaaaac ccaaaacaac ctgacagaag ggcaaccgct 1020tagggagtgt gctgtgactt gtaggtatga caaggaaaca gaattgaaca tcgtgacaca 1080ggctagggac agacctacaa ctctgacagg ttgcaagaaa ggcaagaatt tctctttcgc 1140aggtgttata ctggatgggc cctgtaactt taaagtatcg gttgaagatg tgctgttcaa 1200ggagcacgat tgcggcaaca tgctgcaaga gaccgcgata cagctactcg atggggcaac 1260caacaccatt gagggagcaa gggtagggac ggccaagttg acaacctggt tagggaagca 1320attagggatc cttggtaaga agttggagaa caaaagcaaa gcatggtttg gtgcacatgc 1380agcaagtcca tactgcggag tggagaggaa gatcggttac gtatggtata caaaaaactg 1440cactccagct tgccttccaa gaaacactag aataataggc cccgggaaat ttgataccaa 1500cgccgaagat ggaaaaatac tccatgagat gggggggcac ctctcagaat ttgtcctatt 1560gtccttggtg gttctgtctg actttgcccc ggaaaccgcg agcgtcatct acttggttct 1620acattttgcg atcccgcaaa gccacgttga tgtagacaca tgcgacaaga accagctgaa 1680tttaacggta gcaaccacag tagcagaggt cataccaggg acagtgtgga acctagggaa 1740gtatgtctgc ataagaccag actggtggcc atatgagacg acgacagtct tcgtcataga 1800ggaagcaggg caagtaatca aattgatgct aagggccatc agagacttaa ctaggatatg 1860gaatgctgcc actaccacag ctttcttaat ctttttagta aaagcactga ggggacaact 1920aatccaaggg ctattgtggc tgatgctaat aacaggagca cagggcttcc ctgaatgcaa 1980agagggcttc caatatgcca tatctaaaga caggaaaatg gggttattgg ggccagagag 2040cttaactaca acatggcacc tccccaccaa aaaaatagtg gattccatgg tgcatgtatg 2100gtgtgaagga aaagacttga aaatattaaa aatgtgcaca aaggaagaga ggtatctagt 2160ggctgtgcac gagagagcct tatcaaccag tgccgagttt atgcagatca gtgatgggac 2220aataggccca gacgtgatag atatgcctga tgactttgag tttggactct gcccttgtga 2280ctcaaaacca gtgataaagg gcaaatttaa tgccagctta ctgaatggac cagctttcca 2340gatggtatgc ccacaggggt ggactggtac aatagaatgc accctagcga accaagacac 2400cttggacaca actgtcatta ggacatatag aagaactacc ccatttcagc ggagaaaatg 2460gtgtacctat gaaaaaataa taggggaaga tatctatgaa tgcattctag gtggaaactg 2520gacatgcata accggtgacc atagcaggtt gaaagacgga cctatcaaga agtgtaagtg 2580gtgtggccat gacttcgtca actcagaggg gctaccacac tacccaatag gcaagtgcat 2640gctcatcaac gagagtgggt acaggtatgt agatgacacc tcttgcgata ggggtggtgt 2700agccatagtt ccatctggca ccgtaaagtg tagaataggt aacgtcacgg tgcaagttat 2760cgctactaac aatgatctgg gacccatgcc ttgcagccca gctgaagtga tagcaagtga 2820aggaccagtg gaaaagactg catgcacatt caactattca aggactctac ctaataagta 2880ttatgagcca agggaccggt acttccaaca atacatgtta aaaggggagt ggcaatattg 2940gttcgacctg gattctgtag accaccacaa agactacttc tcagagttca taatcatagc 3000agtggtcgcc ttgttgggtg gtaagtacgt actgtggctc ttgataacat acacaatact 3060gtctgagcag atggctatgg gtgctggagt gaatactgaa gagatagtca tgataggcaa 3120tttgctgaca gacagtgata ttgaggttgt ggtttatttc cttcttctgt acttaatagt 3180taaagaggaa ctggcgagga aatggattat actggtatac cacatccttg tagccaaccc 3240tatgaaaaca attggggtcg tcttactaat gctaggggga gtggtgaagg ccagcagaat 3300caatgctgat gaccaaagtg ctatggaccc atgctttctt ctcgtgacag gcgtagtggc 3360tgttttgatg atcgctagaa gagaacctgc cacattacca ctgattgtag cattgctagc 3420aataagaaca tcaggattcc tactgcccgc tagcattgat gtaactgtag cagtagtatt 3480aattgtactt ttgttggcta gctacataac agactacttt agatataaaa agtggcttca 3540actcttattt agtctgatag ctggtatctt tattataagg agcttaaaac atatcaacca 3600gatggaggta ccagaaatat ctatgccaag ttggagacct ctagctctgg tccttttcta 3660tataacatct acagcaataa ccactaattg ggacattgac ttagcaggct tcctgctgca 3720atgggcgcca gcagtgatca tgatggctac catgtgggca gactttttga ctctgatcat 3780agtcctgccc agttacgagt tatctaagct ttacttccta aagaacgtca ggacagacgt 3840ggaaaagaac tggctcggca aagtgaaata cagacagatc agttcagttt atgacatctg 3900tgacagtgag gaagcagtgt acctatttcc atcaaggcat aagagtggaa gcaggccaga 3960tttcatatta ccttttttga aagccgtgtt aataagctgc atcagcagcc aatggcaagt 4020ggtttacatt tcttacctaa tactggaaat tacatactat atgcacagga aaatcataga 4080tgaggtgtca ggaggagcaa attttctatc aagactcata gcagccatca tagaattaaa 4140ttgggccata gatgatgagg aatgtaaagg actgaagaaa ctgtatctct tgtcagggag 4200agcgaagaat ttgatagtta aacataaggt aagaaatgaa gccgtccaca gatggtttgg 4260tgaggaggaa atatacgggg cacccaaggt gatcactatc ataaaagcta gtaccctaag 4320taaaaacagg cactgcataa tctgcacgat ctgtgaaggg aaagaatgga atggagccaa 4380ctgcccaaag tgtggaagac aaggaaagcc cataacatgt ggaatgacac tcgcagactt 4440tgaggagaaa cattacaaaa agatatttat aagagaagaa tcttcttgtc ctgtgccttt 4500tgatccttct tgccattgta attattttcg ccacgatggg cctttcagga aagagtataa 4560gggttacgtc caatacacag ccagaggaca actctttctg aggaacctac caattctagc 4620gacgaagatg aagctattaa tggtgggaaa cctcggcgca gaaattggcg acctggaaca 4680tctaggatgg gtactgagag ggccagccgt gtgcaaaaaa attaccaacc atgagaagtg 4740ccacgtaaac atcatggata agctaactgc attttttgga atcatgccta gaggcacgac 4800ccctagggca cctgtgaggt tccccacagc actactaaaa gtgagaaggg ggctagagac 4860gggatgggct tacacgcacc aaggagggat cagctcggta gaccatgtca cagccggaaa 4920ggatttacta gtgtgtgaca gtatgggcag gaccagggtt gtctgtcata gtaacaataa 4980gatgactgat gagactgagt atggcatcaa gaccgactca gggtgtcccg aaggtgcgag 5040gtgttacgtg ctaaacccag aagctgttaa catttctggc acaaaaggag ctatggtaca 5100cctccagaaa acgggggggg agttcacatg tgtcactgcc tcagggaccc cggctttctt 5160cgatctgaaa aatctaaaag gctggtccgg gctaccaatt tttgaagcat ccagtggcag 5220ggtggttggt agggtgaaag tcggcaagaa tgaggattcc aagcccacca aactaatgag 5280cggaatccag acagtgtcta agaaccagac agacctagcg gacatcgtaa aaaaattgac 5340tagtatgaac agaggagagt tcaaacagat aacattagcc actggggcag gaaaaactac 5400ggaactgcca aggtccgtca tagaggagat agggaggcac aaaagggtct tagtcctgat 5460accattgaga gcagcagcag agtcagtgta tcagtatatg agagtgaagt acccaagtat 5520atctttcaat ttgagaatag gagatatgaa ggaaggtgac atggccactg gtatcaccta 5580cgcctcatat gggtactttt gtcagcttcc tcagcccaaa ctgagagctg ccatggtaga 5640gtactcatat atattcttag atgagtacca ctgtgctaca cccgagcaat tagcaataat 5700tggaaagata cacaggtttg ctgaaaatct tagagtggta gcaatgacag caaccccagc 5760tggaacggtc acaacgactg gtcagaaaca ccctatagag gagttcatag ccccagaggt 5820gatgaaaggt gaagatctag gtagtgaata cttggatatt gcagggttga agataccgac 5880tgaagagatg aaaggcaaca tgctcgtgtt cgcgccaact aggaacatgg cagtagaaac 5940agctaagaaa ttgaaggcta agggatacaa ctctggatac tattacagtg gggaaaaccc 6000agagaacttg agggtggtaa cctcgcaatc cccgtatgtg gtagtagcca ccaatgccat 6060agagtcaggt gtgacattac cagacttaga cacagttgta gacactggac taaagtgtga 6120gaagagggtg aggatttctt caaaaatgcc cttcattgta acaggactta agagaatggc 6180agtcacaatc ggagagcaag cccagcgcag gggtagagta ggaagagtca agccaggtag 6240gtactatagg agtcaagaaa cagcttcagg gtcaaaagat taccattacg acctactgca 6300agcccagagg tacggaatag aagatggaat taatgtaaca aagtcattca gggagatgaa 6360ctatgattgg agcctttacg aagaggacag cttgatgata actcaactcg aggtccttaa 6420caacctcctt atatcagaag acctgcctgc cgcagtgaag aacatcatgg cccggaccga 6480tcacccagaa cccatacaac tggcctataa cagttatgaa aaccaaattc cagtgctgtt 6540cccaaagatc aaaaatggtg aggtgacaga cagttatgag aattacacat atctcaatgc 6600aagaaaatta ggagaggacg tgccggcata tgtgtacgcc acagaggatg aggatctagc 6660agtggatctt ctgggtatgg attggccgga cccaggcaac caacaggtgg tagagacagg 6720gagggcatta aaacaagtaa ctggcttatc cacagcagaa aacgccctct tgatagccct 6780attcggctac gtcgggtacc agacactttc aaaaaggcac atacccatga ttactgacat 6840ctatacactt gaagaccaca ggcttgagga cacaacccac ctccagtttg ccccaaacgc 6900tataaggacc gacggcaagg actcagagtt gaaggaatta gctgtgggag accttgataa 6960atatgtggac gcactggtag actactccaa acaagggatg aaattcatca aagtccaagc 7020tgaaaaggtc agagactccc agtctacgaa ggaaggcttg caaaccatta aggagtatgt 7080ggataagttt atacaatcac taacagagaa taaggaggag atcatcaggt atggactatg 7140gggagttcac acggcactct acaaaagctt ggcagcgaga ctggggcatg aaacagcttt 7200tgcaacttta gtggtaaaat ggttggcttt tgggggcgaa acggtatctg ctcacatcaa 7260gcaagtagca gttgatctag tagtatatta tatcatcaac aaaccatctt ttcctggaga 7320tacagagacc caacaagagg ggaggaagtt tgtggctagt ctttttatat ctgcactagc 7380aacatacaca tataaaacct ggaattacaa caatctgcaa cgggttgtcg aacctgcctt 7440agcttacctc ccatatgcta caagtgcctt gaagttgttc acacccacaa gattagagag 7500tgtggtcata ctcagttcta caatttacaa gacatacctc tctataagga agggtaagag 7560tgacggcttg ttaggtacag gcataagtgc agccatggag atcttaaacc aaaacccaat 7620ctcagtaggt atatctgtga tgctgggggt aggtgccatc gccgcccata atgcaataga 7680atctagtgaa cagaaaagaa ctttgctgat gaaggtcttt gtaaaaaact tcttagacca 7740agcagcaaca gatgagctag tcaaagagaa ccctgaaaaa ataatcatgg ctctatttga 7800agcagtccag accataggaa accccctaag actcatctac catctgtacg gggtgtacta 7860taaggggtgg gaagcaaaag aactcgcaga gaaaactgct ggccgcaact tattcacatt 7920gatcatgttt gaggcctttg agcttttagg tatggactca gaaggaaaga taagaaactt 7980gtcaggcaac tacatactgg acttaatctt caacttgcat aataaattaa acaaggggct 8040caaaaaacta gtccttgggt gggctcctgc acctttgagc tgtgattgga caccaagtga 8100tgagagaata agcctacctc ataacaacta cttaagggta gaaaccaggt gtccttgtgg 8160ctatgagatg aaggcaataa aaaatgttgc tggtaaattg acaaaagttg aagaaaaggg 8220gtccttccta tgcaggaata gattagggag aggacctcca aacttcaaag taacaaagtt 8280ctatgatgat aacttgatag aagtcaagcc agtagctagg ctagaaggcc aggtggacct 8340ctattacaag ggagtaacag ctaagttaga ctacaacaat gggaaagtac tgttagctac

8400caacaagtgg gaggtggacc acgctttcct gaccagacta gtaaagaagc acacagggat 8460aggttttaaa ggtgcatatt tgggtgaccg accagaccat caagatcttg tcgatagaga 8520ttgtgcaact ataacgaaga actcagtaca gttcctaaaa atgaagaagg gttgcgcttt 8580cacatatgac ctaacaatct ctaaccttgt caggcttatt gaactagtcc ataagaataa 8640tttacaagaa agagagatcc ctaccgtgac agtaactact tggcttgcat attcttttgt 8700caatgaagac ctggggacta tcaagcctgt attgggggag aaagtcatcc cagaaccccc 8760cgaggagttg agtctccaac ccaccgtgag actagtcacc actgaaacag caataaccat 8820aacaggggag gctgaagtga tgacgacagg gatcacacca gtggtagaga tgaaagaaga 8880acctcagctg gaccaccagt caactaccct aaaggtaggg ttgaaggaag gggaatatcc 8940agggccagga gttaacccta accatttagc agaggtgata gatgagaaag atgacaggcc 9000ttttgtccta atcatcggta acaaaggttc tacctcgaac agagcaagaa cggccaagaa 9060tatacggctg tacaaaggaa acaacccaag agagatcagg gatctgatga gccaaggaag 9120aatattgacg gttgctctaa aagagttgga cccggaatta aaagaattag tagattacaa 9180ggggaccttt ctcaataggg aagctttaga agccctaagc ttaggtaagc caatcaagag 9240gaaaaccaca acagcaatga tcaggaggtt aatagagcca gaggttgagg aggaactacc 9300agattggttc caagcggaag aacccctatt tttggaagca aaaatacaga atgacttata 9360ccacctaatt ggcagtgtag atagtataaa aagcaaagca aaggaattag gggccacaga 9420taacacaaag atagtgaagg aagttggggc taggacctat acgatgaaat tgagcagctg 9480gagcacacaa gttacaaaaa aacagatgag tctagcccct ctctttgaag agctgttatt 9540aaagtgccct ccatgtagta aaatttcaaa gggacatatg gtgtcagcat accaactggc 9600tcaaggaaac tgggaacccc tcgggtgtgg ggtctatatg ggaaccatac cagctaggcg 9660tctcaagatc cacccttatg aggcttacct taaactcaaa gagctggtgg aagttgaatc 9720ttcgagggcc actgcaaaag aatccatcat aagagaacat aacacctgga tcctgcggaa 9780ggtgagacat gaagggaacc taagaaccaa atcaatgatc aaccctggga aaatatcaga 9840tcagctatgc agagatggac acaaaagaaa catatataat aagatcatag gctcaacaat 9900ggcctctgct ggtattaggc tggagaaact gccagtagtc cgagcccaaa ctgacacaac 9960cagtttccac caagccataa gagaaaaaat tgataaaaca gaaaacaagc agacccctga 10020attgcatgaa gaactaatga aggtcttcga ctgcttaaag atcccagagc tgaaggaatc 10080gtatgatgaa gtttcatggg aacaattaga agccgggata aaccgtaagg gtgcagcagg 10140ctatctagag agcaagaaca taggggaagt cctagacaca gagaaacaca tagtagagca 10200gctgatcaag gatctgagga aggggaagaa gattaggtac tatgaaacag ccatccccaa 10260gaatgagaag agagacgtca gcgacgactg ggaagccgga gagttcgttg atgaaaagaa 10320accaagagta atccagtacc cggacgccaa ggtgagactg gccattacaa aagtgatgta 10380caaatgggta aagcaaaaac cagtggtgat acccggctat gaaggtaaaa cacctctatt 10440tgacatattc aacaaagtga agaaggaatg ggattcattc caggaccccg tagcagtgag 10500ctttgacacc aaagcgtggg atacacaagt caccagtaga gacctaatgt tgataaagga 10560tatccagaaa tattatttca agagaagtat acacaaattt ttagatacaa taacagaaca 10620catggtggag gtacctgtca ttacagcaga cggtgaagtt tacataagga atggtcagag 10680gggtagtggc caacccgaca caagtgctgg taatagtatg ttgaatgtcc taaccatgat 10740atatgctttc tgtaaaagta caggcatacc ttacagggga ttcagcagag tggcaagaat 10800ccatgtgtgt ggtgatgatg gctttttgat aacagagaga ggactgggac tgaaattctc 10860tgagaagggt atgcagatat tacatgaggc cgggaagccc cagaaaataa ctgaagggga 10920caaaatgaaa gtggcataca gattcgagga catagagttt tgttcccata ctcccgtgcc 10980agtcagatgg gcagataaca ccagtagtta catggcaggg aggagcacag ccactatact 11040agctaagatg gcaaccaggc tggattccag cggagagagg ggtagcacag cttatgagaa 11100ggccgtagcc ttcagcttcc ttttgatgta ctcatggaat cccgtagtta gaaggatctg 11160cttactggtg ttgtcacagt ttccagaaat atccccatcc aaaaacacaa tatactacta 11220ccaaggggat cccatagctg cgtacagaga agtgataggg aaacagctgt gtgaactgaa 11280aagaacagga tttgagaagc tggctggtct gaatttgagt atgaccactc taggcatctg 11340gacaaaacat actagtaaaa gactaatcca agcctgtgta gaaataggta agagagaagg 11400tacctggtta gttaatgctg acagactgat tgcaggaaag actgggaagt tttacatccc 11460aagcactggt gtcactctgt tgggaaaaca ctatgaggaa attaacttaa agcaaaaggc 11520ggcacaaccg ccgatagagg gggttgacag atataagttg ggccccatag ttaatgttat 11580cttgagaagg ctgagggtga tgctgatgac agttgccagc ggaagctggt gaatccgtcc 11640ggagcgtcgt gccctcactc aaggttttta attgtaaata ttgtaaatag acagctaaga 11700tatttattgt agttggatag taatgcagtg atagtaaata ccccaattta acactacctc 11760caatgcacta agcactttag ctgtgtgagg ttaactcgac gtccacggtt ggactaggga 11820agacctctaa cagcccc 1183753913PRTWildtyp BVDV XIKE-A 5Met Glu Leu Phe Ser Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys1 5 10 15Pro Ala Gly Val Val Glu Pro Val Tyr Asp Val Asn Gly Arg Pro Leu 20 25 30Phe Gly Glu Ser Ser Asp Leu His Pro Gln Ser Thr Leu Lys Leu Pro 35 40 45His Gln Arg Gly Ser Ala Asn Ile Leu Thr Asn Ala Arg Ser Leu Pro 50 55 60Arg Lys Gly Asp Cys Arg Arg Gly Asn Val Tyr Gly Pro Val Ser Gly65 70 75 80Ile Tyr Ile Lys Pro Gly Pro Ile Tyr Tyr Gln Asp Tyr Val Gly Pro 85 90 95Val Tyr His Arg Ala Pro Leu Glu Leu Cys Arg Glu Ala Ser Met Cys 100 105 110Glu Thr Thr Arg Arg Val Gly Arg Val Thr Gly Ser Asp Gly Lys Leu 115 120 125Tyr His Ile Tyr Ile Cys Ile Asp Gly Cys Ile Leu Leu Lys Arg Ala 130 135 140Thr Arg Asn Gln Pro Glu Val Leu Lys Trp Val Tyr Asn Arg Leu Asn145 150 155 160Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Glu Gly Ser Lys Gly Ala 165 170 175Thr Ser Lys Lys Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met 180 185 190Lys Ile Ala Pro Lys Glu Thr Glu Lys Asp Cys Lys Thr Arg Pro Pro 195 200 205Asp Ala Thr Ile Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys 210 215 220Gly Lys Val Arg Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys225 230 235 240Asn Lys Pro Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala 245 250 255Trp Ala Ile Leu Ala Ala Val Leu Leu Gln Leu Val Thr Gly Glu Asn 260 265 270Ile Thr Gln Trp Asn Leu Met Asp Asn Gly Thr Glu Gly Ile Gln Gln 275 280 285Ala Met Phe Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro 290 295 300Glu Lys Ile Cys Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu305 310 315 320Leu Lys Glu Ile Val Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr 325 330 335Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys 340 345 350Asn Trp Phe His Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln 355 360 365Asn Asn Leu Thr Glu Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys 370 375 380Arg Tyr Asp Lys Glu Thr Glu Leu Asn Ile Val Thr Gln Ala Arg Asp385 390 395 400Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe 405 410 415Ala Gly Val Ile Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu 420 425 430Asp Val Leu Phe Lys Glu His Asp Cys Gly Asn Met Leu Gln Glu Thr 435 440 445Ala Ile Gln Leu Leu Asp Gly Ala Thr Asn Thr Ile Glu Gly Ala Arg 450 455 460Val Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile465 470 475 480Leu Gly Lys Lys Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His 485 490 495Ala Ala Ser Pro Tyr Cys Gly Val Glu Arg Lys Ile Gly Tyr Val Trp 500 505 510Tyr Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Arg Asn Thr Arg Ile 515 520 525Ile Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu 530 535 540His Glu Met Gly Gly His Leu Ser Glu Phe Val Leu Leu Ser Leu Val545 550 555 560Val Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser Val Ile Tyr Leu Val 565 570 575Leu His Phe Ala Ile Pro Gln Ser His Val Asp Val Asp Thr Cys Asp 580 585 590Lys Asn Gln Leu Asn Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile 595 600 605Pro Gly Thr Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp 610 615 620Trp Trp Pro Tyr Glu Thr Thr Thr Val Phe Val Ile Glu Glu Ala Gly625 630 635 640Gln Val Ile Lys Leu Met Leu Arg Ala Ile Arg Asp Leu Thr Arg Ile 645 650 655Trp Asn Ala Ala Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala 660 665 670Leu Arg Gly Gln Leu Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr 675 680 685Gly Ala Gln Gly Phe Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile 690 695 700Ser Lys Asp Arg Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr705 710 715 720Thr Trp His Leu Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val 725 730 735Trp Cys Glu Gly Lys Asp Leu Lys Ile Leu Lys Met Cys Thr Lys Glu 740 745 750Glu Arg Tyr Leu Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala 755 760 765Glu Phe Met Gln Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp 770 775 780Met Pro Asp Asp Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro785 790 795 800Val Ile Lys Gly Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe 805 810 815Gln Met Val Cys Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Thr Leu 820 825 830Ala Asn Gln Asp Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg 835 840 845Thr Thr Pro Phe Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile 850 855 860Gly Glu Asp Ile Tyr Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile865 870 875 880Thr Gly Asp His Ser Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys 885 890 895Trp Cys Gly His Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro 900 905 910Ile Gly Lys Cys Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp 915 920 925Asp Thr Ser Cys Asp Arg Gly Gly Val Ala Ile Val Pro Ser Gly Thr 930 935 940Val Lys Cys Arg Ile Gly Asn Val Thr Val Gln Val Ile Ala Thr Asn945 950 955 960Asn Asp Leu Gly Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser 965 970 975Glu Gly Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr 980 985 990Leu Pro Asn Lys Tyr Tyr Glu Pro Arg Asp Arg Tyr Phe Gln Gln Tyr 995 1000 1005Met Leu Lys Gly Glu Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val 1010 1015 1020Asp His His Lys Asp Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val 1025 1030 1035Val Ala Leu Leu Gly Gly Lys Tyr Val Leu Trp Leu Leu Ile Thr 1040 1045 1050Tyr Thr Ile Leu Ser Glu Gln Met Ala Met Gly Ala Gly Val Asn 1055 1060 1065Thr Glu Glu Ile Val Met Ile Gly Asn Leu Leu Thr Asp Ser Asp 1070 1075 1080Ile Glu Val Val Val Tyr Phe Leu Leu Leu Tyr Leu Ile Val Lys 1085 1090 1095Glu Glu Leu Ala Arg Lys Trp Ile Ile Leu Val Tyr His Ile Leu 1100 1105 1110Val Ala Asn Pro Met Lys Thr Ile Gly Val Val Leu Leu Met Leu 1115 1120 1125Gly Gly Val Val Lys Ala Ser Arg Ile Asn Ala Asp Asp Gln Ser 1130 1135 1140Ala Met Asp Pro Cys Phe Leu Leu Val Thr Gly Val Val Ala Val 1145 1150 1155Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu Ile Val 1160 1165 1170Ala Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala Ser 1175 1180 1185Ile Asp Val Thr Val Ala Val Val Leu Ile Val Leu Leu Leu Ala 1190 1195 1200Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu Gln Leu 1205 1210 1215Leu Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu Lys 1220 1225 1230His Ile Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser Trp 1235 1240 1245Arg Pro Leu Ala Leu Val Leu Phe Tyr Ile Thr Ser Thr Ala Ile 1250 1255 1260Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu Leu Gln Trp 1265 1270 1275Ala Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe Leu 1280 1285 1290Thr Leu Ile Ile Val Leu Pro Ser Tyr Glu Leu Ser Lys Leu Tyr 1295 1300 1305Phe Leu Lys Asn Val Arg Thr Asp Val Glu Lys Asn Trp Leu Gly 1310 1315 1320Lys Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys Asp 1325 1330 1335Ser Glu Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser Gly 1340 1345 1350Ser Arg Pro Asp Phe Ile Leu Pro Phe Leu Lys Ala Val Leu Ile 1355 1360 1365Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile Ser Tyr Leu 1370 1375 1380Ile Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp Glu 1385 1390 1395Val Ser Gly Gly Ala Asn Phe Leu Ser Arg Leu Ile Ala Ala Ile 1400 1405 1410Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu Glu Cys Lys Gly Leu 1415 1420 1425Lys Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile Val 1430 1435 1440Lys His Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly Glu 1445 1450 1455Glu Glu Ile Tyr Gly Ala Pro Lys Val Ile Thr Ile Ile Lys Ala 1460 1465 1470Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys Thr Ile Cys 1475 1480 1485Glu Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly Arg 1490 1495 1500Gln Gly Lys Pro Ile Thr Cys Gly Met Thr Leu Ala Asp Phe Glu 1505 1510 1515Glu Lys His Tyr Lys Lys Ile Phe Ile Arg Glu Glu Ser Ser Cys 1520 1525 1530Pro Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg His 1535 1540 1545Asp Gly Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr Thr 1550 1555 1560Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala Thr 1565 1570 1575Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala Glu Ile Gly 1580 1585 1590Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys 1595 1600 1605Lys Lys Ile Thr Asn His Glu Lys Cys His Val Asn Ile Met Asp 1610 1615 1620Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr Pro 1625 1630 1635Arg Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg Arg 1640 1645 1650Gly Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile Ser 1655 1660 1665Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys Asp 1670 1675 1680Ser Met Gly Arg Thr Arg Val Val Cys His Ser Asn Asn Lys Met 1685 1690 1695Thr Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys Pro 1700 1705 1710Glu Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn Ile 1715 1720 1725Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly Gly 1730 1735 1740Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp 1745 1750 1755Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala 1760 1765 1770Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn Glu 1775 1780 1785Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val Ser 1790 1795 1800Lys Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr Ser 1805 1810 1815Met Asn Arg Gly Glu Phe Lys Gln Ile Thr Leu Ala Thr Gly Ala 1820 1825 1830Gly Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile Gly 1835 1840 1845Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala Ala 1850 1855 1860Glu Ser Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile Ser 1865 1870 1875Phe Asn Leu Arg Ile Gly Asp Met Lys Glu

Gly Asp Met Ala Thr 1880 1885 1890Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Leu Pro Gln 1895 1900 1905Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe Leu 1910 1915 1920Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile Gly 1925 1930 1935Lys Ile His Arg Phe Ala Glu Asn Leu Arg Val Val Ala Met Thr 1940 1945 1950Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His Pro 1955 1960 1965Ile Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp Leu 1970 1975 1980Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Thr Glu 1985 1990 1995Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr Arg Asn Met 2000 2005 2010Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn Ser 2015 2020 2025Gly Tyr Tyr Tyr Ser Gly Glu Asn Pro Glu Asn Leu Arg Val Val 2030 2035 2040Thr Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile Glu 2045 2050 2055Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr Gly 2060 2065 2070Leu Lys Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro Phe 2075 2080 2085Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu Gln 2090 2095 2100Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg Tyr 2105 2110 2115Tyr Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His Tyr 2120 2125 2130Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile Asn 2135 2140 2145Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu Tyr 2150 2155 2160Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn Asn 2165 2170 2175Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile Met 2180 2185 2190Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn Ser 2195 2200 2205Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile Lys Asn Gly 2210 2215 2220Glu Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala Arg 2225 2230 2235Lys Leu Gly Glu Asp Val Pro Ala Tyr Val Tyr Ala Thr Glu Asp 2240 2245 2250Glu Asp Leu Ala Val Asp Leu Leu Gly Met Asp Trp Pro Asp Pro 2255 2260 2265Gly Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln Val 2270 2275 2280Thr Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu Phe 2285 2290 2295Gly Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg His Ile Pro Met 2300 2305 2310Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu Glu Asp Thr 2315 2320 2325Thr His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly Lys 2330 2335 2340Asp Ser Glu Leu Lys Glu Leu Ala Val Gly Asp Leu Asp Lys Tyr 2345 2350 2355Val Asp Ala Leu Val Asp Tyr Ser Lys Gln Gly Met Lys Phe Ile 2360 2365 2370Lys Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys Glu 2375 2380 2385Gly Leu Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln Ser 2390 2395 2400Leu Thr Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp Gly 2405 2410 2415Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg Leu Gly His 2420 2425 2430Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe Gly 2435 2440 2445Gly Glu Thr Val Ser Ala His Ile Lys Gln Val Ala Val Asp Leu 2450 2455 2460Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser Phe Pro Gly Asp Thr 2465 2470 2475Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe Ile 2480 2485 2490Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn Asn 2495 2500 2505Leu Gln Arg Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr Ala 2510 2515 2520Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu Glu Ser Val 2525 2530 2535Val Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile Arg 2540 2545 2550Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala Ala 2555 2560 2565Met Glu Ile Leu Asn Gln Asn Pro Ile Ser Val Gly Ile Ser Val 2570 2575 2580Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu Ser 2585 2590 2595Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn 2600 2605 2610Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn Pro 2615 2620 2625Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile Gly 2630 2635 2640Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr Lys 2645 2650 2655Gly Trp Glu Ala Lys Glu Leu Ala Glu Lys Thr Ala Gly Arg Asn 2660 2665 2670Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly Met 2675 2680 2685Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile Leu 2690 2695 2700Asp Leu Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu Lys 2705 2710 2715Lys Leu Val Leu Gly Trp Ala Pro Ala Pro Leu Ser Cys Asp Trp 2720 2725 2730Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn Asn Tyr Leu 2735 2740 2745Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala Ile 2750 2755 2760Lys Asn Val Ala Gly Lys Leu Thr Lys Val Glu Glu Lys Gly Ser 2765 2770 2775Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly Pro Pro Asn Phe Lys 2780 2785 2790Val Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro Val 2795 2800 2805Ala Arg Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val Thr 2810 2815 2820Ala Lys Leu Asp Tyr Asn Asn Gly Lys Val Leu Leu Ala Thr Asn 2825 2830 2835Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu Val Lys Lys 2840 2845 2850His Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg Pro 2855 2860 2865Asp His Gln Asp Leu Val Asp Arg Asp Cys Ala Thr Ile Thr Lys 2870 2875 2880Asn Ser Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe Thr 2885 2890 2895Tyr Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu Val 2900 2905 2910His Lys Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr Val 2915 2920 2925Thr Thr Trp Leu Ala Tyr Ser Phe Val Asn Glu Asp Leu Gly Thr 2930 2935 2940Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu Pro Pro Glu 2945 2950 2955Glu Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu Thr 2960 2965 2970Ala Ile Thr Ile Thr Gly Glu Ala Glu Val Met Thr Thr Gly Ile 2975 2980 2985Thr Pro Val Val Glu Met Lys Glu Glu Pro Gln Leu Asp His Gln 2990 2995 3000Ser Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro Gly 3005 3010 3015Pro Gly Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu Lys 3020 3025 3030Asp Asp Arg Pro Phe Val Leu Ile Ile Gly Asn Lys Gly Ser Thr 3035 3040 3045Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu Tyr Lys Gly 3050 3055 3060Asn Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg Ile 3065 3070 3075Leu Thr Val Ala Leu Lys Glu Leu Asp Pro Glu Leu Lys Glu Leu 3080 3085 3090Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg Glu Ala Leu Glu Ala 3095 3100 3105Leu Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala Met 3110 3115 3120Ile Arg Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro Asp 3125 3130 3135Trp Phe Gln Ala Glu Glu Pro Leu Phe Leu Glu Ala Lys Ile Gln 3140 3145 3150Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser Ile Lys Ser 3155 3160 3165Lys Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val Lys 3170 3175 3180Glu Val Gly Ala Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp Ser 3185 3190 3195Thr Gln Val Thr Lys Lys Gln Met Ser Leu Ala Pro Leu Phe Glu 3200 3205 3210Glu Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys Gly 3215 3220 3225His Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro 3230 3235 3240Leu Gly Cys Gly Val Tyr Met Gly Thr Ile Pro Ala Arg Arg Leu 3245 3250 3255Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Glu Leu Val 3260 3265 3270Glu Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile Arg 3275 3280 3285Glu His Asn Thr Trp Ile Leu Arg Lys Val Arg His Glu Gly Asn 3290 3295 3300Leu Arg Thr Lys Ser Met Ile Asn Pro Gly Lys Ile Ser Asp Gln 3305 3310 3315Leu Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile Ile 3320 3325 3330Gly Ser Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro 3335 3340 3345Val Val Arg Ala Gln Thr Asp Thr Thr Ser Phe His Gln Ala Ile 3350 3355 3360Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr Pro Glu Leu 3365 3370 3375His Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro Glu 3380 3385 3390Leu Lys Glu Ser Tyr Asp Glu Val Ser Trp Glu Gln Leu Glu Ala 3395 3400 3405Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr Leu Glu Ser Lys Asn 3410 3415 3420Ile Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln Leu 3425 3430 3435Ile Lys Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu Thr 3440 3445 3450Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Glu 3455 3460 3465Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr 3470 3475 3480Pro Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr Lys 3485 3490 3495Trp Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr Glu Gly Lys 3500 3505 3510Thr Pro Leu Phe Asp Ile Phe Asn Lys Val Lys Lys Glu Trp Asp 3515 3520 3525Ser Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp 3530 3535 3540Asp Thr Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp Ile 3545 3550 3555Gln Lys Tyr Tyr Phe Lys Arg Ser Ile His Lys Phe Leu Asp Thr 3560 3565 3570Ile Thr Glu His Met Val Glu Val Pro Val Ile Thr Ala Asp Gly 3575 3580 3585Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp 3590 3595 3600Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile Tyr 3605 3610 3615Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr Arg Gly Phe Ser Arg 3620 3625 3630Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr 3635 3640 3645Glu Arg Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln Ile 3650 3655 3660Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp Lys 3665 3670 3675Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser His 3680 3685 3690Thr Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr Met 3695 3700 3705Ala Gly Arg Ser Thr Ala Thr Ile Leu Ala Lys Met Ala Thr Arg 3710 3715 3720Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr Ala Tyr Glu Lys Ala 3725 3730 3735Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val Val 3740 3745 3750Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile Ser 3755 3760 3765Pro Ser Lys Asn Thr Ile Tyr Tyr Tyr Gln Gly Asp Pro Ile Ala 3770 3775 3780Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu Leu Lys Arg 3785 3790 3795Thr Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr Thr 3800 3805 3810Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Leu Ile Gln Ala 3815 3820 3825Cys Val Glu Ile Gly Lys Arg Glu Gly Thr Trp Leu Val Asn Ala 3830 3835 3840Asp Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro Ser 3845 3850 3855Thr Gly Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn Leu 3860 3865 3870Lys Gln Lys Ala Ala Gln Pro Pro Ile Glu Gly Val Asp Arg Tyr 3875 3880 3885Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg Leu Arg Val 3890 3895 3900Met Leu Met Thr Val Ala Ser Gly Ser Trp 3905 391063749PRTArtificial SequenceMutated BVDV XIKE-A-NdN 6Met Glu Leu Phe Ser Asp Glu Gly Ser Lys Gly Ala Thr Ser Lys Lys1 5 10 15Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met Lys Ile Ala Pro 20 25 30Lys Glu Thr Glu Lys Asp Cys Lys Thr Arg Pro Pro Asp Ala Thr Ile 35 40 45Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys Gly Lys Val Arg 50 55 60Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro Pro65 70 75 80Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Ile Leu 85 90 95Ala Ala Val Leu Leu Gln Leu Val Thr Gly Glu Asn Ile Thr Gln Trp 100 105 110Asn Leu Met Asp Asn Gly Thr Glu Gly Ile Gln Gln Ala Met Phe Leu 115 120 125Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile Cys 130 135 140Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu Leu Lys Glu Ile145 150 155 160Val Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr Thr Cys Cys Arg 165 170 175Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Phe His 180 185 190Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln Asn Asn Leu Thr 195 200 205Glu Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys 210 215 220Glu Thr Glu Leu Asn Ile Val Thr Gln Ala Arg Asp Arg Pro Thr Thr225 230 235 240Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Val Ile 245 250 255Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu Asp Val Leu Phe 260 265 270Lys Glu His Asp Cys Gly Asn Met Leu Gln Glu Thr Ala Ile Gln Leu 275 280 285Leu Asp Gly Ala Thr Asn Thr Ile Glu Gly Ala Arg Val Gly Thr Ala 290 295 300Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile Leu Gly Lys Lys305 310 315 320Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His Ala Ala Ser Pro 325 330 335Tyr Cys Gly Val Glu Arg Lys Ile Gly Tyr Val Trp Tyr Thr Lys Asn 340 345 350Cys Thr Pro Ala Cys Leu Pro Arg Asn Thr Arg Ile Ile Gly Pro Gly 355 360 365Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His Glu Met Gly 370 375 380Gly His Leu Ser Glu Phe Val Leu Leu Ser Leu Val Val Leu Ser Asp385 390 395 400Phe Ala Pro Glu Thr Ala Ser Val Ile Tyr Leu Val Leu His Phe

Ala 405 410 415Ile Pro Gln Ser His Val Asp Val Asp Thr Cys Asp Lys Asn Gln Leu 420 425 430Asn Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile Pro Gly Thr Val 435 440 445Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp Trp Trp Pro Tyr 450 455 460Glu Thr Thr Thr Val Phe Val Ile Glu Glu Ala Gly Gln Val Ile Lys465 470 475 480Leu Met Leu Arg Ala Ile Arg Asp Leu Thr Arg Ile Trp Asn Ala Ala 485 490 495Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala Leu Arg Gly Gln 500 505 510Leu Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr Gly Ala Gln Gly 515 520 525Phe Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile Ser Lys Asp Arg 530 535 540Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr Thr Trp His Leu545 550 555 560Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val Trp Cys Glu Gly 565 570 575Lys Asp Leu Lys Ile Leu Lys Met Cys Thr Lys Glu Glu Arg Tyr Leu 580 585 590Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala Glu Phe Met Gln 595 600 605Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp Met Pro Asp Asp 610 615 620Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Lys Gly625 630 635 640Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys 645 650 655Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Thr Leu Ala Asn Gln Asp 660 665 670Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg Thr Thr Pro Phe 675 680 685Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile 690 695 700Tyr Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile Thr Gly Asp His705 710 715 720Ser Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys Trp Cys Gly His 725 730 735Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys Cys 740 745 750Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys 755 760 765Asp Arg Gly Gly Val Ala Ile Val Pro Ser Gly Thr Val Lys Cys Arg 770 775 780Ile Gly Asn Val Thr Val Gln Val Ile Ala Thr Asn Asn Asp Leu Gly785 790 795 800Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu Gly Pro Val 805 810 815Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr Leu Pro Asn Lys 820 825 830Tyr Tyr Glu Pro Arg Asp Arg Tyr Phe Gln Gln Tyr Met Leu Lys Gly 835 840 845Glu Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val Asp His His Lys Asp 850 855 860Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val Val Ala Leu Leu Gly Gly865 870 875 880Lys Tyr Val Leu Trp Leu Leu Ile Thr Tyr Thr Ile Leu Ser Glu Gln 885 890 895Met Ala Met Gly Ala Gly Val Asn Thr Glu Glu Ile Val Met Ile Gly 900 905 910Asn Leu Leu Thr Asp Ser Asp Ile Glu Val Val Val Tyr Phe Leu Leu 915 920 925Leu Tyr Leu Ile Val Lys Glu Glu Leu Ala Arg Lys Trp Ile Ile Leu 930 935 940Val Tyr His Ile Leu Val Ala Asn Pro Met Lys Thr Ile Gly Val Val945 950 955 960Leu Leu Met Leu Gly Gly Val Val Lys Ala Ser Arg Ile Asn Ala Asp 965 970 975Asp Gln Ser Ala Met Asp Pro Cys Phe Leu Leu Val Thr Gly Val Val 980 985 990Ala Val Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu Ile 995 1000 1005Val Ala Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala 1010 1015 1020Ser Ile Asp Val Thr Val Ala Val Val Leu Ile Val Leu Leu Leu 1025 1030 1035Ala Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu Gln 1040 1045 1050Leu Leu Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu 1055 1060 1065Lys His Ile Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser 1070 1075 1080Trp Arg Pro Leu Ala Leu Val Leu Phe Tyr Ile Thr Ser Thr Ala 1085 1090 1095Ile Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu Leu Gln 1100 1105 1110Trp Ala Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe 1115 1120 1125Leu Thr Leu Ile Ile Val Leu Pro Ser Tyr Glu Leu Ser Lys Leu 1130 1135 1140Tyr Phe Leu Lys Asn Val Arg Thr Asp Val Glu Lys Asn Trp Leu 1145 1150 1155Gly Lys Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys 1160 1165 1170Asp Ser Glu Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser 1175 1180 1185Gly Ser Arg Pro Asp Phe Ile Leu Pro Phe Leu Lys Ala Val Leu 1190 1195 1200Ile Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile Ser Tyr 1205 1210 1215Leu Ile Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp 1220 1225 1230Glu Val Ser Gly Gly Ala Asn Phe Leu Ser Arg Leu Ile Ala Ala 1235 1240 1245Ile Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu Glu Cys Lys Gly 1250 1255 1260Leu Lys Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile 1265 1270 1275Val Lys His Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly 1280 1285 1290Glu Glu Glu Ile Tyr Gly Ala Pro Lys Val Ile Thr Ile Ile Lys 1295 1300 1305Ala Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys Thr Ile 1310 1315 1320Cys Glu Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly 1325 1330 1335Arg Gln Gly Lys Pro Ile Thr Cys Gly Met Thr Leu Ala Asp Phe 1340 1345 1350Glu Glu Lys His Tyr Lys Lys Ile Phe Ile Arg Glu Glu Ser Ser 1355 1360 1365Cys Pro Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg 1370 1375 1380His Asp Gly Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr 1385 1390 1395Thr Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala 1400 1405 1410Thr Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala Glu Ile 1415 1420 1425Gly Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val 1430 1435 1440Cys Lys Lys Ile Thr Asn His Glu Lys Cys His Val Asn Ile Met 1445 1450 1455Asp Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr 1460 1465 1470Pro Arg Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg 1475 1480 1485Arg Gly Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile 1490 1495 1500Ser Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys 1505 1510 1515Asp Ser Met Gly Arg Thr Arg Val Val Cys His Ser Asn Asn Lys 1520 1525 1530Met Thr Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys 1535 1540 1545Pro Glu Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn 1550 1555 1560Ile Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly 1565 1570 1575Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe 1580 1585 1590Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu 1595 1600 1605Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn 1610 1615 1620Glu Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val 1625 1630 1635Ser Lys Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr 1640 1645 1650Ser Met Asn Arg Gly Glu Phe Lys Gln Ile Thr Leu Ala Thr Gly 1655 1660 1665Ala Gly Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile 1670 1675 1680Gly Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala 1685 1690 1695Ala Glu Ser Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile 1700 1705 1710Ser Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala 1715 1720 1725Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Leu Pro 1730 1735 1740Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe 1745 1750 1755Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile 1760 1765 1770Gly Lys Ile His Arg Phe Ala Glu Asn Leu Arg Val Val Ala Met 1775 1780 1785Thr Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His 1790 1795 1800Pro Ile Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp 1805 1810 1815Leu Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Thr 1820 1825 1830Glu Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr Arg Asn 1835 1840 1845Met Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn 1850 1855 1860Ser Gly Tyr Tyr Tyr Ser Gly Glu Asn Pro Glu Asn Leu Arg Val 1865 1870 1875Val Thr Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile 1880 1885 1890Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr 1895 1900 1905Gly Leu Lys Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro 1910 1915 1920Phe Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu 1925 1930 1935Gln Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg 1940 1945 1950Tyr Tyr Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His 1955 1960 1965Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile 1970 1975 1980Asn Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu 1985 1990 1995Tyr Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn 2000 2005 2010Asn Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile 2015 2020 2025Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn 2030 2035 2040Ser Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile Lys Asn 2045 2050 2055Gly Glu Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala 2060 2065 2070Arg Lys Leu Gly Glu Asp Val Pro Ala Tyr Val Tyr Ala Thr Glu 2075 2080 2085Asp Glu Asp Leu Ala Val Asp Leu Leu Gly Met Asp Trp Pro Asp 2090 2095 2100Pro Gly Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln 2105 2110 2115Val Thr Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu 2120 2125 2130Phe Gly Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg His Ile Pro 2135 2140 2145Met Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu Glu Asp 2150 2155 2160Thr Thr His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly 2165 2170 2175Lys Asp Ser Glu Leu Lys Glu Leu Ala Val Gly Asp Leu Asp Lys 2180 2185 2190Tyr Val Asp Ala Leu Val Asp Tyr Ser Lys Gln Gly Met Lys Phe 2195 2200 2205Ile Lys Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys 2210 2215 2220Glu Gly Leu Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln 2225 2230 2235Ser Leu Thr Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp 2240 2245 2250Gly Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg Leu Gly 2255 2260 2265His Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe 2270 2275 2280Gly Gly Glu Thr Val Ser Ala His Ile Lys Gln Val Ala Val Asp 2285 2290 2295Leu Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser Phe Pro Gly Asp 2300 2305 2310Thr Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe 2315 2320 2325Ile Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn 2330 2335 2340Asn Leu Gln Arg Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr 2345 2350 2355Ala Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu Glu Ser 2360 2365 2370Val Val Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile 2375 2380 2385Arg Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala 2390 2395 2400Ala Met Glu Ile Leu Asn Gln Asn Pro Ile Ser Val Gly Ile Ser 2405 2410 2415Val Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu 2420 2425 2430Ser Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys 2435 2440 2445Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn 2450 2455 2460Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile 2465 2470 2475Gly Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr 2480 2485 2490Lys Gly Trp Glu Ala Lys Glu Leu Ala Glu Lys Thr Ala Gly Arg 2495 2500 2505Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly 2510 2515 2520Met Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile 2525 2530 2535Leu Asp Leu Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu 2540 2545 2550Lys Lys Leu Val Leu Gly Trp Ala Pro Ala Pro Leu Ser Cys Asp 2555 2560 2565Trp Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn Asn Tyr 2570 2575 2580Leu Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala 2585 2590 2595Ile Lys Asn Val Ala Gly Lys Leu Thr Lys Val Glu Glu Lys Gly 2600 2605 2610Ser Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly Pro Pro Asn Phe 2615 2620 2625Lys Val Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro 2630 2635 2640Val Ala Arg Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val 2645 2650 2655Thr Ala Lys Leu Asp Tyr Asn Asn Gly Lys Val Leu Leu Ala Thr 2660 2665 2670Asn Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu Val Lys 2675 2680 2685Lys His Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg 2690 2695 2700Pro Asp His Gln Asp Leu Val Asp Arg Asp Cys Ala Thr Ile Thr 2705 2710 2715Lys Asn Ser Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe 2720 2725 2730Thr Tyr Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu 2735 2740 2745Val His Lys Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr 2750 2755 2760Val Thr Thr Trp Leu Ala Tyr Ser Phe Val Asn Glu Asp Leu Gly 2765 2770 2775Thr Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu Pro Pro 2780 2785 2790Glu Glu Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu 2795 2800 2805Thr Ala Ile Thr Ile Thr Gly Glu Ala Glu Val Met Thr Thr Gly 2810 2815 2820Ile Thr Pro Val Val Glu Met Lys Glu Glu Pro Gln Leu Asp His 2825 2830 2835Gln Ser Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro 2840 2845

2850Gly Pro Gly Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu 2855 2860 2865Lys Asp Asp Arg Pro Phe Val Leu Ile Ile Gly Asn Lys Gly Ser 2870 2875 2880Thr Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu Tyr Lys 2885 2890 2895Gly Asn Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg 2900 2905 2910Ile Leu Thr Val Ala Leu Lys Glu Leu Asp Pro Glu Leu Lys Glu 2915 2920 2925Leu Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg Glu Ala Leu Glu 2930 2935 2940Ala Leu Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala 2945 2950 2955Met Ile Arg Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro 2960 2965 2970Asp Trp Phe Gln Ala Glu Glu Pro Leu Phe Leu Glu Ala Lys Ile 2975 2980 2985Gln Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser Ile Lys 2990 2995 3000Ser Lys Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val 3005 3010 3015Lys Glu Val Gly Ala Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp 3020 3025 3030Ser Thr Gln Val Thr Lys Lys Gln Met Ser Leu Ala Pro Leu Phe 3035 3040 3045Glu Glu Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys 3050 3055 3060Gly His Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu 3065 3070 3075Pro Leu Gly Cys Gly Val Tyr Met Gly Thr Ile Pro Ala Arg Arg 3080 3085 3090Leu Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Glu Leu 3095 3100 3105Val Glu Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile 3110 3115 3120Arg Glu His Asn Thr Trp Ile Leu Arg Lys Val Arg His Glu Gly 3125 3130 3135Asn Leu Arg Thr Lys Ser Met Ile Asn Pro Gly Lys Ile Ser Asp 3140 3145 3150Gln Leu Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile 3155 3160 3165Ile Gly Ser Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu 3170 3175 3180Pro Val Val Arg Ala Gln Thr Asp Thr Thr Ser Phe His Gln Ala 3185 3190 3195Ile Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr Pro Glu 3200 3205 3210Leu His Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro 3215 3220 3225Glu Leu Lys Glu Ser Tyr Asp Glu Val Ser Trp Glu Gln Leu Glu 3230 3235 3240Ala Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr Leu Glu Ser Lys 3245 3250 3255Asn Ile Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln 3260 3265 3270Leu Ile Lys Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu 3275 3280 3285Thr Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp 3290 3295 3300Glu Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln 3305 3310 3315Tyr Pro Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr 3320 3325 3330Lys Trp Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr Glu Gly 3335 3340 3345Lys Thr Pro Leu Phe Asp Ile Phe Asn Lys Val Lys Lys Glu Trp 3350 3355 3360Asp Ser Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala 3365 3370 3375Trp Asp Thr Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp 3380 3385 3390Ile Gln Lys Tyr Tyr Phe Lys Arg Ser Ile His Lys Phe Leu Asp 3395 3400 3405Thr Ile Thr Glu His Met Val Glu Val Pro Val Ile Thr Ala Asp 3410 3415 3420Gly Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro 3425 3430 3435Asp Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile 3440 3445 3450Tyr Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr Arg Gly Phe Ser 3455 3460 3465Arg Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile 3470 3475 3480Thr Glu Arg Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln 3485 3490 3495Ile Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp 3500 3505 3510Lys Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser 3515 3520 3525His Thr Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr 3530 3535 3540Met Ala Gly Arg Ser Thr Ala Thr Ile Leu Ala Lys Met Ala Thr 3545 3550 3555Arg Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr Ala Tyr Glu Lys 3560 3565 3570Ala Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val 3575 3580 3585Val Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile 3590 3595 3600Ser Pro Ser Lys Asn Thr Ile Tyr Tyr Tyr Gln Gly Asp Pro Ile 3605 3610 3615Ala Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu Leu Lys 3620 3625 3630Arg Thr Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr 3635 3640 3645Thr Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Leu Ile Gln 3650 3655 3660Ala Cys Val Glu Ile Gly Lys Arg Glu Gly Thr Trp Leu Val Asn 3665 3670 3675Ala Asp Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro 3680 3685 3690Ser Thr Gly Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn 3695 3700 3705Leu Lys Gln Lys Ala Ala Gln Pro Pro Ile Glu Gly Val Asp Arg 3710 3715 3720Tyr Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg Leu Arg 3725 3730 3735Val Met Leu Met Thr Val Ala Ser Gly Ser Trp 3740 374573912PRTArtificial SequenceMutated BVDV XIKE-B 7Met Glu Leu Phe Ser Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys1 5 10 15Pro Ala Gly Val Val Glu Pro Val Tyr Asp Val Asn Gly Arg Pro Leu 20 25 30Phe Gly Glu Ser Ser Asp Leu His Pro Gln Ser Thr Leu Lys Leu Pro 35 40 45His Gln Arg Gly Ser Ala Asn Ile Leu Thr Asn Ala Arg Ser Leu Pro 50 55 60Arg Lys Gly Asp Cys Arg Arg Gly Asn Val Tyr Gly Pro Val Ser Gly65 70 75 80Ile Tyr Ile Lys Pro Gly Pro Ile Tyr Tyr Gln Asp Tyr Val Gly Pro 85 90 95Val Tyr His Arg Ala Pro Leu Glu Leu Cys Arg Glu Ala Ser Met Cys 100 105 110Glu Thr Thr Arg Arg Val Gly Arg Val Thr Gly Ser Asp Gly Lys Leu 115 120 125Tyr His Ile Tyr Ile Cys Ile Asp Gly Cys Ile Leu Leu Lys Arg Ala 130 135 140Thr Arg Asn Gln Pro Glu Val Leu Lys Trp Val Tyr Asn Arg Leu Asn145 150 155 160Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Glu Gly Ser Lys Gly Ala 165 170 175Thr Ser Lys Lys Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met 180 185 190Lys Ile Ala Pro Lys Glu Thr Glu Lys Asp Cys Lys Thr Arg Pro Pro 195 200 205Asp Ala Thr Ile Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys 210 215 220Gly Lys Val Arg Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys225 230 235 240Asn Lys Pro Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala 245 250 255Trp Ala Ile Leu Ala Ala Val Leu Leu Gln Leu Val Thr Gly Glu Asn 260 265 270Ile Thr Gln Trp Asn Leu Met Asp Asn Gly Thr Glu Gly Ile Gln Gln 275 280 285Ala Met Phe Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro 290 295 300Glu Lys Ile Cys Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu305 310 315 320Leu Lys Glu Ile Val Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr 325 330 335Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys Gly Trp Cys Asn 340 345 350Trp Phe His Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln Asn 355 360 365Asn Leu Thr Glu Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys Arg 370 375 380Tyr Asp Lys Glu Thr Glu Leu Asn Ile Val Thr Gln Ala Arg Asp Arg385 390 395 400Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala 405 410 415Gly Val Ile Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu Asp 420 425 430Val Leu Phe Lys Glu His Asp Cys Gly Asn Met Leu Gln Glu Thr Ala 435 440 445Ile Gln Leu Leu Asp Gly Ala Thr Asn Thr Ile Glu Gly Ala Arg Val 450 455 460Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile Leu465 470 475 480Gly Lys Lys Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His Ala 485 490 495Ala Ser Pro Tyr Cys Gly Val Glu Arg Lys Ile Gly Tyr Val Trp Tyr 500 505 510Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Arg Asn Thr Arg Ile Ile 515 520 525Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His 530 535 540Glu Met Gly Gly His Leu Ser Glu Phe Val Leu Leu Ser Leu Val Val545 550 555 560Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser Val Ile Tyr Leu Val Leu 565 570 575His Phe Ala Ile Pro Gln Ser His Val Asp Val Asp Thr Cys Asp Lys 580 585 590Asn Gln Leu Asn Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile Pro 595 600 605Gly Thr Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp Trp 610 615 620Trp Pro Tyr Glu Thr Thr Thr Val Phe Val Ile Glu Glu Ala Gly Gln625 630 635 640Val Ile Lys Leu Met Leu Arg Ala Ile Arg Asp Leu Thr Arg Ile Trp 645 650 655Asn Ala Ala Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala Leu 660 665 670Arg Gly Gln Leu Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr Gly 675 680 685Ala Gln Gly Phe Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile Ser 690 695 700Lys Asp Arg Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr Thr705 710 715 720Trp His Leu Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val Trp 725 730 735Cys Glu Gly Lys Asp Leu Lys Ile Leu Lys Met Cys Thr Lys Glu Glu 740 745 750Arg Tyr Leu Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala Glu 755 760 765Phe Met Gln Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp Met 770 775 780Pro Asp Asp Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val785 790 795 800Ile Lys Gly Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln 805 810 815Met Val Cys Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Thr Leu Ala 820 825 830Asn Gln Asp Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg Thr 835 840 845Thr Pro Phe Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly 850 855 860Glu Asp Ile Tyr Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile Thr865 870 875 880Gly Asp His Ser Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys Trp 885 890 895Cys Gly His Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro Ile 900 905 910Gly Lys Cys Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp 915 920 925Thr Ser Cys Asp Arg Gly Gly Val Ala Ile Val Pro Ser Gly Thr Val 930 935 940Lys Cys Arg Ile Gly Asn Val Thr Val Gln Val Ile Ala Thr Asn Asn945 950 955 960Asp Leu Gly Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu 965 970 975Gly Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr Leu 980 985 990Pro Asn Lys Tyr Tyr Glu Pro Arg Asp Arg Tyr Phe Gln Gln Tyr Met 995 1000 1005Leu Lys Gly Glu Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val Asp 1010 1015 1020His His Lys Asp Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val Val 1025 1030 1035Ala Leu Leu Gly Gly Lys Tyr Val Leu Trp Leu Leu Ile Thr Tyr 1040 1045 1050Thr Ile Leu Ser Glu Gln Met Ala Met Gly Ala Gly Val Asn Thr 1055 1060 1065Glu Glu Ile Val Met Ile Gly Asn Leu Leu Thr Asp Ser Asp Ile 1070 1075 1080Glu Val Val Val Tyr Phe Leu Leu Leu Tyr Leu Ile Val Lys Glu 1085 1090 1095Glu Leu Ala Arg Lys Trp Ile Ile Leu Val Tyr His Ile Leu Val 1100 1105 1110Ala Asn Pro Met Lys Thr Ile Gly Val Val Leu Leu Met Leu Gly 1115 1120 1125Gly Val Val Lys Ala Ser Arg Ile Asn Ala Asp Asp Gln Ser Ala 1130 1135 1140Met Asp Pro Cys Phe Leu Leu Val Thr Gly Val Val Ala Val Leu 1145 1150 1155Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu Ile Val Ala 1160 1165 1170Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala Ser Ile 1175 1180 1185Asp Val Thr Val Ala Val Val Leu Ile Val Leu Leu Leu Ala Ser 1190 1195 1200Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu Gln Leu Leu 1205 1210 1215Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu Lys His 1220 1225 1230Ile Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser Trp Arg 1235 1240 1245Pro Leu Ala Leu Val Leu Phe Tyr Ile Thr Ser Thr Ala Ile Thr 1250 1255 1260Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu Leu Gln Trp Ala 1265 1270 1275Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe Leu Thr 1280 1285 1290Leu Ile Ile Val Leu Pro Ser Tyr Glu Leu Ser Lys Leu Tyr Phe 1295 1300 1305Leu Lys Asn Val Arg Thr Asp Val Glu Lys Asn Trp Leu Gly Lys 1310 1315 1320Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys Asp Ser 1325 1330 1335Glu Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser Gly Ser 1340 1345 1350Arg Pro Asp Phe Ile Leu Pro Phe Leu Lys Ala Val Leu Ile Ser 1355 1360 1365Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile Ser Tyr Leu Ile 1370 1375 1380Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp Glu Val 1385 1390 1395Ser Gly Gly Ala Asn Phe Leu Ser Arg Leu Ile Ala Ala Ile Ile 1400 1405 1410Glu Leu Asn Trp Ala Ile Asp Asp Glu Glu Cys Lys Gly Leu Lys 1415 1420 1425Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile Val Lys 1430 1435 1440His Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly Glu Glu 1445 1450 1455Glu Ile Tyr Gly Ala Pro Lys Val Ile Thr Ile Ile Lys Ala Ser 1460 1465 1470Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys Thr Ile Cys Glu 1475 1480 1485Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly Arg Gln 1490 1495 1500Gly Lys Pro Ile Thr Cys Gly Met Thr Leu Ala Asp Phe Glu Glu 1505 1510 1515Lys His Tyr Lys Lys Ile Phe Ile Arg Glu Glu Ser Ser Cys Pro 1520 1525 1530Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg His Asp 1535 1540 1545Gly Pro

Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr Thr Ala 1550 1555 1560Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala Thr Lys 1565 1570 1575Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala Glu Ile Gly Asp 1580 1585 1590Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys Lys 1595 1600 1605Lys Ile Thr Asn His Glu Lys Cys His Val Asn Ile Met Asp Lys 1610 1615 1620Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr Pro Arg 1625 1630 1635Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg Arg Gly 1640 1645 1650Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile Ser Ser 1655 1660 1665Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys Asp Ser 1670 1675 1680Met Gly Arg Thr Arg Val Val Cys His Ser Asn Asn Lys Met Thr 1685 1690 1695Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys Pro Glu 1700 1705 1710Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn Ile Ser 1715 1720 1725Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly Gly Glu 1730 1735 1740Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp Leu 1745 1750 1755Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala Ser 1760 1765 1770Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn Glu Asp 1775 1780 1785Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val Ser Lys 1790 1795 1800Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr Ser Met 1805 1810 1815Asn Arg Gly Glu Phe Lys Gln Ile Thr Leu Ala Thr Gly Ala Gly 1820 1825 1830Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile Gly Arg 1835 1840 1845His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala Ala Glu 1850 1855 1860Ser Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile Ser Phe 1865 1870 1875Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala Thr Gly 1880 1885 1890Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Leu Pro Gln Pro 1895 1900 1905Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe Leu Asp 1910 1915 1920Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile Gly Lys 1925 1930 1935Ile His Arg Phe Ala Glu Asn Leu Arg Val Val Ala Met Thr Ala 1940 1945 1950Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His Pro Ile 1955 1960 1965Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp Leu Gly 1970 1975 1980Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Thr Glu Glu 1985 1990 1995Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr Arg Asn Met Ala 2000 2005 2010Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn Ser Gly 2015 2020 2025Tyr Tyr Tyr Ser Gly Glu Asn Pro Glu Asn Leu Arg Val Val Thr 2030 2035 2040Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile Glu Ser 2045 2050 2055Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr Gly Leu 2060 2065 2070Lys Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro Phe Ile 2075 2080 2085Val Thr Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu Gln Ala 2090 2095 2100Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg Tyr Tyr 2105 2110 2115Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His Tyr Asp 2120 2125 2130Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile Asn Val 2135 2140 2145Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu Tyr Glu 2150 2155 2160Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn Asn Leu 2165 2170 2175Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile Met Ala 2180 2185 2190Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn Ser Tyr 2195 2200 2205Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile Lys Asn Gly Glu 2210 2215 2220Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala Arg Lys 2225 2230 2235Leu Gly Glu Asp Val Pro Ala Tyr Val Tyr Ala Thr Glu Asp Glu 2240 2245 2250Asp Leu Ala Val Asp Leu Leu Gly Met Asp Trp Pro Asp Pro Gly 2255 2260 2265Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln Val Thr 2270 2275 2280Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu Phe Gly 2285 2290 2295Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg His Ile Pro Met Ile 2300 2305 2310Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu Glu Asp Thr Thr 2315 2320 2325His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly Lys Asp 2330 2335 2340Ser Glu Leu Lys Glu Leu Ala Val Gly Asp Leu Asp Lys Tyr Val 2345 2350 2355Asp Ala Leu Val Asp Tyr Ser Lys Gln Gly Met Lys Phe Ile Lys 2360 2365 2370Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys Glu Gly 2375 2380 2385Leu Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln Ser Leu 2390 2395 2400Thr Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp Gly Val 2405 2410 2415His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg Leu Gly His Glu 2420 2425 2430Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe Gly Gly 2435 2440 2445Glu Thr Val Ser Ala His Ile Lys Gln Val Ala Val Asp Leu Val 2450 2455 2460Val Tyr Tyr Ile Ile Asn Lys Pro Ser Phe Pro Gly Asp Thr Glu 2465 2470 2475Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe Ile Ser 2480 2485 2490Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn Asn Leu 2495 2500 2505Gln Arg Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr Ala Thr 2510 2515 2520Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu Glu Ser Val Val 2525 2530 2535Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile Arg Lys 2540 2545 2550Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala Ala Met 2555 2560 2565Glu Ile Leu Asn Gln Asn Pro Ile Ser Val Gly Ile Ser Val Met 2570 2575 2580Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu Ser Ser 2585 2590 2595Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn Phe 2600 2605 2610Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn Pro Glu 2615 2620 2625Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile Gly Asn 2630 2635 2640Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr Lys Gly 2645 2650 2655Trp Glu Ala Lys Glu Leu Ala Glu Lys Thr Ala Gly Arg Asn Leu 2660 2665 2670Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly Met Asp 2675 2680 2685Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile Leu Asp 2690 2695 2700Leu Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu Lys Lys 2705 2710 2715Leu Val Leu Gly Trp Ala Pro Ala Pro Leu Ser Cys Asp Trp Thr 2720 2725 2730Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn Asn Tyr Leu Arg 2735 2740 2745Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala Ile Lys 2750 2755 2760Asn Val Ala Gly Lys Leu Thr Lys Val Glu Glu Lys Gly Ser Phe 2765 2770 2775Leu Cys Arg Asn Arg Leu Gly Arg Gly Pro Pro Asn Phe Lys Val 2780 2785 2790Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro Val Ala 2795 2800 2805Arg Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val Thr Ala 2810 2815 2820Lys Leu Asp Tyr Asn Asn Gly Lys Val Leu Leu Ala Thr Asn Lys 2825 2830 2835Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu Val Lys Lys His 2840 2845 2850Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg Pro Asp 2855 2860 2865His Gln Asp Leu Val Asp Arg Asp Cys Ala Thr Ile Thr Lys Asn 2870 2875 2880Ser Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe Thr Tyr 2885 2890 2895Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu Val His 2900 2905 2910Lys Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr Val Thr 2915 2920 2925Thr Trp Leu Ala Tyr Ser Phe Val Asn Glu Asp Leu Gly Thr Ile 2930 2935 2940Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu Pro Pro Glu Glu 2945 2950 2955Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu Thr Ala 2960 2965 2970Ile Thr Ile Thr Gly Glu Ala Glu Val Met Thr Thr Gly Ile Thr 2975 2980 2985Pro Val Val Glu Met Lys Glu Glu Pro Gln Leu Asp His Gln Ser 2990 2995 3000Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro Gly Pro 3005 3010 3015Gly Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu Lys Asp 3020 3025 3030Asp Arg Pro Phe Val Leu Ile Ile Gly Asn Lys Gly Ser Thr Ser 3035 3040 3045Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu Tyr Lys Gly Asn 3050 3055 3060Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg Ile Leu 3065 3070 3075Thr Val Ala Leu Lys Glu Leu Asp Pro Glu Leu Lys Glu Leu Val 3080 3085 3090Asp Tyr Lys Gly Thr Phe Leu Asn Arg Glu Ala Leu Glu Ala Leu 3095 3100 3105Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala Met Ile 3110 3115 3120Arg Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro Asp Trp 3125 3130 3135Phe Gln Ala Glu Glu Pro Leu Phe Leu Glu Ala Lys Ile Gln Asn 3140 3145 3150Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser Ile Lys Ser Lys 3155 3160 3165Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val Lys Glu 3170 3175 3180Val Gly Ala Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp Ser Thr 3185 3190 3195Gln Val Thr Lys Lys Gln Met Ser Leu Ala Pro Leu Phe Glu Glu 3200 3205 3210Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys Gly His 3215 3220 3225Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro Leu 3230 3235 3240Gly Cys Gly Val Tyr Met Gly Thr Ile Pro Ala Arg Arg Leu Lys 3245 3250 3255Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Glu Leu Val Glu 3260 3265 3270Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile Arg Glu 3275 3280 3285His Asn Thr Trp Ile Leu Arg Lys Val Arg His Glu Gly Asn Leu 3290 3295 3300Arg Thr Lys Ser Met Ile Asn Pro Gly Lys Ile Ser Asp Gln Leu 3305 3310 3315Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile Ile Gly 3320 3325 3330Ser Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro Val 3335 3340 3345Val Arg Ala Gln Thr Asp Thr Thr Ser Phe His Gln Ala Ile Arg 3350 3355 3360Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr Pro Glu Leu His 3365 3370 3375Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro Glu Leu 3380 3385 3390Lys Glu Ser Tyr Asp Glu Val Ser Trp Glu Gln Leu Glu Ala Gly 3395 3400 3405Ile Asn Arg Lys Gly Ala Ala Gly Tyr Leu Glu Ser Lys Asn Ile 3410 3415 3420Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln Leu Ile 3425 3430 3435Lys Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu Thr Ala 3440 3445 3450Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Glu Ala 3455 3460 3465Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr Pro 3470 3475 3480Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr Lys Trp 3485 3490 3495Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr Glu Gly Lys Thr 3500 3505 3510Pro Leu Phe Asp Ile Phe Asn Lys Val Lys Lys Glu Trp Asp Ser 3515 3520 3525Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp Asp 3530 3535 3540Thr Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp Ile Gln 3545 3550 3555Lys Tyr Tyr Phe Lys Arg Ser Ile His Lys Phe Leu Asp Thr Ile 3560 3565 3570Thr Glu His Met Val Glu Val Pro Val Ile Thr Ala Asp Gly Glu 3575 3580 3585Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp Thr 3590 3595 3600Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile Tyr Ala 3605 3610 3615Phe Cys Lys Ser Thr Gly Ile Pro Tyr Arg Gly Phe Ser Arg Val 3620 3625 3630Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr Glu 3635 3640 3645Arg Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln Ile Leu 3650 3655 3660His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp Lys Met 3665 3670 3675Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser His Thr 3680 3685 3690Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr Met Ala 3695 3700 3705Gly Arg Ser Thr Ala Thr Ile Leu Ala Lys Met Ala Thr Arg Leu 3710 3715 3720Asp Ser Ser Gly Glu Arg Gly Ser Thr Ala Tyr Glu Lys Ala Val 3725 3730 3735Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val Val Arg 3740 3745 3750Arg Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile Ser Pro 3755 3760 3765Ser Lys Asn Thr Ile Tyr Tyr Tyr Gln Gly Asp Pro Ile Ala Ala 3770 3775 3780Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu Leu Lys Arg Thr 3785 3790 3795Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr Thr Leu 3800 3805 3810Gly Ile Trp Thr Lys His Thr Ser Lys Arg Leu Ile Gln Ala Cys 3815 3820 3825Val Glu Ile Gly Lys Arg Glu Gly Thr Trp Leu Val Asn Ala Asp 3830 3835 3840Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro Ser Thr 3845 3850 3855Gly Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn Leu Lys 3860 3865 3870Gln Lys Ala Ala Gln Pro Pro Ile Glu Gly Val Asp Arg Tyr Lys 3875 3880 3885Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg Leu Arg Val Met 3890 3895 3900Leu Met Thr Val Ala Ser Gly Ser Trp 3905 391083748PRTArtificial SequenceMutated BVDV XIKE-B-NdN 8Met Glu Leu Phe Ser Asp Glu Gly Ser Lys Gly Ala Thr Ser Lys Lys1 5 10 15Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met Lys Ile Ala Pro 20 25 30Lys Glu Thr Glu Lys Asp Cys Lys Thr Arg Pro Pro Asp Ala Thr Ile 35 40 45Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys Gly Lys Val Arg 50 55 60Gly Lys Asn Thr Gln Asp Gly Leu Tyr

His Asn Lys Asn Lys Pro Pro65 70 75 80Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Ile Leu 85 90 95Ala Ala Val Leu Leu Gln Leu Val Thr Gly Glu Asn Ile Thr Gln Trp 100 105 110Asn Leu Met Asp Asn Gly Thr Glu Gly Ile Gln Gln Ala Met Phe Leu 115 120 125Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile Cys 130 135 140Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu Leu Lys Glu Ile145 150 155 160Val Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr Thr Cys Cys Arg 165 170 175Leu Gln Arg His Glu Trp Asn Lys Gly Trp Cys Asn Trp Phe His Ile 180 185 190Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln Asn Asn Leu Thr Glu 195 200 205Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys Glu 210 215 220Thr Glu Leu Asn Ile Val Thr Gln Ala Arg Asp Arg Pro Thr Thr Leu225 230 235 240Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Val Ile Leu 245 250 255Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu Asp Val Leu Phe Lys 260 265 270Glu His Asp Cys Gly Asn Met Leu Gln Glu Thr Ala Ile Gln Leu Leu 275 280 285Asp Gly Ala Thr Asn Thr Ile Glu Gly Ala Arg Val Gly Thr Ala Lys 290 295 300Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile Leu Gly Lys Lys Leu305 310 315 320Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His Ala Ala Ser Pro Tyr 325 330 335Cys Gly Val Glu Arg Lys Ile Gly Tyr Val Trp Tyr Thr Lys Asn Cys 340 345 350Thr Pro Ala Cys Leu Pro Arg Asn Thr Arg Ile Ile Gly Pro Gly Lys 355 360 365Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His Glu Met Gly Gly 370 375 380His Leu Ser Glu Phe Val Leu Leu Ser Leu Val Val Leu Ser Asp Phe385 390 395 400Ala Pro Glu Thr Ala Ser Val Ile Tyr Leu Val Leu His Phe Ala Ile 405 410 415Pro Gln Ser His Val Asp Val Asp Thr Cys Asp Lys Asn Gln Leu Asn 420 425 430Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile Pro Gly Thr Val Trp 435 440 445Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp Trp Trp Pro Tyr Glu 450 455 460Thr Thr Thr Val Phe Val Ile Glu Glu Ala Gly Gln Val Ile Lys Leu465 470 475 480Met Leu Arg Ala Ile Arg Asp Leu Thr Arg Ile Trp Asn Ala Ala Thr 485 490 495Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala Leu Arg Gly Gln Leu 500 505 510Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr Gly Ala Gln Gly Phe 515 520 525Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile Ser Lys Asp Arg Lys 530 535 540Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr Thr Trp His Leu Pro545 550 555 560Thr Lys Lys Ile Val Asp Ser Met Val His Val Trp Cys Glu Gly Lys 565 570 575Asp Leu Lys Ile Leu Lys Met Cys Thr Lys Glu Glu Arg Tyr Leu Val 580 585 590Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala Glu Phe Met Gln Ile 595 600 605Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp Met Pro Asp Asp Phe 610 615 620Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Lys Gly Lys625 630 635 640Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys Pro 645 650 655Gln Gly Trp Thr Gly Thr Ile Glu Cys Thr Leu Ala Asn Gln Asp Thr 660 665 670Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg Thr Thr Pro Phe Gln 675 680 685Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile Tyr 690 695 700Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile Thr Gly Asp His Ser705 710 715 720Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys Trp Cys Gly His Asp 725 730 735Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys Cys Met 740 745 750Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys Asp 755 760 765Arg Gly Gly Val Ala Ile Val Pro Ser Gly Thr Val Lys Cys Arg Ile 770 775 780Gly Asn Val Thr Val Gln Val Ile Ala Thr Asn Asn Asp Leu Gly Pro785 790 795 800Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu Gly Pro Val Glu 805 810 815Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr Leu Pro Asn Lys Tyr 820 825 830Tyr Glu Pro Arg Asp Arg Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu 835 840 845Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val Asp His His Lys Asp Tyr 850 855 860Phe Ser Glu Phe Ile Ile Ile Ala Val Val Ala Leu Leu Gly Gly Lys865 870 875 880Tyr Val Leu Trp Leu Leu Ile Thr Tyr Thr Ile Leu Ser Glu Gln Met 885 890 895Ala Met Gly Ala Gly Val Asn Thr Glu Glu Ile Val Met Ile Gly Asn 900 905 910Leu Leu Thr Asp Ser Asp Ile Glu Val Val Val Tyr Phe Leu Leu Leu 915 920 925Tyr Leu Ile Val Lys Glu Glu Leu Ala Arg Lys Trp Ile Ile Leu Val 930 935 940Tyr His Ile Leu Val Ala Asn Pro Met Lys Thr Ile Gly Val Val Leu945 950 955 960Leu Met Leu Gly Gly Val Val Lys Ala Ser Arg Ile Asn Ala Asp Asp 965 970 975Gln Ser Ala Met Asp Pro Cys Phe Leu Leu Val Thr Gly Val Val Ala 980 985 990Val Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu Ile Val 995 1000 1005Ala Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala Ser 1010 1015 1020Ile Asp Val Thr Val Ala Val Val Leu Ile Val Leu Leu Leu Ala 1025 1030 1035Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu Gln Leu 1040 1045 1050Leu Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu Lys 1055 1060 1065His Ile Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser Trp 1070 1075 1080Arg Pro Leu Ala Leu Val Leu Phe Tyr Ile Thr Ser Thr Ala Ile 1085 1090 1095Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu Leu Gln Trp 1100 1105 1110Ala Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe Leu 1115 1120 1125Thr Leu Ile Ile Val Leu Pro Ser Tyr Glu Leu Ser Lys Leu Tyr 1130 1135 1140Phe Leu Lys Asn Val Arg Thr Asp Val Glu Lys Asn Trp Leu Gly 1145 1150 1155Lys Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys Asp 1160 1165 1170Ser Glu Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser Gly 1175 1180 1185Ser Arg Pro Asp Phe Ile Leu Pro Phe Leu Lys Ala Val Leu Ile 1190 1195 1200Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile Ser Tyr Leu 1205 1210 1215Ile Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp Glu 1220 1225 1230Val Ser Gly Gly Ala Asn Phe Leu Ser Arg Leu Ile Ala Ala Ile 1235 1240 1245Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu Glu Cys Lys Gly Leu 1250 1255 1260Lys Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile Val 1265 1270 1275Lys His Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly Glu 1280 1285 1290Glu Glu Ile Tyr Gly Ala Pro Lys Val Ile Thr Ile Ile Lys Ala 1295 1300 1305Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys Thr Ile Cys 1310 1315 1320Glu Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly Arg 1325 1330 1335Gln Gly Lys Pro Ile Thr Cys Gly Met Thr Leu Ala Asp Phe Glu 1340 1345 1350Glu Lys His Tyr Lys Lys Ile Phe Ile Arg Glu Glu Ser Ser Cys 1355 1360 1365Pro Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg His 1370 1375 1380Asp Gly Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr Thr 1385 1390 1395Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala Thr 1400 1405 1410Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala Glu Ile Gly 1415 1420 1425Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys 1430 1435 1440Lys Lys Ile Thr Asn His Glu Lys Cys His Val Asn Ile Met Asp 1445 1450 1455Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr Pro 1460 1465 1470Arg Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg Arg 1475 1480 1485Gly Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile Ser 1490 1495 1500Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys Asp 1505 1510 1515Ser Met Gly Arg Thr Arg Val Val Cys His Ser Asn Asn Lys Met 1520 1525 1530Thr Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys Pro 1535 1540 1545Glu Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn Ile 1550 1555 1560Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly Gly 1565 1570 1575Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp 1580 1585 1590Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala 1595 1600 1605Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn Glu 1610 1615 1620Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val Ser 1625 1630 1635Lys Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr Ser 1640 1645 1650Met Asn Arg Gly Glu Phe Lys Gln Ile Thr Leu Ala Thr Gly Ala 1655 1660 1665Gly Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile Gly 1670 1675 1680Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala Ala 1685 1690 1695Glu Ser Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile Ser 1700 1705 1710Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala Thr 1715 1720 1725Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Leu Pro Gln 1730 1735 1740Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe Leu 1745 1750 1755Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile Gly 1760 1765 1770Lys Ile His Arg Phe Ala Glu Asn Leu Arg Val Val Ala Met Thr 1775 1780 1785Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His Pro 1790 1795 1800Ile Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp Leu 1805 1810 1815Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Thr Glu 1820 1825 1830Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr Arg Asn Met 1835 1840 1845Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn Ser 1850 1855 1860Gly Tyr Tyr Tyr Ser Gly Glu Asn Pro Glu Asn Leu Arg Val Val 1865 1870 1875Thr Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile Glu 1880 1885 1890Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr Gly 1895 1900 1905Leu Lys Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro Phe 1910 1915 1920Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu Gln 1925 1930 1935Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg Tyr 1940 1945 1950Tyr Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His Tyr 1955 1960 1965Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile Asn 1970 1975 1980Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu Tyr 1985 1990 1995Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn Asn 2000 2005 2010Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile Met 2015 2020 2025Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn Ser 2030 2035 2040Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile Lys Asn Gly 2045 2050 2055Glu Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala Arg 2060 2065 2070Lys Leu Gly Glu Asp Val Pro Ala Tyr Val Tyr Ala Thr Glu Asp 2075 2080 2085Glu Asp Leu Ala Val Asp Leu Leu Gly Met Asp Trp Pro Asp Pro 2090 2095 2100Gly Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln Val 2105 2110 2115Thr Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu Phe 2120 2125 2130Gly Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg His Ile Pro Met 2135 2140 2145Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu Glu Asp Thr 2150 2155 2160Thr His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly Lys 2165 2170 2175Asp Ser Glu Leu Lys Glu Leu Ala Val Gly Asp Leu Asp Lys Tyr 2180 2185 2190Val Asp Ala Leu Val Asp Tyr Ser Lys Gln Gly Met Lys Phe Ile 2195 2200 2205Lys Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys Glu 2210 2215 2220Gly Leu Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln Ser 2225 2230 2235Leu Thr Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp Gly 2240 2245 2250Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg Leu Gly His 2255 2260 2265Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe Gly 2270 2275 2280Gly Glu Thr Val Ser Ala His Ile Lys Gln Val Ala Val Asp Leu 2285 2290 2295Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser Phe Pro Gly Asp Thr 2300 2305 2310Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe Ile 2315 2320 2325Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn Asn 2330 2335 2340Leu Gln Arg Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr Ala 2345 2350 2355Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu Glu Ser Val 2360 2365 2370Val Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile Arg 2375 2380 2385Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala Ala 2390 2395 2400Met Glu Ile Leu Asn Gln Asn Pro Ile Ser Val Gly Ile Ser Val 2405 2410 2415Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu Ser 2420 2425 2430Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn 2435 2440 2445Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn Pro 2450 2455 2460Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile Gly 2465 2470 2475Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr Lys 2480 2485 2490Gly Trp Glu Ala Lys Glu Leu Ala Glu Lys Thr Ala Gly Arg Asn 2495 2500 2505Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly Met

2510 2515 2520Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile Leu 2525 2530 2535Asp Leu Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu Lys 2540 2545 2550Lys Leu Val Leu Gly Trp Ala Pro Ala Pro Leu Ser Cys Asp Trp 2555 2560 2565Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn Asn Tyr Leu 2570 2575 2580Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala Ile 2585 2590 2595Lys Asn Val Ala Gly Lys Leu Thr Lys Val Glu Glu Lys Gly Ser 2600 2605 2610Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly Pro Pro Asn Phe Lys 2615 2620 2625Val Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro Val 2630 2635 2640Ala Arg Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val Thr 2645 2650 2655Ala Lys Leu Asp Tyr Asn Asn Gly Lys Val Leu Leu Ala Thr Asn 2660 2665 2670Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu Val Lys Lys 2675 2680 2685His Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg Pro 2690 2695 2700Asp His Gln Asp Leu Val Asp Arg Asp Cys Ala Thr Ile Thr Lys 2705 2710 2715Asn Ser Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe Thr 2720 2725 2730Tyr Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu Val 2735 2740 2745His Lys Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr Val 2750 2755 2760Thr Thr Trp Leu Ala Tyr Ser Phe Val Asn Glu Asp Leu Gly Thr 2765 2770 2775Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu Pro Pro Glu 2780 2785 2790Glu Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu Thr 2795 2800 2805Ala Ile Thr Ile Thr Gly Glu Ala Glu Val Met Thr Thr Gly Ile 2810 2815 2820Thr Pro Val Val Glu Met Lys Glu Glu Pro Gln Leu Asp His Gln 2825 2830 2835Ser Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro Gly 2840 2845 2850Pro Gly Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu Lys 2855 2860 2865Asp Asp Arg Pro Phe Val Leu Ile Ile Gly Asn Lys Gly Ser Thr 2870 2875 2880Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu Tyr Lys Gly 2885 2890 2895Asn Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg Ile 2900 2905 2910Leu Thr Val Ala Leu Lys Glu Leu Asp Pro Glu Leu Lys Glu Leu 2915 2920 2925Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg Glu Ala Leu Glu Ala 2930 2935 2940Leu Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala Met 2945 2950 2955Ile Arg Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro Asp 2960 2965 2970Trp Phe Gln Ala Glu Glu Pro Leu Phe Leu Glu Ala Lys Ile Gln 2975 2980 2985Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser Ile Lys Ser 2990 2995 3000Lys Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val Lys 3005 3010 3015Glu Val Gly Ala Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp Ser 3020 3025 3030Thr Gln Val Thr Lys Lys Gln Met Ser Leu Ala Pro Leu Phe Glu 3035 3040 3045Glu Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys Gly 3050 3055 3060His Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro 3065 3070 3075Leu Gly Cys Gly Val Tyr Met Gly Thr Ile Pro Ala Arg Arg Leu 3080 3085 3090Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Glu Leu Val 3095 3100 3105Glu Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile Arg 3110 3115 3120Glu His Asn Thr Trp Ile Leu Arg Lys Val Arg His Glu Gly Asn 3125 3130 3135Leu Arg Thr Lys Ser Met Ile Asn Pro Gly Lys Ile Ser Asp Gln 3140 3145 3150Leu Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile Ile 3155 3160 3165Gly Ser Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro 3170 3175 3180Val Val Arg Ala Gln Thr Asp Thr Thr Ser Phe His Gln Ala Ile 3185 3190 3195Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr Pro Glu Leu 3200 3205 3210His Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro Glu 3215 3220 3225Leu Lys Glu Ser Tyr Asp Glu Val Ser Trp Glu Gln Leu Glu Ala 3230 3235 3240Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr Leu Glu Ser Lys Asn 3245 3250 3255Ile Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln Leu 3260 3265 3270Ile Lys Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu Thr 3275 3280 3285Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Glu 3290 3295 3300Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr 3305 3310 3315Pro Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr Lys 3320 3325 3330Trp Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr Glu Gly Lys 3335 3340 3345Thr Pro Leu Phe Asp Ile Phe Asn Lys Val Lys Lys Glu Trp Asp 3350 3355 3360Ser Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp 3365 3370 3375Asp Thr Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp Ile 3380 3385 3390Gln Lys Tyr Tyr Phe Lys Arg Ser Ile His Lys Phe Leu Asp Thr 3395 3400 3405Ile Thr Glu His Met Val Glu Val Pro Val Ile Thr Ala Asp Gly 3410 3415 3420Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp 3425 3430 3435Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile Tyr 3440 3445 3450Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr Arg Gly Phe Ser Arg 3455 3460 3465Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr 3470 3475 3480Glu Arg Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln Ile 3485 3490 3495Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp Lys 3500 3505 3510Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser His 3515 3520 3525Thr Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr Met 3530 3535 3540Ala Gly Arg Ser Thr Ala Thr Ile Leu Ala Lys Met Ala Thr Arg 3545 3550 3555Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr Ala Tyr Glu Lys Ala 3560 3565 3570Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val Val 3575 3580 3585Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile Ser 3590 3595 3600Pro Ser Lys Asn Thr Ile Tyr Tyr Tyr Gln Gly Asp Pro Ile Ala 3605 3610 3615Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu Leu Lys Arg 3620 3625 3630Thr Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr Thr 3635 3640 3645Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Leu Ile Gln Ala 3650 3655 3660Cys Val Glu Ile Gly Lys Arg Glu Gly Thr Trp Leu Val Asn Ala 3665 3670 3675Asp Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro Ser 3680 3685 3690Thr Gly Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn Leu 3695 3700 3705Lys Gln Lys Ala Ala Gln Pro Pro Ile Glu Gly Val Asp Arg Tyr 3710 3715 3720Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg Leu Arg Val 3725 3730 3735Met Leu Met Thr Val Ala Ser Gly Ser Trp 3740 374593913PRTArtificial SequenceXIKE-C BVDV-Sequence 9Met Glu Leu Phe Ser Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys1 5 10 15Pro Ala Gly Val Val Glu Pro Val Tyr Asp Val Asn Gly Arg Pro Leu 20 25 30Phe Gly Glu Ser Ser Asp Leu His Pro Gln Ser Thr Leu Lys Leu Pro 35 40 45His Gln Arg Gly Ser Ala Asn Ile Leu Thr Asn Ala Arg Ser Leu Pro 50 55 60Arg Lys Gly Asp Cys Arg Arg Gly Asn Val Tyr Gly Pro Val Ser Gly65 70 75 80Ile Tyr Ile Lys Pro Gly Pro Ile Tyr Tyr Gln Asp Tyr Val Gly Pro 85 90 95Val Tyr His Arg Ala Pro Leu Glu Leu Cys Arg Glu Ala Ser Met Cys 100 105 110Glu Thr Thr Arg Arg Val Gly Arg Val Thr Gly Ser Asp Gly Lys Leu 115 120 125Tyr His Ile Tyr Ile Cys Ile Asp Gly Cys Ile Leu Leu Lys Arg Ala 130 135 140Thr Arg Asn Gln Pro Glu Val Leu Lys Trp Val Tyr Asn Arg Leu Asn145 150 155 160Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Glu Gly Ser Lys Gly Ala 165 170 175Thr Ser Lys Lys Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met 180 185 190Lys Ile Ala Pro Lys Glu Thr Glu Lys Asp Cys Lys Thr Arg Pro Pro 195 200 205Asp Ala Thr Ile Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys 210 215 220Gly Lys Val Arg Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys225 230 235 240Asn Lys Pro Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala 245 250 255Trp Ala Ile Leu Ala Ala Val Leu Leu Gln Leu Val Thr Gly Glu Asn 260 265 270Ile Thr Gln Trp Asn Leu Met Asp Asn Gly Thr Glu Gly Ile Gln Gln 275 280 285Ala Met Phe Leu Arg Gly Val Asn Arg Ser Leu Leu Gly Ile Trp Pro 290 295 300Glu Lys Ile Cys Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu305 310 315 320Leu Lys Glu Ile Val Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr 325 330 335Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys 340 345 350Asn Trp Phe His Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln 355 360 365Asn Asn Leu Thr Glu Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys 370 375 380Arg Tyr Asp Lys Glu Thr Glu Leu Asn Ile Val Thr Gln Ala Arg Asp385 390 395 400Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe 405 410 415Ala Gly Val Ile Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu 420 425 430Asp Val Leu Phe Lys Glu His Asp Cys Gly Asn Met Leu Gln Glu Thr 435 440 445Ala Ile Gln Leu Leu Asp Gly Ala Thr Asn Thr Ile Glu Gly Ala Arg 450 455 460Val Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile465 470 475 480Leu Gly Lys Lys Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His 485 490 495Ala Ala Ser Pro Tyr Cys Gly Val Glu Arg Lys Ile Gly Tyr Val Trp 500 505 510Tyr Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Arg Asn Thr Arg Ile 515 520 525Ile Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu 530 535 540His Glu Met Gly Gly His Leu Ser Glu Phe Val Leu Leu Ser Leu Val545 550 555 560Val Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser Val Ile Tyr Leu Val 565 570 575Leu His Phe Ala Ile Pro Gln Ser His Val Asp Val Asp Thr Cys Asp 580 585 590Lys Asn Gln Leu Asn Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile 595 600 605Pro Gly Thr Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp 610 615 620Trp Trp Pro Tyr Glu Thr Thr Thr Val Phe Val Ile Glu Glu Ala Gly625 630 635 640Gln Val Ile Lys Leu Met Leu Arg Ala Ile Arg Asp Leu Thr Arg Ile 645 650 655Trp Asn Ala Ala Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala 660 665 670Leu Arg Gly Gln Leu Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr 675 680 685Gly Ala Gln Gly Phe Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile 690 695 700Ser Lys Asp Arg Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr705 710 715 720Thr Trp His Leu Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val 725 730 735Trp Cys Glu Gly Lys Asp Leu Lys Ile Leu Lys Met Cys Thr Lys Glu 740 745 750Glu Arg Tyr Leu Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala 755 760 765Glu Phe Met Gln Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp 770 775 780Met Pro Asp Asp Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro785 790 795 800Val Ile Lys Gly Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe 805 810 815Gln Met Val Cys Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Thr Leu 820 825 830Ala Asn Gln Asp Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg 835 840 845Thr Thr Pro Phe Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile 850 855 860Gly Glu Asp Ile Tyr Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile865 870 875 880Thr Gly Asp His Ser Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys 885 890 895Trp Cys Gly His Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro 900 905 910Ile Gly Lys Cys Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp 915 920 925Asp Thr Ser Cys Asp Arg Gly Gly Val Ala Ile Val Pro Ser Gly Thr 930 935 940Val Lys Cys Arg Ile Gly Asn Val Thr Val Gln Val Ile Ala Thr Asn945 950 955 960Asn Asp Leu Gly Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser 965 970 975Glu Gly Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr 980 985 990Leu Pro Asn Lys Tyr Tyr Glu Pro Arg Asp Arg Tyr Phe Gln Gln Tyr 995 1000 1005Met Leu Lys Gly Glu Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val 1010 1015 1020Asp His His Lys Asp Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val 1025 1030 1035Val Ala Leu Leu Gly Gly Lys Tyr Val Leu Trp Leu Leu Ile Thr 1040 1045 1050Tyr Thr Ile Leu Ser Glu Gln Met Ala Met Gly Ala Gly Val Asn 1055 1060 1065Thr Glu Glu Ile Val Met Ile Gly Asn Leu Leu Thr Asp Ser Asp 1070 1075 1080Ile Glu Val Val Val Tyr Phe Leu Leu Leu Tyr Leu Ile Val Lys 1085 1090 1095Glu Glu Leu Ala Arg Lys Trp Ile Ile Leu Val Tyr His Ile Leu 1100 1105 1110Val Ala Asn Pro Met Lys Thr Ile Gly Val Val Leu Leu Met Leu 1115 1120 1125Gly Gly Val Val Lys Ala Ser Arg Ile Asn Ala Asp Asp Gln Ser 1130 1135 1140Ala Met Asp Pro Cys Phe Leu Leu Val Thr Gly Val Val Ala Val 1145 1150 1155Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu Ile Val 1160 1165 1170Ala Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala Ser 1175 1180 1185Ile Asp Val Thr Val Ala Val Val Leu Ile Val Leu Leu Leu Ala 1190 1195 1200Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu Gln Leu 1205 1210

1215Leu Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu Lys 1220 1225 1230His Ile Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser Trp 1235 1240 1245Arg Pro Leu Ala Leu Val Leu Phe Tyr Ile Thr Ser Thr Ala Ile 1250 1255 1260Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu Leu Gln Trp 1265 1270 1275Ala Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe Leu 1280 1285 1290Thr Leu Ile Ile Val Leu Pro Ser Tyr Glu Leu Ser Lys Leu Tyr 1295 1300 1305Phe Leu Lys Asn Val Arg Thr Asp Val Glu Lys Asn Trp Leu Gly 1310 1315 1320Lys Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys Asp 1325 1330 1335Ser Glu Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser Gly 1340 1345 1350Ser Arg Pro Asp Phe Ile Leu Pro Phe Leu Lys Ala Val Leu Ile 1355 1360 1365Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile Ser Tyr Leu 1370 1375 1380Ile Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp Glu 1385 1390 1395Val Ser Gly Gly Ala Asn Phe Leu Ser Arg Leu Ile Ala Ala Ile 1400 1405 1410Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu Glu Cys Lys Gly Leu 1415 1420 1425Lys Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile Val 1430 1435 1440Lys His Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly Glu 1445 1450 1455Glu Glu Ile Tyr Gly Ala Pro Lys Val Ile Thr Ile Ile Lys Ala 1460 1465 1470Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys Thr Ile Cys 1475 1480 1485Glu Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly Arg 1490 1495 1500Gln Gly Lys Pro Ile Thr Cys Gly Met Thr Leu Ala Asp Phe Glu 1505 1510 1515Glu Lys His Tyr Lys Lys Ile Phe Ile Arg Glu Glu Ser Ser Cys 1520 1525 1530Pro Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg His 1535 1540 1545Asp Gly Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr Thr 1550 1555 1560Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala Thr 1565 1570 1575Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala Glu Ile Gly 1580 1585 1590Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys 1595 1600 1605Lys Lys Ile Thr Asn His Glu Lys Cys His Val Asn Ile Met Asp 1610 1615 1620Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr Pro 1625 1630 1635Arg Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg Arg 1640 1645 1650Gly Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile Ser 1655 1660 1665Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys Asp 1670 1675 1680Ser Met Gly Arg Thr Arg Val Val Cys His Ser Asn Asn Lys Met 1685 1690 1695Thr Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys Pro 1700 1705 1710Glu Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn Ile 1715 1720 1725Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly Gly 1730 1735 1740Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp 1745 1750 1755Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala 1760 1765 1770Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn Glu 1775 1780 1785Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val Ser 1790 1795 1800Lys Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr Ser 1805 1810 1815Met Asn Arg Gly Glu Phe Lys Gln Ile Thr Leu Ala Thr Gly Ala 1820 1825 1830Gly Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile Gly 1835 1840 1845Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala Ala 1850 1855 1860Glu Ser Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile Ser 1865 1870 1875Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala Thr 1880 1885 1890Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Leu Pro Gln 1895 1900 1905Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe Leu 1910 1915 1920Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile Gly 1925 1930 1935Lys Ile His Arg Phe Ala Glu Asn Leu Arg Val Val Ala Met Thr 1940 1945 1950Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His Pro 1955 1960 1965Ile Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp Leu 1970 1975 1980Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Thr Glu 1985 1990 1995Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr Arg Asn Met 2000 2005 2010Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn Ser 2015 2020 2025Gly Tyr Tyr Tyr Ser Gly Glu Asn Pro Glu Asn Leu Arg Val Val 2030 2035 2040Thr Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile Glu 2045 2050 2055Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr Gly 2060 2065 2070Leu Lys Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro Phe 2075 2080 2085Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu Gln 2090 2095 2100Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg Tyr 2105 2110 2115Tyr Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His Tyr 2120 2125 2130Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile Asn 2135 2140 2145Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu Tyr 2150 2155 2160Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn Asn 2165 2170 2175Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile Met 2180 2185 2190Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn Ser 2195 2200 2205Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile Lys Asn Gly 2210 2215 2220Glu Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala Arg 2225 2230 2235Lys Leu Gly Glu Asp Val Pro Ala Tyr Val Tyr Ala Thr Glu Asp 2240 2245 2250Glu Asp Leu Ala Val Asp Leu Leu Gly Met Asp Trp Pro Asp Pro 2255 2260 2265Gly Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln Val 2270 2275 2280Thr Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu Phe 2285 2290 2295Gly Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg His Ile Pro Met 2300 2305 2310Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu Glu Asp Thr 2315 2320 2325Thr His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly Lys 2330 2335 2340Asp Ser Glu Leu Lys Glu Leu Ala Val Gly Asp Leu Asp Lys Tyr 2345 2350 2355Val Asp Ala Leu Val Asp Tyr Ser Lys Gln Gly Met Lys Phe Ile 2360 2365 2370Lys Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys Glu 2375 2380 2385Gly Leu Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln Ser 2390 2395 2400Leu Thr Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp Gly 2405 2410 2415Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg Leu Gly His 2420 2425 2430Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe Gly 2435 2440 2445Gly Glu Thr Val Ser Ala His Ile Lys Gln Val Ala Val Asp Leu 2450 2455 2460Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser Phe Pro Gly Asp Thr 2465 2470 2475Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe Ile 2480 2485 2490Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn Asn 2495 2500 2505Leu Gln Arg Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr Ala 2510 2515 2520Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu Glu Ser Val 2525 2530 2535Val Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile Arg 2540 2545 2550Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala Ala 2555 2560 2565Met Glu Ile Leu Asn Gln Asn Pro Ile Ser Val Gly Ile Ser Val 2570 2575 2580Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu Ser 2585 2590 2595Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn 2600 2605 2610Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn Pro 2615 2620 2625Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile Gly 2630 2635 2640Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr Lys 2645 2650 2655Gly Trp Glu Ala Lys Glu Leu Ala Glu Lys Thr Ala Gly Arg Asn 2660 2665 2670Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly Met 2675 2680 2685Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile Leu 2690 2695 2700Asp Leu Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu Lys 2705 2710 2715Lys Leu Val Leu Gly Trp Ala Pro Ala Pro Leu Ser Cys Asp Trp 2720 2725 2730Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn Asn Tyr Leu 2735 2740 2745Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala Ile 2750 2755 2760Lys Asn Val Ala Gly Lys Leu Thr Lys Val Glu Glu Lys Gly Ser 2765 2770 2775Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly Pro Pro Asn Phe Lys 2780 2785 2790Val Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro Val 2795 2800 2805Ala Arg Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val Thr 2810 2815 2820Ala Lys Leu Asp Tyr Asn Asn Gly Lys Val Leu Leu Ala Thr Asn 2825 2830 2835Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu Val Lys Lys 2840 2845 2850His Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg Pro 2855 2860 2865Asp His Gln Asp Leu Val Asp Arg Asp Cys Ala Thr Ile Thr Lys 2870 2875 2880Asn Ser Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe Thr 2885 2890 2895Tyr Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu Val 2900 2905 2910His Lys Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr Val 2915 2920 2925Thr Thr Trp Leu Ala Tyr Ser Phe Val Asn Glu Asp Leu Gly Thr 2930 2935 2940Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu Pro Pro Glu 2945 2950 2955Glu Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu Thr 2960 2965 2970Ala Ile Thr Ile Thr Gly Glu Ala Glu Val Met Thr Thr Gly Ile 2975 2980 2985Thr Pro Val Val Glu Met Lys Glu Glu Pro Gln Leu Asp His Gln 2990 2995 3000Ser Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro Gly 3005 3010 3015Pro Gly Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu Lys 3020 3025 3030Asp Asp Arg Pro Phe Val Leu Ile Ile Gly Asn Lys Gly Ser Thr 3035 3040 3045Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu Tyr Lys Gly 3050 3055 3060Asn Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg Ile 3065 3070 3075Leu Thr Val Ala Leu Lys Glu Leu Asp Pro Glu Leu Lys Glu Leu 3080 3085 3090Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg Glu Ala Leu Glu Ala 3095 3100 3105Leu Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala Met 3110 3115 3120Ile Arg Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro Asp 3125 3130 3135Trp Phe Gln Ala Glu Glu Pro Leu Phe Leu Glu Ala Lys Ile Gln 3140 3145 3150Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser Ile Lys Ser 3155 3160 3165Lys Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val Lys 3170 3175 3180Glu Val Gly Ala Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp Ser 3185 3190 3195Thr Gln Val Thr Lys Lys Gln Met Ser Leu Ala Pro Leu Phe Glu 3200 3205 3210Glu Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys Gly 3215 3220 3225His Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro 3230 3235 3240Leu Gly Cys Gly Val Tyr Met Gly Thr Ile Pro Ala Arg Arg Leu 3245 3250 3255Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Glu Leu Val 3260 3265 3270Glu Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile Arg 3275 3280 3285Glu His Asn Thr Trp Ile Leu Arg Lys Val Arg His Glu Gly Asn 3290 3295 3300Leu Arg Thr Lys Ser Met Ile Asn Pro Gly Lys Ile Ser Asp Gln 3305 3310 3315Leu Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile Ile 3320 3325 3330Gly Ser Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro 3335 3340 3345Val Val Arg Ala Gln Thr Asp Thr Thr Ser Phe His Gln Ala Ile 3350 3355 3360Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr Pro Glu Leu 3365 3370 3375His Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro Glu 3380 3385 3390Leu Lys Glu Ser Tyr Asp Glu Val Ser Trp Glu Gln Leu Glu Ala 3395 3400 3405Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr Leu Glu Ser Lys Asn 3410 3415 3420Ile Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln Leu 3425 3430 3435Ile Lys Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu Thr 3440 3445 3450Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Glu 3455 3460 3465Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr 3470 3475 3480Pro Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr Lys 3485 3490 3495Trp Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr Glu Gly Lys 3500 3505 3510Thr Pro Leu Phe Asp Ile Phe Asn Lys Val Lys Lys Glu Trp Asp 3515 3520 3525Ser Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp 3530 3535 3540Asp Thr Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp Ile 3545 3550 3555Gln Lys Tyr Tyr Phe Lys Arg Ser Ile His Lys Phe Leu Asp Thr 3560 3565 3570Ile Thr Glu His Met Val Glu Val Pro Val Ile Thr Ala Asp Gly 3575 3580 3585Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp 3590 3595 3600Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile Tyr 3605 3610 3615Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr Arg Gly Phe Ser Arg 3620 3625 3630Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr 3635 3640 3645Glu Arg Gly Leu Gly Leu

Lys Phe Ser Glu Lys Gly Met Gln Ile 3650 3655 3660Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp Lys 3665 3670 3675Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser His 3680 3685 3690Thr Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr Met 3695 3700 3705Ala Gly Arg Ser Thr Ala Thr Ile Leu Ala Lys Met Ala Thr Arg 3710 3715 3720Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr Ala Tyr Glu Lys Ala 3725 3730 3735Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val Val 3740 3745 3750Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile Ser 3755 3760 3765Pro Ser Lys Asn Thr Ile Tyr Tyr Tyr Gln Gly Asp Pro Ile Ala 3770 3775 3780Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu Leu Lys Arg 3785 3790 3795Thr Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr Thr 3800 3805 3810Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Leu Ile Gln Ala 3815 3820 3825Cys Val Glu Ile Gly Lys Arg Glu Gly Thr Trp Leu Val Asn Ala 3830 3835 3840Asp Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro Ser 3845 3850 3855Thr Gly Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn Leu 3860 3865 3870Lys Gln Lys Ala Ala Gln Pro Pro Ile Glu Gly Val Asp Arg Tyr 3875 3880 3885Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg Leu Arg Val 3890 3895 3900Met Leu Met Thr Val Ala Ser Gly Ser Trp 3905 39101012332DNAArtificial SequenceXIKE-C BVDV-Sequence 10gtatacgaga ttagctaaag aactcgtata tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga gttgttttca aatgaacttt tatacaaaac 420atataaacaa aaaccagcag gcgtcgtgga acctgtttac gacgtcaacg ggcgcccact 480gtttggagag agcagtgact tgcacccgca gtcaacacta aaactaccac accaacgagg 540cagcgccaac atcctgacca atgctaggtc cctaccgcgg aaaggtgact gccggagagg 600taatgtgtat ggaccggtga gtggcatcta tatcaaacca ggaccgatct actaccagga 660ttatgtgggc cccgtctatc atagagcccc actggaacta tgtagggagg caagtatgtg 720cgaaacaact aggagagttg gcagagtgac cggtagtgat gggaaattat atcatatcta 780catctgcata gatgggtgta tcctcctgaa gagggcgact aggaaccaac cagaagtcct 840gaaatgggta tacaacagat taaattgtcc tttatgggtc accagctgct ccgatgaagg 900gagcaagggt gctacaagta agaagcagcc taagccagat aggatagaaa aaggtaagat 960gaaaatagcc ccaaaagaga cagaaaaaga ttgcaaaacc agaccccccg acgcgactat 1020agtagtagaa ggggttaagt accaggtgaa gaaaaaagga aaggtaaggg gaaaaaatac 1080tcaagatggg ttatatcaca acaagaataa gccccctgaa tcaagaaaaa aattggaaaa 1140ggcactgctg gcttgggcca tcttagcagc ggtcctgctt cagctggtaa caggagagaa 1200tatcacccag tggaacttga tggacaacgg caccgaggga atacagcaag cgatgttcct 1260aagaggggtg aacaggagtc tattaggaat ttggccagag aaaatttgca ccggagtacc 1320aactcactta gcaacagact atgagcttaa agagatagtg gggatgatgg acgcgagtga 1380gaagaccaac tacacgtgtt gcaggttgca aagacatgag tggaataaac atggttggtg 1440taactggttt catatagaac cgtggatatg gttgatgaac aaaacccaaa acaacctgac 1500agaagggcaa ccgcttaggg agtgtgctgt gacttgtagg tatgacaagg aaacagaatt 1560gaacatcgtg acacaggcta gggacagacc tacaactctg acaggttgca agaaaggcaa 1620gaatttctct ttcgcaggtg ttatactgga tgggccctgt aactttaaag tatcggttga 1680agatgtgctg ttcaaggagc acgattgcgg caacatgctg caagagaccg cgatacagct 1740actcgatggg gcaaccaaca ccattgaggg agcaagggta gggacggcca agttgacaac 1800ctggttaggg aagcaattag ggatccttgg taagaagttg gagaacaaaa gcaaagcatg 1860gtttggtgca catgcagcaa gtccatactg cggagtggag aggaagatcg gttacgtatg 1920gtatacaaaa aactgcactc cagcttgcct tccaagaaac actagaataa taggccccgg 1980gaaatttgat accaacgccg aagatggaaa aatactccat gagatggggg ggcacctctc 2040agaatttgtc ctattgtcct tggtggttct gtctgacttt gccccggaaa ccgcgagcgt 2100catctacttg gttctacatt ttgcgatccc gcaaagccac gttgatgtag acacatgcga 2160caagaaccag ctgaatttaa cggtagcaac cacagtagca gaggtcatac cagggacagt 2220gtggaaccta gggaagtatg tctgcataag accagactgg tggccatatg agacgacgac 2280agtcttcgtc atagaggaag cagggcaagt aatcaaattg atgctaaggg ccatcagaga 2340cttaactagg atatggaatg ctgccactac cacagctttc ttaatctttt tagtaaaagc 2400actgagggga caactaatcc aagggctatt gtggctgatg ctaataacag gagcacaggg 2460cttccctgaa tgcaaagagg gcttccaata tgccatatct aaagacagga aaatggggtt 2520attggggcca gagagcttaa ctacaacatg gcacctcccc accaaaaaaa tagtggattc 2580catggtgcat gtatggtgtg aaggaaaaga cttgaaaata ttaaaaatgt gcacaaagga 2640agagaggtat ctagtggctg tgcacgagag agccttatca accagtgccg agtttatgca 2700gatcagtgat gggacaatag gcccagacgt gatagatatg cctgatgact ttgagtttgg 2760actctgccct tgtgactcaa aaccagtgat aaagggcaaa tttaatgcca gcttactgaa 2820tggaccagct ttccagatgg tatgcccaca ggggtggact ggtacaatag aatgcaccct 2880agcgaaccaa gacaccttgg acacaactgt cattaggaca tatagaagaa ctaccccatt 2940tcagcggaga aaatggtgta cctatgaaaa aataataggg gaagatatct atgaatgcat 3000tctaggtgga aactggacat gcataaccgg tgaccatagc aggttgaaag acggacctat 3060caagaagtgt aagtggtgtg gccatgactt cgtcaactca gaggggctac cacactaccc 3120aataggcaag tgcatgctca tcaacgagag tgggtacagg tatgtagatg acacctcttg 3180cgataggggt ggtgtagcca tagttccatc tggcaccgta aagtgtagaa taggtaacgt 3240cacggtgcaa gttatcgcta ctaacaatga tctgggaccc atgccttgca gcccagctga 3300agtgatagca agtgaaggac cagtggaaaa gactgcatgc acattcaact attcaaggac 3360tctacctaat aagtattatg agccaaggga ccggtacttc caacaataca tgttaaaagg 3420ggagtggcaa tattggttcg acctggattc tgtagaccac cacaaagact acttctcaga 3480gttcataatc atagcagtgg tcgccttgtt gggtggtaag tacgtactgt ggctcttgat 3540aacatacaca atactgtctg agcagatggc tatgggtgct ggagtgaata ctgaagagat 3600agtcatgata ggcaatttgc tgacagacag tgatattgag gttgtggttt atttccttct 3660tctgtactta atagttaaag aggaactggc gaggaaatgg attatactgg tataccacat 3720ccttgtagcc aaccctatga aaacaattgg ggtcgtctta ctaatgctag ggggagtggt 3780gaaggccagc agaatcaatg ctgatgacca aagtgctatg gacccatgct ttcttctcgt 3840gacaggcgta gtggctgttt tgatgatcgc tagaagagaa cctgccacat taccactgat 3900tgtagcattg ctagcaataa gaacatcagg attcctactg cccgctagca ttgatgtaac 3960tgtagcagta gtattaattg tacttttgtt ggctagctac ataacagact actttagata 4020taaaaagtgg cttcaactct tatttagtct gatagctggt atctttatta taaggagctt 4080aaaacatatc aaccagatgg aggtaccaga aatatctatg ccaagttgga gacctctagc 4140tctggtcctt ttctatataa catctacagc aataaccact aattgggaca ttgacttagc 4200aggcttcctg ctgcaatggg cgccagcagt gatcatgatg gctaccatgt gggcagactt 4260tttgactctg atcatagtcc tgcccagtta cgagttatct aagctttact tcctaaagaa 4320cgtcaggaca gacgtggaaa agaactggct cggcaaagtg aaatacagac agatcagttc 4380agtttatgac atctgtgaca gtgaggaagc agtgtaccta tttccatcaa ggcataagag 4440tggaagcagg ccagatttca tattaccttt tttgaaagcc gtgttaataa gctgcatcag 4500cagccaatgg caagtggttt acatttctta cctaatactg gaaattacat actatatgca 4560caggaaaatc atagatgagg tgtcaggagg agcaaatttt ctatcaagac tcatagcagc 4620catcatagaa ttaaattggg ccatagatga tgaggaatgt aaaggactga agaaactgta 4680tctcttgtca gggagagcga agaatttgat agttaaacat aaggtaagaa atgaagccgt 4740ccacagatgg tttggtgagg aggaaatata cggggcaccc aaggtgatca ctatcataaa 4800agctagtacc ctaagtaaaa acaggcactg cataatctgc acgatctgtg aagggaaaga 4860atggaatgga gccaactgcc caaagtgtgg aagacaagga aagcccataa catgtggaat 4920gacactcgca gactttgagg agaaacatta caaaaagata tttataagag aagaatcttc 4980ttgtcctgtg ccttttgatc cttcttgcca ttgtaattat tttcgccacg atgggccttt 5040caggaaagag tataagggtt acgtccaata cacagccaga ggacaactct ttctgaggaa 5100cctaccaatt ctagcgacga agatgaagct attaatggtg ggaaacctcg gcgcagaaat 5160tggcgacctg gaacatctag gatgggtact gagagggcca gccgtgtgca aaaaaattac 5220caaccatgag aagtgccacg taaacatcat ggataagcta actgcatttt ttggaatcat 5280gcctagaggc acgaccccta gggcacctgt gaggttcccc acagcactac taaaagtgag 5340aagggggcta gagacgggat gggcttacac gcaccaagga gggatcagct cggtagacca 5400tgtcacagcc ggaaaggatt tactagtgtg tgacagtatg ggcaggacca gggttgtctg 5460tcatagtaac aataagatga ctgatgagac tgagtatggc atcaagaccg actcagggtg 5520tcccgaaggt gcgaggtgtt acgtgctaaa cccagaagct gttaacattt ctggcacaaa 5580aggagctatg gtacacctcc agaaaacggg gggggagttc acatgtgtca ctgcctcagg 5640gaccccggct ttcttcgatc tgaaaaatct aaaaggctgg tccgggctac caatttttga 5700agcatccagt ggcagggtgg ttggtagggt gaaagtcggc aagaatgagg attccaagcc 5760caccaaacta atgagcggaa tccagacagt gtctaagaac cagacagacc tagcggacat 5820cgtaaaaaaa ttgactagta tgaacagagg agagttcaaa cagataacat tagccactgg 5880ggcaggaaaa actacggaac tgccaaggtc cgtcatagag gagataggga ggcacaaaag 5940ggtcttagtc ctgataccat tgagagcagc agcagagtca gtgtatcagt atatgagagt 6000gaagtaccca agtatatctt tcaatttgag aataggagat atgaaggaag gtgacatggc 6060cactggtatc acctacgcct catatgggta cttttgtcag cttcctcagc ccaaactgag 6120agctgccatg gtagagtact catatatatt cttagatgag taccactgtg ctacacccga 6180gcaattagca ataattggaa agatacacag gtttgctgaa aatcttagag tggtagcaat 6240gacagcaacc ccagctggaa cggtcacaac gactggtcag aaacacccta tagaggagtt 6300catagcccca gaggtgatga aaggtgaaga tctaggtagt gaatacttgg atattgcagg 6360gttgaagata ccgactgaag agatgaaagg caacatgctc gtgttcgcgc caactaggaa 6420catggcagta gaaacagcta agaaattgaa ggctaaggga tacaactctg gatactatta 6480cagtggggaa aacccagaga acttgagggt ggtaacctcg caatccccgt atgtggtagt 6540agccaccaat gccatagagt caggtgtgac attaccagac ttagacacag ttgtagacac 6600tggactaaag tgtgagaaga gggtgaggat ttcttcaaaa atgcccttca ttgtaacagg 6660acttaagaga atggcagtca caatcggaga gcaagcccag cgcaggggta gagtaggaag 6720agtcaagcca ggtaggtact ataggagtca agaaacagct tcagggtcaa aagattacca 6780ttacgaccta ctgcaagccc agaggtacgg aatagaagat ggaattaatg taacaaagtc 6840attcagggag atgaactatg attggagcct ttacgaagag gacagcttga tgataactca 6900actcgaggtc cttaacaacc tccttatatc agaagacctg cctgccgcag tgaagaacat 6960catggcccgg accgatcacc cagaacccat acaactggcc tataacagtt atgaaaacca 7020aattccagtg ctgttcccaa agatcaaaaa tggtgaggtg acagacagtt atgagaatta 7080cacatatctc aatgcaagaa aattaggaga ggacgtgccg gcatatgtgt acgccacaga 7140ggatgaggat ctagcagtgg atcttctggg tatggattgg ccggacccag gcaaccaaca 7200ggtggtagag acagggaggg cattaaaaca agtaactggc ttatccacag cagaaaacgc 7260cctcttgata gccctattcg gctacgtcgg gtaccagaca ctttcaaaaa ggcacatacc 7320catgattact gacatctata cacttgaaga ccacaggctt gaggacacaa cccacctcca 7380gtttgcccca aacgctataa ggaccgacgg caaggactca gagttgaagg aattagctgt 7440gggagacctt gataaatatg tggacgcact ggtagactac tccaaacaag ggatgaaatt 7500catcaaagtc caagctgaaa aggtcagaga ctcccagtct acgaaggaag gcttgcaaac 7560cattaaggag tatgtggata agtttataca atcactaaca gagaataagg aggagatcat 7620caggtatgga ctatggggag ttcacacggc actctacaaa agcttggcag cgagactggg 7680gcatgaaaca gcttttgcaa ctttagtggt aaaatggttg gcttttgggg gcgaaacggt 7740atctgctcac atcaagcaag tagcagttga tctagtagta tattatatca tcaacaaacc 7800atcttttcct ggagatacag agacccaaca agaggggagg aagtttgtgg ctagtctttt 7860tatatctgca ctagcaacat acacatataa aacctggaat tacaacaatc tgcaacgggt 7920tgtcgaacct gccttagctt acctcccata tgctacaagt gccttgaagt tgttcacacc 7980cacaagatta gagagtgtgg tcatactcag ttctacaatt tacaagacat acctctctat 8040aaggaagggt aagagtgacg gcttgttagg tacaggcata agtgcagcca tggagatctt 8100aaaccaaaac ccaatctcag taggtatatc tgtgatgctg ggggtaggtg ccatcgccgc 8160ccataatgca atagaatcta gtgaacagaa aagaactttg ctgatgaagg tctttgtaaa 8220aaacttctta gaccaagcag caacagatga gctagtcaaa gagaaccctg aaaaaataat 8280catggctcta tttgaagcag tccagaccat aggaaacccc ctaagactca tctaccatct 8340gtacggggtg tactataagg ggtgggaagc aaaagaactc gcagagaaaa ctgctggccg 8400caacttattc acattgatca tgtttgaggc ctttgagctt ttaggtatgg actcagaagg 8460aaagataaga aacttgtcag gcaactacat actggactta atcttcaact tgcataataa 8520attaaacaag gggctcaaaa aactagtcct tgggtgggct cctgcacctt tgagctgtga 8580ttggacacca agtgatgaga gaataagcct acctcataac aactacttaa gggtagaaac 8640caggtgtcct tgtggctatg agatgaaggc aataaaaaat gttgctggta aattgacaaa 8700agttgaagaa aaggggtcct tcctatgcag gaatagatta gggagaggac ctccaaactt 8760caaagtaaca aagttctatg atgataactt gatagaagtc aagccagtag ctaggctaga 8820aggccaggtg gacctctatt acaagggagt aacagctaag ttagactaca acaatgggaa 8880agtactgtta gctaccaaca agtgggaggt ggaccacgct ttcctgacca gactagtaaa 8940gaagcacaca gggataggtt ttaaaggtgc atatttgggt gaccgaccag accatcaaga 9000tcttgtcgat agagattgtg caactataac gaagaactca gtacagttcc taaaaatgaa 9060gaagggttgc gctttcacat atgacctaac aatctctaac cttgtcaggc ttattgaact 9120agtccataag aataatttac aagaaagaga gatccctacc gtgacagtaa ctacttggct 9180tgcatattct tttgtcaatg aagacctggg gactatcaag cctgtattgg gggagaaagt 9240catcccagaa ccccccgagg agttgagtct ccaacccacc gtgagactag tcaccactga 9300aacagcaata accataacag gggaggctga agtgatgacg acagggatca caccagtggt 9360agagatgaaa gaagaacctc agctggacca ccagtcaact accctaaagg tagggttgaa 9420ggaaggggaa tatccagggc caggagttaa ccctaaccat ttagcagagg tgatagatga 9480gaaagatgac aggccttttg tcctaatcat cggtaacaaa ggttctacct cgaacagagc 9540aagaacggcc aagaatatac ggctgtacaa aggaaacaac ccaagagaga tcagggatct 9600gatgagccaa ggaagaatat tgacggttgc tctaaaagag ttggacccgg aattaaaaga 9660attagtagat tacaagggga cctttctcaa tagggaagct ttagaagccc taagcttagg 9720taagccaatc aagaggaaaa ccacaacagc aatgatcagg aggttaatag agccagaggt 9780tgaggaggaa ctaccagatt ggttccaagc ggaagaaccc ctatttttgg aagcaaaaat 9840acagaatgac ttataccacc taattggcag tgtagatagt ataaaaagca aagcaaagga 9900attaggggcc acagataaca caaagatagt gaaggaagtt ggggctagga cctatacgat 9960gaaattgagc agctggagca cacaagttac aaaaaaacag atgagtctag cccctctctt 10020tgaagagctg ttattaaagt gccctccatg tagtaaaatt tcaaagggac atatggtgtc 10080agcataccaa ctggctcaag gaaactggga acccctcggg tgtggggtct atatgggaac 10140cataccagct aggcgtctca agatccaccc ttatgaggct taccttaaac tcaaagagct 10200ggtggaagtt gaatcttcga gggccactgc aaaagaatcc atcataagag aacataacac 10260ctggatcctg cggaaggtga gacatgaagg gaacctaaga accaaatcaa tgatcaaccc 10320tgggaaaata tcagatcagc tatgcagaga tggacacaaa agaaacatat ataataagat 10380cataggctca acaatggcct ctgctggtat taggctggag aaactgccag tagtccgagc 10440ccaaactgac acaaccagtt tccaccaagc cataagagaa aaaattgata aaacagaaaa 10500caagcagacc cctgaattgc atgaagaact aatgaaggtc ttcgactgct taaagatccc 10560agagctgaag gaatcgtatg atgaagtttc atgggaacaa ttagaagccg ggataaaccg 10620taagggtgca gcaggctatc tagagagcaa gaacataggg gaagtcctag acacagagaa 10680acacatagta gagcagctga tcaaggatct gaggaagggg aagaagatta ggtactatga 10740aacagccatc cccaagaatg agaagagaga cgtcagcgac gactgggaag ccggagagtt 10800cgttgatgaa aagaaaccaa gagtaatcca gtacccggac gccaaggtga gactggccat 10860tacaaaagtg atgtacaaat gggtaaagca aaaaccagtg gtgatacccg gctatgaagg 10920taaaacacct ctatttgaca tattcaacaa agtgaagaag gaatgggatt cattccagga 10980ccccgtagca gtgagctttg acaccaaagc gtgggataca caagtcacca gtagagacct 11040aatgttgata aaggatatcc agaaatatta tttcaagaga agtatacaca aatttttaga 11100tacaataaca gaacacatgg tggaggtacc tgtcattaca gcagacggtg aagtttacat 11160aaggaatggt cagaggggta gtggccaacc cgacacaagt gctggtaata gtatgttgaa 11220tgtcctaacc atgatatatg ctttctgtaa aagtacaggc ataccttaca ggggattcag 11280cagagtggca agaatccatg tgtgtggtga tgatggcttt ttgataacag agagaggact 11340gggactgaaa ttctctgaga agggtatgca gatattacat gaggccggga agccccagaa 11400aataactgaa ggggacaaaa tgaaagtggc atacagattc gaggacatag agttttgttc 11460ccatactccc gtgccagtca gatgggcaga taacaccagt agttacatgg cagggaggag 11520cacagccact atactagcta agatggcaac caggctggat tccagcggag agaggggtag 11580cacagcttat gagaaggccg tagccttcag cttccttttg atgtactcat ggaatcccgt 11640agttagaagg atctgcttac tggtgttgtc acagtttcca gaaatatccc catccaaaaa 11700cacaatatac tactaccaag gggatcccat agctgcgtac agagaagtga tagggaaaca 11760gctgtgtgaa ctgaaaagaa caggatttga gaagctggct ggtctgaatt tgagtatgac 11820cactctaggc atctggacaa aacatactag taaaagacta atccaagcct gtgtagaaat 11880aggtaagaga gaaggtacct ggttagttaa tgctgacaga ctgattgcag gaaagactgg 11940gaagttttac atcccaagca ctggtgtcac tctgttggga aaacactatg aggaaattaa 12000cttaaagcaa aaggcggcac aaccgccgat agagggggtt gacagatata agttgggccc 12060catagttaat gttatcttga gaaggctgag ggtgatgctg atgacagttg ccagcggaag 12120ctggtgaatc cgtccggagc gtcgtgccct cactcaaggt ttttaattgt aaatattgta 12180aatagacagc taagatattt attgtagttg gatagtaatg cagtgatagt aaatacccca 12240atttaacact acctccaatg cactaagcac tttagctgtg tgaggttaac tcgacgtcca 12300cggttggact agggaagacc tctaacagcc cc 123321111840DNAArtificial SequenceXIKE-C-NdN BVDV-Sequence 11gtatacgaga ttagctaaag aactcgtata tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga gttgttttcc gatgaaggga gcaagggtgc 420tacaagtaag aagcagccta agccagatag gatagaaaaa ggtaagatga aaatagcccc 480aaaagagaca gaaaaagatt gcaaaaccag accccccgac gcgactatag tagtagaagg 540ggttaagtac caggtgaaga aaaaaggaaa ggtaagggga aaaaatactc aagatgggtt 600atatcacaac aagaataagc cccctgaatc aagaaaaaaa ttggaaaagg cactgctggc 660ttgggccatc ttagcagcgg tcctgcttca gctggtaaca ggagagaata tcacccagtg 720gaacttgatg gacaacggca ccgagggaat acagcaagcg atgttcctaa gaggggtgaa 780caggagtcta ttaggaattt ggccagagaa aatttgcacc ggagtaccaa ctcacttagc 840aacagactat gagcttaaag agatagtggg gatgatggac gcgagtgaga agaccaacta 900cacgtgttgc aggttgcaaa gacatgagtg gaataaacat ggttggtgta actggtttca 960tatagaaccg

tggatatggt tgatgaacaa aacccaaaac aacctgacag aagggcaacc 1020gcttagggag tgtgctgtga cttgtaggta tgacaaggaa acagaattga acatcgtgac 1080acaggctagg gacagaccta caactctgac aggttgcaag aaaggcaaga atttctcttt 1140cgcaggtgtt atactggatg ggccctgtaa ctttaaagta tcggttgaag atgtgctgtt 1200caaggagcac gattgcggca acatgctgca agagaccgcg atacagctac tcgatggggc 1260aaccaacacc attgagggag caagggtagg gacggccaag ttgacaacct ggttagggaa 1320gcaattaggg atccttggta agaagttgga gaacaaaagc aaagcatggt ttggtgcaca 1380tgcagcaagt ccatactgcg gagtggagag gaagatcggt tacgtatggt atacaaaaaa 1440ctgcactcca gcttgccttc caagaaacac tagaataata ggccccggga aatttgatac 1500caacgccgaa gatggaaaaa tactccatga gatggggggg cacctctcag aatttgtcct 1560attgtccttg gtggttctgt ctgactttgc cccggaaacc gcgagcgtca tctacttggt 1620tctacatttt gcgatcccgc aaagccacgt tgatgtagac acatgcgaca agaaccagct 1680gaatttaacg gtagcaacca cagtagcaga ggtcatacca gggacagtgt ggaacctagg 1740gaagtatgtc tgcataagac cagactggtg gccatatgag acgacgacag tcttcgtcat 1800agaggaagca gggcaagtaa tcaaattgat gctaagggcc atcagagact taactaggat 1860atggaatgct gccactacca cagctttctt aatcttttta gtaaaagcac tgaggggaca 1920actaatccaa gggctattgt ggctgatgct aataacagga gcacagggct tccctgaatg 1980caaagagggc ttccaatatg ccatatctaa agacaggaaa atggggttat tggggccaga 2040gagcttaact acaacatggc acctccccac caaaaaaata gtggattcca tggtgcatgt 2100atggtgtgaa ggaaaagact tgaaaatatt aaaaatgtgc acaaaggaag agaggtatct 2160agtggctgtg cacgagagag ccttatcaac cagtgccgag tttatgcaga tcagtgatgg 2220gacaataggc ccagacgtga tagatatgcc tgatgacttt gagtttggac tctgcccttg 2280tgactcaaaa ccagtgataa agggcaaatt taatgccagc ttactgaatg gaccagcttt 2340ccagatggta tgcccacagg ggtggactgg tacaatagaa tgcaccctag cgaaccaaga 2400caccttggac acaactgtca ttaggacata tagaagaact accccatttc agcggagaaa 2460atggtgtacc tatgaaaaaa taatagggga agatatctat gaatgcattc taggtggaaa 2520ctggacatgc ataaccggtg accatagcag gttgaaagac ggacctatca agaagtgtaa 2580gtggtgtggc catgacttcg tcaactcaga ggggctacca cactacccaa taggcaagtg 2640catgctcatc aacgagagtg ggtacaggta tgtagatgac acctcttgcg ataggggtgg 2700tgtagccata gttccatctg gcaccgtaaa gtgtagaata ggtaacgtca cggtgcaagt 2760tatcgctact aacaatgatc tgggacccat gccttgcagc ccagctgaag tgatagcaag 2820tgaaggacca gtggaaaaga ctgcatgcac attcaactat tcaaggactc tacctaataa 2880gtattatgag ccaagggacc ggtacttcca acaatacatg ttaaaagggg agtggcaata 2940ttggttcgac ctggattctg tagaccacca caaagactac ttctcagagt tcataatcat 3000agcagtggtc gccttgttgg gtggtaagta cgtactgtgg ctcttgataa catacacaat 3060actgtctgag cagatggcta tgggtgctgg agtgaatact gaagagatag tcatgatagg 3120caatttgctg acagacagtg atattgaggt tgtggtttat ttccttcttc tgtacttaat 3180agttaaagag gaactggcga ggaaatggat tatactggta taccacatcc ttgtagccaa 3240ccctatgaaa acaattgggg tcgtcttact aatgctaggg ggagtggtga aggccagcag 3300aatcaatgct gatgaccaaa gtgctatgga cccatgcttt cttctcgtga caggcgtagt 3360ggctgttttg atgatcgcta gaagagaacc tgccacatta ccactgattg tagcattgct 3420agcaataaga acatcaggat tcctactgcc cgctagcatt gatgtaactg tagcagtagt 3480attaattgta cttttgttgg ctagctacat aacagactac tttagatata aaaagtggct 3540tcaactctta tttagtctga tagctggtat ctttattata aggagcttaa aacatatcaa 3600ccagatggag gtaccagaaa tatctatgcc aagttggaga cctctagctc tggtcctttt 3660ctatataaca tctacagcaa taaccactaa ttgggacatt gacttagcag gcttcctgct 3720gcaatgggcg ccagcagtga tcatgatggc taccatgtgg gcagactttt tgactctgat 3780catagtcctg cccagttacg agttatctaa gctttacttc ctaaagaacg tcaggacaga 3840cgtggaaaag aactggctcg gcaaagtgaa atacagacag atcagttcag tttatgacat 3900ctgtgacagt gaggaagcag tgtacctatt tccatcaagg cataagagtg gaagcaggcc 3960agatttcata ttaccttttt tgaaagccgt gttaataagc tgcatcagca gccaatggca 4020agtggtttac atttcttacc taatactgga aattacatac tatatgcaca ggaaaatcat 4080agatgaggtg tcaggaggag caaattttct atcaagactc atagcagcca tcatagaatt 4140aaattgggcc atagatgatg aggaatgtaa aggactgaag aaactgtatc tcttgtcagg 4200gagagcgaag aatttgatag ttaaacataa ggtaagaaat gaagccgtcc acagatggtt 4260tggtgaggag gaaatatacg gggcacccaa ggtgatcact atcataaaag ctagtaccct 4320aagtaaaaac aggcactgca taatctgcac gatctgtgaa gggaaagaat ggaatggagc 4380caactgccca aagtgtggaa gacaaggaaa gcccataaca tgtggaatga cactcgcaga 4440ctttgaggag aaacattaca aaaagatatt tataagagaa gaatcttctt gtcctgtgcc 4500ttttgatcct tcttgccatt gtaattattt tcgccacgat gggcctttca ggaaagagta 4560taagggttac gtccaataca cagccagagg acaactcttt ctgaggaacc taccaattct 4620agcgacgaag atgaagctat taatggtggg aaacctcggc gcagaaattg gcgacctgga 4680acatctagga tgggtactga gagggccagc cgtgtgcaaa aaaattacca accatgagaa 4740gtgccacgta aacatcatgg ataagctaac tgcatttttt ggaatcatgc ctagaggcac 4800gacccctagg gcacctgtga ggttccccac agcactacta aaagtgagaa gggggctaga 4860gacgggatgg gcttacacgc accaaggagg gatcagctcg gtagaccatg tcacagccgg 4920aaaggattta ctagtgtgtg acagtatggg caggaccagg gttgtctgtc atagtaacaa 4980taagatgact gatgagactg agtatggcat caagaccgac tcagggtgtc ccgaaggtgc 5040gaggtgttac gtgctaaacc cagaagctgt taacatttct ggcacaaaag gagctatggt 5100acacctccag aaaacggggg gggagttcac atgtgtcact gcctcaggga ccccggcttt 5160cttcgatctg aaaaatctaa aaggctggtc cgggctacca atttttgaag catccagtgg 5220cagggtggtt ggtagggtga aagtcggcaa gaatgaggat tccaagccca ccaaactaat 5280gagcggaatc cagacagtgt ctaagaacca gacagaccta gcggacatcg taaaaaaatt 5340gactagtatg aacagaggag agttcaaaca gataacatta gccactgggg caggaaaaac 5400tacggaactg ccaaggtccg tcatagagga gatagggagg cacaaaaggg tcttagtcct 5460gataccattg agagcagcag cagagtcagt gtatcagtat atgagagtga agtacccaag 5520tatatctttc aatttgagaa taggagatat gaaggaaggt gacatggcca ctggtatcac 5580ctacgcctca tatgggtact tttgtcagct tcctcagccc aaactgagag ctgccatggt 5640agagtactca tatatattct tagatgagta ccactgtgct acacccgagc aattagcaat 5700aattggaaag atacacaggt ttgctgaaaa tcttagagtg gtagcaatga cagcaacccc 5760agctggaacg gtcacaacga ctggtcagaa acaccctata gaggagttca tagccccaga 5820ggtgatgaaa ggtgaagatc taggtagtga atacttggat attgcagggt tgaagatacc 5880gactgaagag atgaaaggca acatgctcgt gttcgcgcca actaggaaca tggcagtaga 5940aacagctaag aaattgaagg ctaagggata caactctgga tactattaca gtggggaaaa 6000cccagagaac ttgagggtgg taacctcgca atccccgtat gtggtagtag ccaccaatgc 6060catagagtca ggtgtgacat taccagactt agacacagtt gtagacactg gactaaagtg 6120tgagaagagg gtgaggattt cttcaaaaat gcccttcatt gtaacaggac ttaagagaat 6180ggcagtcaca atcggagagc aagcccagcg caggggtaga gtaggaagag tcaagccagg 6240taggtactat aggagtcaag aaacagcttc agggtcaaaa gattaccatt acgacctact 6300gcaagcccag aggtacggaa tagaagatgg aattaatgta acaaagtcat tcagggagat 6360gaactatgat tggagccttt acgaagagga cagcttgatg ataactcaac tcgaggtcct 6420taacaacctc cttatatcag aagacctgcc tgccgcagtg aagaacatca tggcccggac 6480cgatcaccca gaacccatac aactggccta taacagttat gaaaaccaaa ttccagtgct 6540gttcccaaag atcaaaaatg gtgaggtgac agacagttat gagaattaca catatctcaa 6600tgcaagaaaa ttaggagagg acgtgccggc atatgtgtac gccacagagg atgaggatct 6660agcagtggat cttctgggta tggattggcc ggacccaggc aaccaacagg tggtagagac 6720agggagggca ttaaaacaag taactggctt atccacagca gaaaacgccc tcttgatagc 6780cctattcggc tacgtcgggt accagacact ttcaaaaagg cacataccca tgattactga 6840catctataca cttgaagacc acaggcttga ggacacaacc cacctccagt ttgccccaaa 6900cgctataagg accgacggca aggactcaga gttgaaggaa ttagctgtgg gagaccttga 6960taaatatgtg gacgcactgg tagactactc caaacaaggg atgaaattca tcaaagtcca 7020agctgaaaag gtcagagact cccagtctac gaaggaaggc ttgcaaacca ttaaggagta 7080tgtggataag tttatacaat cactaacaga gaataaggag gagatcatca ggtatggact 7140atggggagtt cacacggcac tctacaaaag cttggcagcg agactggggc atgaaacagc 7200ttttgcaact ttagtggtaa aatggttggc ttttgggggc gaaacggtat ctgctcacat 7260caagcaagta gcagttgatc tagtagtata ttatatcatc aacaaaccat cttttcctgg 7320agatacagag acccaacaag aggggaggaa gtttgtggct agtcttttta tatctgcact 7380agcaacatac acatataaaa cctggaatta caacaatctg caacgggttg tcgaacctgc 7440cttagcttac ctcccatatg ctacaagtgc cttgaagttg ttcacaccca caagattaga 7500gagtgtggtc atactcagtt ctacaattta caagacatac ctctctataa ggaagggtaa 7560gagtgacggc ttgttaggta caggcataag tgcagccatg gagatcttaa accaaaaccc 7620aatctcagta ggtatatctg tgatgctggg ggtaggtgcc atcgccgccc ataatgcaat 7680agaatctagt gaacagaaaa gaactttgct gatgaaggtc tttgtaaaaa acttcttaga 7740ccaagcagca acagatgagc tagtcaaaga gaaccctgaa aaaataatca tggctctatt 7800tgaagcagtc cagaccatag gaaaccccct aagactcatc taccatctgt acggggtgta 7860ctataagggg tgggaagcaa aagaactcgc agagaaaact gctggccgca acttattcac 7920attgatcatg tttgaggcct ttgagctttt aggtatggac tcagaaggaa agataagaaa 7980cttgtcaggc aactacatac tggacttaat cttcaacttg cataataaat taaacaaggg 8040gctcaaaaaa ctagtccttg ggtgggctcc tgcacctttg agctgtgatt ggacaccaag 8100tgatgagaga ataagcctac ctcataacaa ctacttaagg gtagaaacca ggtgtccttg 8160tggctatgag atgaaggcaa taaaaaatgt tgctggtaaa ttgacaaaag ttgaagaaaa 8220ggggtccttc ctatgcagga atagattagg gagaggacct ccaaacttca aagtaacaaa 8280gttctatgat gataacttga tagaagtcaa gccagtagct aggctagaag gccaggtgga 8340cctctattac aagggagtaa cagctaagtt agactacaac aatgggaaag tactgttagc 8400taccaacaag tgggaggtgg accacgcttt cctgaccaga ctagtaaaga agcacacagg 8460gataggtttt aaaggtgcat atttgggtga ccgaccagac catcaagatc ttgtcgatag 8520agattgtgca actataacga agaactcagt acagttccta aaaatgaaga agggttgcgc 8580tttcacatat gacctaacaa tctctaacct tgtcaggctt attgaactag tccataagaa 8640taatttacaa gaaagagaga tccctaccgt gacagtaact acttggcttg catattcttt 8700tgtcaatgaa gacctgggga ctatcaagcc tgtattgggg gagaaagtca tcccagaacc 8760ccccgaggag ttgagtctcc aacccaccgt gagactagtc accactgaaa cagcaataac 8820cataacaggg gaggctgaag tgatgacgac agggatcaca ccagtggtag agatgaaaga 8880agaacctcag ctggaccacc agtcaactac cctaaaggta gggttgaagg aaggggaata 8940tccagggcca ggagttaacc ctaaccattt agcagaggtg atagatgaga aagatgacag 9000gccttttgtc ctaatcatcg gtaacaaagg ttctacctcg aacagagcaa gaacggccaa 9060gaatatacgg ctgtacaaag gaaacaaccc aagagagatc agggatctga tgagccaagg 9120aagaatattg acggttgctc taaaagagtt ggacccggaa ttaaaagaat tagtagatta 9180caaggggacc tttctcaata gggaagcttt agaagcccta agcttaggta agccaatcaa 9240gaggaaaacc acaacagcaa tgatcaggag gttaatagag ccagaggttg aggaggaact 9300accagattgg ttccaagcgg aagaacccct atttttggaa gcaaaaatac agaatgactt 9360ataccaccta attggcagtg tagatagtat aaaaagcaaa gcaaaggaat taggggccac 9420agataacaca aagatagtga aggaagttgg ggctaggacc tatacgatga aattgagcag 9480ctggagcaca caagttacaa aaaaacagat gagtctagcc cctctctttg aagagctgtt 9540attaaagtgc cctccatgta gtaaaatttc aaagggacat atggtgtcag cataccaact 9600ggctcaagga aactgggaac ccctcgggtg tggggtctat atgggaacca taccagctag 9660gcgtctcaag atccaccctt atgaggctta ccttaaactc aaagagctgg tggaagttga 9720atcttcgagg gccactgcaa aagaatccat cataagagaa cataacacct ggatcctgcg 9780gaaggtgaga catgaaggga acctaagaac caaatcaatg atcaaccctg ggaaaatatc 9840agatcagcta tgcagagatg gacacaaaag aaacatatat aataagatca taggctcaac 9900aatggcctct gctggtatta ggctggagaa actgccagta gtccgagccc aaactgacac 9960aaccagtttc caccaagcca taagagaaaa aattgataaa acagaaaaca agcagacccc 10020tgaattgcat gaagaactaa tgaaggtctt cgactgctta aagatcccag agctgaagga 10080atcgtatgat gaagtttcat gggaacaatt agaagccggg ataaaccgta agggtgcagc 10140aggctatcta gagagcaaga acatagggga agtcctagac acagagaaac acatagtaga 10200gcagctgatc aaggatctga ggaaggggaa gaagattagg tactatgaaa cagccatccc 10260caagaatgag aagagagacg tcagcgacga ctgggaagcc ggagagttcg ttgatgaaaa 10320gaaaccaaga gtaatccagt acccggacgc caaggtgaga ctggccatta caaaagtgat 10380gtacaaatgg gtaaagcaaa aaccagtggt gatacccggc tatgaaggta aaacacctct 10440atttgacata ttcaacaaag tgaagaagga atgggattca ttccaggacc ccgtagcagt 10500gagctttgac accaaagcgt gggatacaca agtcaccagt agagacctaa tgttgataaa 10560ggatatccag aaatattatt tcaagagaag tatacacaaa tttttagata caataacaga 10620acacatggtg gaggtacctg tcattacagc agacggtgaa gtttacataa ggaatggtca 10680gaggggtagt ggccaacccg acacaagtgc tggtaatagt atgttgaatg tcctaaccat 10740gatatatgct ttctgtaaaa gtacaggcat accttacagg ggattcagca gagtggcaag 10800aatccatgtg tgtggtgatg atggcttttt gataacagag agaggactgg gactgaaatt 10860ctctgagaag ggtatgcaga tattacatga ggccgggaag ccccagaaaa taactgaagg 10920ggacaaaatg aaagtggcat acagattcga ggacatagag ttttgttccc atactcccgt 10980gccagtcaga tgggcagata acaccagtag ttacatggca gggaggagca cagccactat 11040actagctaag atggcaacca ggctggattc cagcggagag aggggtagca cagcttatga 11100gaaggccgta gccttcagct tccttttgat gtactcatgg aatcccgtag ttagaaggat 11160ctgcttactg gtgttgtcac agtttccaga aatatcccca tccaaaaaca caatatacta 11220ctaccaaggg gatcccatag ctgcgtacag agaagtgata gggaaacagc tgtgtgaact 11280gaaaagaaca ggatttgaga agctggctgg tctgaatttg agtatgacca ctctaggcat 11340ctggacaaaa catactagta aaagactaat ccaagcctgt gtagaaatag gtaagagaga 11400aggtacctgg ttagttaatg ctgacagact gattgcagga aagactggga agttttacat 11460cccaagcact ggtgtcactc tgttgggaaa acactatgag gaaattaact taaagcaaaa 11520ggcggcacaa ccgccgatag agggggttga cagatataag ttgggcccca tagttaatgt 11580tatcttgaga aggctgaggg tgatgctgat gacagttgcc agcggaagct ggtgaatccg 11640tccggagcgt cgtgccctca ctcaaggttt ttaattgtaa atattgtaaa tagacagcta 11700agatatttat tgtagttgga tagtaatgca gtgatagtaa ataccccaat ttaacactac 11760ctccaatgca ctaagcactt tagctgtgtg aggttaactc gacgtccacg gttggactag 11820ggaagacctc taacagcccc 11840123749PRTArtificial SequenceXIKE-C-NdN 12Met Glu Leu Phe Ser Asp Glu Gly Ser Lys Gly Ala Thr Ser Lys Lys1 5 10 15Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met Lys Ile Ala Pro 20 25 30Lys Glu Thr Glu Lys Asp Cys Lys Thr Arg Pro Pro Asp Ala Thr Ile 35 40 45Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys Gly Lys Val Arg 50 55 60Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro Pro65 70 75 80Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Ile Leu 85 90 95Ala Ala Val Leu Leu Gln Leu Val Thr Gly Glu Asn Ile Thr Gln Trp 100 105 110Asn Leu Met Asp Asn Gly Thr Glu Gly Ile Gln Gln Ala Met Phe Leu 115 120 125Arg Gly Val Asn Arg Ser Leu Leu Gly Ile Trp Pro Glu Lys Ile Cys 130 135 140Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu Leu Lys Glu Ile145 150 155 160Val Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr Thr Cys Cys Arg 165 170 175Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Phe His 180 185 190Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln Asn Asn Leu Thr 195 200 205Glu Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys 210 215 220Glu Thr Glu Leu Asn Ile Val Thr Gln Ala Arg Asp Arg Pro Thr Thr225 230 235 240Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Val Ile 245 250 255Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu Asp Val Leu Phe 260 265 270Lys Glu His Asp Cys Gly Asn Met Leu Gln Glu Thr Ala Ile Gln Leu 275 280 285Leu Asp Gly Ala Thr Asn Thr Ile Glu Gly Ala Arg Val Gly Thr Ala 290 295 300Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile Leu Gly Lys Lys305 310 315 320Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His Ala Ala Ser Pro 325 330 335Tyr Cys Gly Val Glu Arg Lys Ile Gly Tyr Val Trp Tyr Thr Lys Asn 340 345 350Cys Thr Pro Ala Cys Leu Pro Arg Asn Thr Arg Ile Ile Gly Pro Gly 355 360 365Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His Glu Met Gly 370 375 380Gly His Leu Ser Glu Phe Val Leu Leu Ser Leu Val Val Leu Ser Asp385 390 395 400Phe Ala Pro Glu Thr Ala Ser Val Ile Tyr Leu Val Leu His Phe Ala 405 410 415Ile Pro Gln Ser His Val Asp Val Asp Thr Cys Asp Lys Asn Gln Leu 420 425 430Asn Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile Pro Gly Thr Val 435 440 445Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp Trp Trp Pro Tyr 450 455 460Glu Thr Thr Thr Val Phe Val Ile Glu Glu Ala Gly Gln Val Ile Lys465 470 475 480Leu Met Leu Arg Ala Ile Arg Asp Leu Thr Arg Ile Trp Asn Ala Ala 485 490 495Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala Leu Arg Gly Gln 500 505 510Leu Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr Gly Ala Gln Gly 515 520 525Phe Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile Ser Lys Asp Arg 530 535 540Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr Thr Trp His Leu545 550 555 560Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val Trp Cys Glu Gly 565 570 575Lys Asp Leu Lys Ile Leu Lys Met Cys Thr Lys Glu Glu Arg Tyr Leu 580 585 590Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala Glu Phe Met Gln 595 600 605Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp Met Pro Asp Asp 610 615 620Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Lys Gly625 630 635 640Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys 645 650 655Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Thr Leu Ala Asn Gln Asp 660 665 670Thr Leu Asp Thr Thr

Val Ile Arg Thr Tyr Arg Arg Thr Thr Pro Phe 675 680 685Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile 690 695 700Tyr Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile Thr Gly Asp His705 710 715 720Ser Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys Trp Cys Gly His 725 730 735Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys Cys 740 745 750Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys 755 760 765Asp Arg Gly Gly Val Ala Ile Val Pro Ser Gly Thr Val Lys Cys Arg 770 775 780Ile Gly Asn Val Thr Val Gln Val Ile Ala Thr Asn Asn Asp Leu Gly785 790 795 800Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu Gly Pro Val 805 810 815Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr Leu Pro Asn Lys 820 825 830Tyr Tyr Glu Pro Arg Asp Arg Tyr Phe Gln Gln Tyr Met Leu Lys Gly 835 840 845Glu Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val Asp His His Lys Asp 850 855 860Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val Val Ala Leu Leu Gly Gly865 870 875 880Lys Tyr Val Leu Trp Leu Leu Ile Thr Tyr Thr Ile Leu Ser Glu Gln 885 890 895Met Ala Met Gly Ala Gly Val Asn Thr Glu Glu Ile Val Met Ile Gly 900 905 910Asn Leu Leu Thr Asp Ser Asp Ile Glu Val Val Val Tyr Phe Leu Leu 915 920 925Leu Tyr Leu Ile Val Lys Glu Glu Leu Ala Arg Lys Trp Ile Ile Leu 930 935 940Val Tyr His Ile Leu Val Ala Asn Pro Met Lys Thr Ile Gly Val Val945 950 955 960Leu Leu Met Leu Gly Gly Val Val Lys Ala Ser Arg Ile Asn Ala Asp 965 970 975Asp Gln Ser Ala Met Asp Pro Cys Phe Leu Leu Val Thr Gly Val Val 980 985 990Ala Val Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu Ile 995 1000 1005Val Ala Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala 1010 1015 1020Ser Ile Asp Val Thr Val Ala Val Val Leu Ile Val Leu Leu Leu 1025 1030 1035Ala Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu Gln 1040 1045 1050Leu Leu Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu 1055 1060 1065Lys His Ile Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser 1070 1075 1080Trp Arg Pro Leu Ala Leu Val Leu Phe Tyr Ile Thr Ser Thr Ala 1085 1090 1095Ile Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu Leu Gln 1100 1105 1110Trp Ala Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe 1115 1120 1125Leu Thr Leu Ile Ile Val Leu Pro Ser Tyr Glu Leu Ser Lys Leu 1130 1135 1140Tyr Phe Leu Lys Asn Val Arg Thr Asp Val Glu Lys Asn Trp Leu 1145 1150 1155Gly Lys Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys 1160 1165 1170Asp Ser Glu Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser 1175 1180 1185Gly Ser Arg Pro Asp Phe Ile Leu Pro Phe Leu Lys Ala Val Leu 1190 1195 1200Ile Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile Ser Tyr 1205 1210 1215Leu Ile Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp 1220 1225 1230Glu Val Ser Gly Gly Ala Asn Phe Leu Ser Arg Leu Ile Ala Ala 1235 1240 1245Ile Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu Glu Cys Lys Gly 1250 1255 1260Leu Lys Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile 1265 1270 1275Val Lys His Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly 1280 1285 1290Glu Glu Glu Ile Tyr Gly Ala Pro Lys Val Ile Thr Ile Ile Lys 1295 1300 1305Ala Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys Thr Ile 1310 1315 1320Cys Glu Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly 1325 1330 1335Arg Gln Gly Lys Pro Ile Thr Cys Gly Met Thr Leu Ala Asp Phe 1340 1345 1350Glu Glu Lys His Tyr Lys Lys Ile Phe Ile Arg Glu Glu Ser Ser 1355 1360 1365Cys Pro Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg 1370 1375 1380His Asp Gly Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr 1385 1390 1395Thr Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala 1400 1405 1410Thr Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala Glu Ile 1415 1420 1425Gly Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val 1430 1435 1440Cys Lys Lys Ile Thr Asn His Glu Lys Cys His Val Asn Ile Met 1445 1450 1455Asp Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr 1460 1465 1470Pro Arg Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg 1475 1480 1485Arg Gly Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile 1490 1495 1500Ser Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys 1505 1510 1515Asp Ser Met Gly Arg Thr Arg Val Val Cys His Ser Asn Asn Lys 1520 1525 1530Met Thr Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys 1535 1540 1545Pro Glu Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn 1550 1555 1560Ile Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly 1565 1570 1575Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe 1580 1585 1590Asp Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu 1595 1600 1605Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn 1610 1615 1620Glu Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val 1625 1630 1635Ser Lys Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr 1640 1645 1650Ser Met Asn Arg Gly Glu Phe Lys Gln Ile Thr Leu Ala Thr Gly 1655 1660 1665Ala Gly Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile 1670 1675 1680Gly Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala 1685 1690 1695Ala Glu Ser Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile 1700 1705 1710Ser Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala 1715 1720 1725Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Leu Pro 1730 1735 1740Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe 1745 1750 1755Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile 1760 1765 1770Gly Lys Ile His Arg Phe Ala Glu Asn Leu Arg Val Val Ala Met 1775 1780 1785Thr Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His 1790 1795 1800Pro Ile Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp 1805 1810 1815Leu Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Thr 1820 1825 1830Glu Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr Arg Asn 1835 1840 1845Met Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn 1850 1855 1860Ser Gly Tyr Tyr Tyr Ser Gly Glu Asn Pro Glu Asn Leu Arg Val 1865 1870 1875Val Thr Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile 1880 1885 1890Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr 1895 1900 1905Gly Leu Lys Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro 1910 1915 1920Phe Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu 1925 1930 1935Gln Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg 1940 1945 1950Tyr Tyr Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His 1955 1960 1965Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile 1970 1975 1980Asn Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu 1985 1990 1995Tyr Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn 2000 2005 2010Asn Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile 2015 2020 2025Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn 2030 2035 2040Ser Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile Lys Asn 2045 2050 2055Gly Glu Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala 2060 2065 2070Arg Lys Leu Gly Glu Asp Val Pro Ala Tyr Val Tyr Ala Thr Glu 2075 2080 2085Asp Glu Asp Leu Ala Val Asp Leu Leu Gly Met Asp Trp Pro Asp 2090 2095 2100Pro Gly Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln 2105 2110 2115Val Thr Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu 2120 2125 2130Phe Gly Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg His Ile Pro 2135 2140 2145Met Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu Glu Asp 2150 2155 2160Thr Thr His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly 2165 2170 2175Lys Asp Ser Glu Leu Lys Glu Leu Ala Val Gly Asp Leu Asp Lys 2180 2185 2190Tyr Val Asp Ala Leu Val Asp Tyr Ser Lys Gln Gly Met Lys Phe 2195 2200 2205Ile Lys Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys 2210 2215 2220Glu Gly Leu Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln 2225 2230 2235Ser Leu Thr Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp 2240 2245 2250Gly Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg Leu Gly 2255 2260 2265His Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe 2270 2275 2280Gly Gly Glu Thr Val Ser Ala His Ile Lys Gln Val Ala Val Asp 2285 2290 2295Leu Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser Phe Pro Gly Asp 2300 2305 2310Thr Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe 2315 2320 2325Ile Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn 2330 2335 2340Asn Leu Gln Arg Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr 2345 2350 2355Ala Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu Glu Ser 2360 2365 2370Val Val Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile 2375 2380 2385Arg Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala 2390 2395 2400Ala Met Glu Ile Leu Asn Gln Asn Pro Ile Ser Val Gly Ile Ser 2405 2410 2415Val Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu 2420 2425 2430Ser Ser Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys 2435 2440 2445Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn 2450 2455 2460Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile 2465 2470 2475Gly Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr 2480 2485 2490Lys Gly Trp Glu Ala Lys Glu Leu Ala Glu Lys Thr Ala Gly Arg 2495 2500 2505Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly 2510 2515 2520Met Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile 2525 2530 2535Leu Asp Leu Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu 2540 2545 2550Lys Lys Leu Val Leu Gly Trp Ala Pro Ala Pro Leu Ser Cys Asp 2555 2560 2565Trp Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn Asn Tyr 2570 2575 2580Leu Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala 2585 2590 2595Ile Lys Asn Val Ala Gly Lys Leu Thr Lys Val Glu Glu Lys Gly 2600 2605 2610Ser Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly Pro Pro Asn Phe 2615 2620 2625Lys Val Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro 2630 2635 2640Val Ala Arg Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val 2645 2650 2655Thr Ala Lys Leu Asp Tyr Asn Asn Gly Lys Val Leu Leu Ala Thr 2660 2665 2670Asn Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu Val Lys 2675 2680 2685Lys His Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg 2690 2695 2700Pro Asp His Gln Asp Leu Val Asp Arg Asp Cys Ala Thr Ile Thr 2705 2710 2715Lys Asn Ser Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe 2720 2725 2730Thr Tyr Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu 2735 2740 2745Val His Lys Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr 2750 2755 2760Val Thr Thr Trp Leu Ala Tyr Ser Phe Val Asn Glu Asp Leu Gly 2765 2770 2775Thr Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu Pro Pro 2780 2785 2790Glu Glu Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu 2795 2800 2805Thr Ala Ile Thr Ile Thr Gly Glu Ala Glu Val Met Thr Thr Gly 2810 2815 2820Ile Thr Pro Val Val Glu Met Lys Glu Glu Pro Gln Leu Asp His 2825 2830 2835Gln Ser Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro 2840 2845 2850Gly Pro Gly Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu 2855 2860 2865Lys Asp Asp Arg Pro Phe Val Leu Ile Ile Gly Asn Lys Gly Ser 2870 2875 2880Thr Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu Tyr Lys 2885 2890 2895Gly Asn Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg 2900 2905 2910Ile Leu Thr Val Ala Leu Lys Glu Leu Asp Pro Glu Leu Lys Glu 2915 2920 2925Leu Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg Glu Ala Leu Glu 2930 2935 2940Ala Leu Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala 2945 2950 2955Met Ile Arg Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro 2960 2965 2970Asp Trp Phe Gln Ala Glu Glu Pro Leu Phe Leu Glu Ala Lys Ile 2975 2980 2985Gln Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser Ile Lys 2990 2995 3000Ser Lys Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val 3005 3010 3015Lys Glu Val Gly Ala Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp 3020 3025 3030Ser Thr Gln Val Thr Lys Lys Gln Met Ser Leu Ala Pro Leu Phe 3035 3040 3045Glu Glu Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys 3050 3055 3060Gly His Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu 3065 3070 3075Pro Leu Gly Cys Gly Val Tyr Met Gly Thr Ile Pro Ala Arg Arg 3080 3085 3090Leu Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Glu Leu 3095 3100 3105Val Glu Val Glu Ser Ser Arg Ala Thr Ala Lys

Glu Ser Ile Ile 3110 3115 3120Arg Glu His Asn Thr Trp Ile Leu Arg Lys Val Arg His Glu Gly 3125 3130 3135Asn Leu Arg Thr Lys Ser Met Ile Asn Pro Gly Lys Ile Ser Asp 3140 3145 3150Gln Leu Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile 3155 3160 3165Ile Gly Ser Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu 3170 3175 3180Pro Val Val Arg Ala Gln Thr Asp Thr Thr Ser Phe His Gln Ala 3185 3190 3195Ile Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr Pro Glu 3200 3205 3210Leu His Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro 3215 3220 3225Glu Leu Lys Glu Ser Tyr Asp Glu Val Ser Trp Glu Gln Leu Glu 3230 3235 3240Ala Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr Leu Glu Ser Lys 3245 3250 3255Asn Ile Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln 3260 3265 3270Leu Ile Lys Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu 3275 3280 3285Thr Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp 3290 3295 3300Glu Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln 3305 3310 3315Tyr Pro Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr 3320 3325 3330Lys Trp Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr Glu Gly 3335 3340 3345Lys Thr Pro Leu Phe Asp Ile Phe Asn Lys Val Lys Lys Glu Trp 3350 3355 3360Asp Ser Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala 3365 3370 3375Trp Asp Thr Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp 3380 3385 3390Ile Gln Lys Tyr Tyr Phe Lys Arg Ser Ile His Lys Phe Leu Asp 3395 3400 3405Thr Ile Thr Glu His Met Val Glu Val Pro Val Ile Thr Ala Asp 3410 3415 3420Gly Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro 3425 3430 3435Asp Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile 3440 3445 3450Tyr Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr Arg Gly Phe Ser 3455 3460 3465Arg Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile 3470 3475 3480Thr Glu Arg Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln 3485 3490 3495Ile Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp 3500 3505 3510Lys Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser 3515 3520 3525His Thr Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr 3530 3535 3540Met Ala Gly Arg Ser Thr Ala Thr Ile Leu Ala Lys Met Ala Thr 3545 3550 3555Arg Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr Ala Tyr Glu Lys 3560 3565 3570Ala Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val 3575 3580 3585Val Arg Arg Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile 3590 3595 3600Ser Pro Ser Lys Asn Thr Ile Tyr Tyr Tyr Gln Gly Asp Pro Ile 3605 3610 3615Ala Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu Leu Lys 3620 3625 3630Arg Thr Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr 3635 3640 3645Thr Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Leu Ile Gln 3650 3655 3660Ala Cys Val Glu Ile Gly Lys Arg Glu Gly Thr Trp Leu Val Asn 3665 3670 3675Ala Asp Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro 3680 3685 3690Ser Thr Gly Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn 3695 3700 3705Leu Lys Gln Lys Ala Ala Gln Pro Pro Ile Glu Gly Val Asp Arg 3710 3715 3720Tyr Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg Leu Arg 3725 3730 3735Val Met Leu Met Thr Val Ala Ser Gly Ser Trp 3740 37451313PRTBovine Viral Diarrhea VirusMISC_FEATURE(1)..(13)Conserved protein sequence within glycoprotein Erns 13Ser Leu His Gly Ile Trp Pro Glu Lys Ile Cys Thr Gly1 5 101420PRTBovine Viral Diarrhea VirusMISC_FEATURE(1)..(20)Conserved protein sequence within glycoprotein Erns 14Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Phe His1 5 10 15Ile Glu Pro Trp 201511PRTBovine Viral Diarrhea VirusMISC_FEATURE(1)..(11)Conserved protein sequence within glycoprotein Erns 15Ser Leu His Gly Ile Trp Pro Glu Lys Ile Cys1 5 101612PRTBovine Viral Diarrhea VirusMISC_FEATURE(1)..(12)Conserved protein sequence within glycoprotein Erns 16Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp1 5 10

Claims

1. An attenuated pestivirus having at least one mutation in the coding sequence for glycoprotein E.sup.ms and at least another mutation in the coding sequence for N.sup.pro, wherein the mutation in the coding sequence for glycoprotein E.sup.ms leads to inactivation of RNase activity residing in E.sup.ms and/or the mutation in the coding sequence for N.sup.pro leads to inactivation of the N.sup.pro.

2. (canceled)

3. The virus according to claim 1, wherein the mutations are selected from the group of deletions, insertion mutations, and substitution mutations.

4-14. (canceled)

15. The virus according to claim 1, wherein the mutation(s) in the coding sequence for glycoprotein E.sup.ms are located in the nucleotide sequence coding for the conserved E.sup.ms sequence SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW.

16. The virus according to claim 1, wherein the mutation(s) in the coding sequence for glycoprotein E.sup.ms located in the nucleotide sequence coding for the conserved E.sup.ms sequence SLHGIWPEKIC and/or RHEWNKHGWCNR.

17. The virus according to claim 1, wherein the mutation(s) in the coding sequence for glycoprotein E.sup.ms are two mutations located in the nucleotide sequence coding for the conserved E.sup.ms sequence SLHGIWPEKIC and/or RHENKHGWCNR.

18. The virus according to claim 1, wherein the mutation in the coding sequence for glycoprotein E.sup.ms is a single mutation located in the conserved E.sup.ms sequence SLHGIWPEKIC or RHEWNKHGWCNR.

19. The virus according to claim 1, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula: [N.sup.pro].sub.x-[PS].sub.y-[C-term] wherein: [N.sup.pro] is the N.sup.pro portion of the polyprotein, wherein x is the number of amino acids of the N.sup.pro present in the polyprotein; [PS] is a processing signal selected from the group consisting of: ubiquitin, LC3, SUMO-1, NEDD8, GATE-16 or GABA(A)RAP), Intein, picornavirus 3C, caridovirus 2A, or p15 of rabbit hemorrhagic disease virus; [C-term] is the complete virus polyprotein except for N.sup.Pro, but including the capsid (C)-protein and any other protein present in the virus polyprotein including the carboxyterminal NS5B; y is 0 or 1, where 0 means [PS] is absent and 1 means [PS] is present; and x is 0 to 12 amino acids if y is 0, or 0 to 168 amino acids if y is 1.

20. (canceled)

21. The virus according to claim 19, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula: [N.sup.pro].sub.1-[PS].sub.0-[C-term].

22. (canceled)

23. The virus according to claim 19, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula: [N.sup.pro].sub.3-[PS].sub.0-[C-term].

24. (canceled)

25. The virus according to claim 19, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula: [N.sup.pro].sub.3-[PS].sub.0-[C-term] and the mutation in the coding sequence for glycoprotein E.sup.ms is a single mutation located in the conserved E.sup.ms sequence SLHGIWPEKIC or RHEWNKHGWCNR.

26. (canceled)

27. The virus according to claim 19, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula: [N.sup.pro].sub.4-[PS].sub.0-[C-term].

28. (canceled)

29. The virus according to claim 19, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula: [N.sup.pro].sub.6-[PS].sub.0-[C-term].

30. (canceled)

31. The virus according to claim 19, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula: [N.sup.pro].sub.4-[PS]O--[C-term*], wherein [C-term]* is [C-term] wherein in the C-protein the amino acid at position 2 is changed from D to N.

32. (canceled)

33. The virus according to claim 19, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein is characterized by the following formula: [N.sup.pro].sub.x-[PS].sub.1-[C-term], wherein PS is ubiquitin or LC3.

34-38. (canceled)

39. The virus according to claim 19, wherein the mutation(s) in the coding sequence for N.sup.pro lead to an encoded polyprotein as characterized by the following formula: [N.sup.pro].sub.22-[PS].sub.1-[C-term], wherein PS is ubiquitin or LC3.

40. (canceled)

41. The virus according to claim 21, wherein the [PS].sub.0 is replaced by [PS].sub.1, and wherein the PS is selected from the group of consisting of: ubiquitin, LC3, SUMO-1, NEDD8, GATE-16, GABA(A)RAP, intein, picornavirus 3C, caridovirus 2A, and p15 of rabbit hemorrhagic disease virus.

42-43. (canceled)

44. A composition comprising the virus according to claim 1 and a solution.

45. (canceled)

46. The composition according to claim 44, which induces an immunological response in an animal.

47. (canceled)

48. The composition according to claim 44, which is a vaccine.

49. (canceled)

50. The composition according to claim 48, further comprising a pharmaceutically acceptable carrier or excipient.

51-54. (canceled)

55. A method for attenuating a pestivirus, wherein at least one mutation in the coding sequence for glycoprotein E.sup.ms and at least another mutation in the coding sequence for N.sup.pro is generated in a pestivirus.

56. (canceled)

57. The method according to claim 55, the method comprising: (a) reversely transcribing a wild type pestivirus to obtain a cDNA; (b) cloning the cDNA; (c) introducing mutations selected from deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein E.sup.ms and the protease N.sup.pro; and (d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of the pestivirus cDNA into RNA in vitro or upon infection of suitable cells.

58-59. (canceled)