Abstract:
A method for determining the capability of an arterivirus to replicate in a permissive cell is disclosed. The method includes determining an amino acid at a position that corresponds to an amino acid of a protein of a porcine reproductive and respiratory syndrome virus. The invention also discloses a method for determining the capability of an arterivirus to replicate in a green monkey cell line. The invention further discloses a method for producing arterivirus in a green monkey cell line wherein the virulence of the arterivirus is maintained and the virus yield is increased. Methods for determining the attenuation of an arterivirus and for attenuating the virulence of the arterivirus by changing amino acids are further disclosed.

Description:
TECHNICAL FIELD  
         [0001]    The invention relates to the field of virology and more particularly, to the field of vaccine production. More specifically, the invention relates to the in vitro propagation of virus and to the adaptation of a virus to a cell line and the attenuation of a virus.  
         BACKGROUND  
         [0002]    Porcine reproductive and respiratory syndrome virus (PRRSV) is a small, enveloped, positive-stranded RNA virus which belongs to the genus arterivirus. PRRSV was isolated in Europe and in the United States of America in 1991 (Collins et al., 1992, Wensvoort et al., 1991). The genus arterivirus also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). The genus arterivirus is the only member in the family of the Arteriviridae. The family Arteriviridae and the Coronaviridae are grouped into the order Nidovirales.  
           [0003]    In vivo, PRRSV reproduces primarily in porcine alveolar lung macrophages and blood monocytes. (Wensvoort et al., 1991). Macrophages from other tissues, such as heart, tonsil, spleen, turbinates and choroid plexus are also susceptible to PRRSV infection.  
           [0004]    In vitro, the porcine virus can be passaged to primary cultures of porcine alveolar lung macrophages and blood monocytes, to cultures of the African green monkey kidney cell line MA-104 and to the derivative cell lines CL2621 and MARC-145. (Collins et al., 1992, Wensvoort et al., 1991). A few cell lines exist that the porcine virus cannot penetrate, such as BHK-21 and Vero cells, but once the genome of these cell lines is brought inside the cells artificially, the porcine virus is able to replicate and produce new virus particles. (Meulenberg et al., 1998).  
           [0005]    PRRSV enters porcine alveolar macrophages and MARC-145 cells via receptor-mediated endocytosis. The uptake of PRRSV by the host cell is a multi-step process in which one or two viral constituents appear to interact with at least one host proteonic factor before virus-containing clathrin-coated pits are formed. (Kreutz, 1998). The entry process of PRRSV into porcine macrophages differs from the binding and internalization of PRRSV into MARC-145 cells. A membrane protein of about 210-kDa has been identified as being a putative receptor for PRRSV on macrophages (Duan et al., 1998) while a heparin-like molecule is suggested to be important for the binding of PRRSV to MARC-145 cells. (Jusa et al., 1997).  
           [0006]    PRRSV has a concise genome structure (for a review, see, Snijder, and Meulenberg, 1998) which is 15,098 nt in length without the poly-A tail and contains 9 open reading frames (ORFs) flanked by a 5′ and 3′ non-translated region (NTR) of 221 and 114 nucleotides, respectively. The two 5′-terminal ORFs designated 1A and 1B (FIGS. 1A and 1B) comprise about 75% of the genomic RNA. The structural region of the RNA contains 7 ORFs designated 2A, 2B, and 3 to 7 (FIGS. 1A and 1B) and occupies the 3′-terminal third of the genome. ORF 1A/B encodes the non-structural proteins (nsps) involved in replication of the RNA genome and the forming of a 3′ nested set of subgenomic RNAs (sgRNAs).  
           [0007]    The first two N-terminal proteins named nsp 1α and nsp 1β, respectively, have been studied for PRRSV. The functions of the other nsps are based on predictions derived from sequence homologies with EAV. Nsp 1α and nsp 1β are papain-like cysteine proteases and together with nsp2 and nsp4, a cysteine protease and a serine protease, respectively, the entire polyprotein ORF1 (a/ab) is assumed to be cleaved into 12 nsps. Nsp9 and 10 are predicted to be the replicase subunits.  
           [0008]    The 3′-terminal end of the genome encodes the structural proteins. The ORFs 2A to 6 encode six structural membrane-associated proteins and the most 3′-terminal ORF encodes the N protein (ORF7). The minor glycoproteins are GP2a (formerly named GP2), GP3 and GP4. The GP2a and the GP4 proteins are class I membrane proteins and have molecular masses of 27-30 and 31-35 kDa, respectively. Both N-glycosylated proteins are constituents of the virion. The other minor N-glycosylated structural protein of 42-50 kDa is GP3 and is encoded by ORF3. The nature of GP3 was unclear until the present disclosure.  
           [0009]    The three major structural proteins are GP5, M and the N protein. GP5, a N-glycosylated protein of 24-26 kDa, is the most variable protein in its sequence. The most conserved protein is the non-glycosylated M protein. The M protein is a 18-19 kDa integral membrane protein and appears to have a function in viral infectivity. The M protein forms a heterodimer with GP5 which is present in the virion. The N protein is a small, highly basic, nucleocapsid protein of 14-15 kDa which interacts with the viral RNA during assembly. (Snijder &amp; Meulenberg, 1998).  
           [0010]    Sequence comparisons and studies involving the antigenic nature of PRRSV strains reveal two distinct groups designated as EU (European) and US (North American) strains. (Wensvoort et al., 1992, Snijder &amp; Meulenberg, 1998). Another indication of the observed diversity between the EU strain and the US strain from the two continents is the fact that EU field isolates initially replicate easier in alveolar lung macrophages, whereas US isolates are recovered easier in the African green monkey kidney cell line MA-104 or derivatives thereof, such as CL2621 or MARC-145 cells. (Bautista et al., 1993).  
           [0011]    The candidate proteins which will adsorb to the host cell surface of permissive cells are GP2a, GP2b, GP3, GP4, GP5, the M protein or a heterodimer of GP5 and the M protein.  
           [0012]    Typically, when viruses are replicated in vitro in cell lines derived from a different species, the viruses tend to attenuate in the process of adapting to the cell line. This adaptation characteristic is widely used in vaccine development and is also true for PRRSV replicated in monkey cell lines (Collins et al. 1992).  
         SUMMARY OF THE INVENTION  
         [0013]    In one embodiment, changes in the PRRSV genome that occur during the adaptation process are related to adaptation to a cell. This aspect is useful because the PRRSV can be adapted to replicate in a permissive cell without further loss of virulence.  
           [0014]    In another embodiment, changes in other sites of the genome are more related to attenuation. This aspect is useful because it enables a person skilled in the art to control the level of attenuation of a PRRSV.  
           [0015]    A method to determine the capability of an arterivirus to replicate in a permissive cell, such as a green monkey cell, comprising determining the amino acid positions that correspond to the amino acid positions 75-107 of GP2a of PRRSV isolate I-1102 is disclosed. In a further aspect of the present invention, a method for determining the amino acid positions that correspond to the amino acid position 88 and/or amino acid position 95 of GP2a of PRRSV isolate I-1102 is disclosed. The knowledge and methods disclosed herein will enable a person of ordinary skill in the art to identify the capability of a PRRS virus to replicate in a permissive cell, such as a green monkey cell.  
           [0016]    Further, a method for increasing the capability of an arterivirus to replicate in a permissive cell, such as a green monkey cell, comprising changing the amino acids at amino acid positions 75-107 is disclosed. In another aspect, the method comprises changing the amino acid at amino acid position 88 from a valine to any other amino acid, such as a phenylalanine, and/or changing the amino acid at amino acid position 95 from a phenylalanine to any other amino acid, such as a leucine is disclosed.  
           [0017]    In a further embodiment, the concomitant amino acid changes in the two sites of GP2a enhance the adaptation of a PRRSV for a permissive cell, such as a green monkey cell. The method is suited for the production of an arterivirus and/or a nucleic acid in a permissive cell and/or cell line, wherein the virulence of the arterivirus is maintained while the virus and/or nucleic acid yield from the permissive cell and/or cell line is increased.  
           [0018]    In another embodiment, a method for determining the attenuation of an Arterivirus comprising determining the amino acids positions that correspond to the amino acid positions 121-148 of GP5 of PRRSV isolate I-1102 is disclosed. The method further comprises determining the amino acid position that corresponds to the amino acid position 136 of GP5 of PRRSV isolate I-1102. A method to control and/or increase the attenuation of an arterivirus comprising changing amino acids at amino acid positions 121-148 of GP5 of PRRSV isolate I-1102 by changing the amino acid at amino acid position 136 from a cysteine to any other amino acid, such as a tyrosine, is disclosed.  
           [0019]    In a further embodiment, a method for determining the attenuation of an Arterivirus comprising determining the amino acids at positions that correspond to the amino acid positions 651-675 and/or amino acid positions 2331-2355 of ORFlab of PRRSV isolate I-1102 is disclosed. The method further comprises determining the amino acid at a position that corresponds to the amino acid position 663 and/or amino acid position 2343 of ORFlab of PRRSV isolate I-1102. A method to control and/or increase the attenuation of an arterivirus comprising changing amino acids at amino acid positions 121-148 of GP5 of PRRSV isolate I-1102 by changing the amino acid at amino acid position 136 from a cysteine to any other amino acid, such as a tyrosine, is disclosed.  
           [0020]    In another embodiment, a method for determining the attenuation of an Arterivirus comprising determining the amino acids positions that correspond to the amino acid positions 651-675 and/or amino acid positions 2331-2355 of ORFlab of PRRSV isolate I-1102 is disclosed. In a further aspect, the method comprises determining the amino acid at positions that corresponds to the amino acid position 663 and/or amino acid position 2343 of ORFlab of PRRSV isolate I-1102.  
           [0021]    A method to control and/or increase the attenuation of an arterivirus comprising changing the amino acids at positions that correspond to the amino acid positions 651-675 and/or amino acid positions 2331-2355 of ORFlab of PRRSV isolate I-1102 is disclosed. In a further aspect, the method comprises changing the amino acid at amino acid position 663 from a glutamic acid to any other amino acid, such a lysine, and/or changing the amino acid at amino acid at position 2343 from a valine to any other amino acid, such as an alanine. The methods also allow for the production of an attenuated arterivirus. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0022]    FIGS.  1 A and  1 B: Schematic representation of amino acid differences between the LV4.2.1 strain, which is adapted to MA-104 cells, and strain pABV437, which is the infectious cDNA clone of the field strain Ter Huurne is shown in (A). An overview of generated full length constructs based on pABV437 to observe the responsible mutations for the adaptation phenotype is shown in (B). The numbers in the bar correspond to the 9 open reading frames.  
         [0023]    [0023]FIG. 2A: Multi-step growth curves on MARC-145 cells. Virus titer is in TDID50/ml.  
         [0024]    [0024]FIG. 2B: Multi-step growth curves on PAMs. Virus titer is in TCID50/ml.  
         [0025]    [0025]FIG. 2C: Immunostaining of infected cells using a PRRSV-specific Mab (122.17) against the nucleocapsid protein. The total amount of virus positive cells was counted per 2 cm 2 .  
         [0026]    [0026]FIG. 3: Mean virus titer per group of pigs after inoculation with vABV688, vABV707, vABV746 and vABV437 as determined by end point dilution. Significance of the differences between the-virus groups (p≦0.05) was determined using the Chi test.  
     
    
     DETAILED DESCRIPTION  
       [0027]    Materials and Methods.  
         [0028]    Cells and Viruses.  
         [0029]    Porcine alveolar lung macrophages (PAMs) were maintained in MCA-RPMI-1640 medium containing 5% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. CL2621-cells were propagated in Eagle&#39;s minimal essential medium supplemented with Hanks salts (Gibco BRL), 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 1.5% sodium bicarbonate and 1% L-glutamine. Serial passage of the recombinant PRRSVs vABV688 and vABV437 was performed as described. (Meulenberg et al, 1998). Challenge virus LV-Ter Huurne, a virulent European wild type isolate of PRRSV, was isolated during the 1991 epizootic from a clinical case of PRRSV in the Netherlands and propagated on PAMs. (Wensvoort et al, 1991). SDSU# 73, a virulent American wild type isolate of PRRSV (passage 3 on CL2621-cells), was kindly provided by Dr. E. Vaughn (Boehringer Ingelheim, Animal Health, Ames, Iowa) and propagated on MA-104 cells. Virus titers (expressed as 50% tissue culture infective doses (TCID 50 ) per ml) were determined on PAMs by end point dilution (Wensvoort et al, 1986) and calculated according to Reed and Muench. (Reed et al, 1938).  
         [0030]    Inoculation of pigs with PRRSV recombinants and test on their in vivo stability.  
         [0031]    Three groups of three 8-week-old Dutch Landrace/Yorkshire (LY) SPF pigs, tested free of antibodies against PRRSV, were inoculated on the same day intranasally with 2 ml of a recombinant virus stock of passage 5 each containing a titer of 105 TCID 50 /ml. The three groups were housed in isolated pens. Experimental procedures and animal management procedures were undertaken according to the Dutch legislation animal experiments. Serum samples were collected at day 0, 2, 4, 7, 9, 11, 14, 16, 18 and 21. At day 21, the pigs were sacrificed.  
         [0032]    Immunization of pigs with vABV688 and challenge with LV-Ter Huurne and SDSU#73.  
         [0033]    For each recombinant virus, two groups of five 8-week-old Dutch LY SPF pigs, lacking antibodies against PRRSV, were immunized intramuscularly (half-way between the pinna of the right ear and the cranial ridge of the right shoulder blade) with 2 ml of a virus stock of 10 5  TCID 50 /ml. All groups were housed in isolated pens. Experimental procedures and animal management procedures were undertaken according to the Dutch legislation animal experiments. In order to determine the transmission of recombinant virus from the inoculated pigs, one naive sentinel pig was introduced into each group of inoculated pigs 24 hours post-vaccination and sacrificed 28 days thereafter. At day 28 post-vaccination, two animals were separated from one group of each mutant and challenged intranasally with 2 ml 10 5  TCID 50 /ml LV-Ter Huurne. Similarly, two animals were separated from the other group and challenged with 2 ml 10 5  TCID 50 /ml SDSU#73. The two challenged animals joined the other three vaccinates after 24 hours. At 28 days after challenge, all pigs were sacrificed.  
         [0034]    A schematic representation of the amino acid changes is depicted in FIGS. 1A and 1B. To confirm the efficacy of challenge, two non-inoculated animals were either inoculated intranasally with 2 ml 10 5  TCID 50 /ml LV-Ter Huurne or SDSU#73. These challenged animals joined three sentinel animals after 24 hours and were monitored for two weeks starting at the moment of challenge. Serum samples were collected for all animals three times a week starting on day 0. During the experiment, the animals were observed daily for signs of disease, i.e., fever (a rectal body temperature higher than 39.7° C.), diarrhea and respiratory distress.  
         [0035]    Analysis of the genetic stability of the recombinant viruses.  
         [0036]    The genetic stability of the recombinant viruses in pigs was tested by inoculation of PAMs with serum from these pigs taken at the last-virus-positive day, followed by sequence analysis of the viral RNA. In short, as soon as cytopathogenic effect (cpe) was detected, the culture supernatant was harvested and the viral RNA was isolated as described (16). The RNA was reverse transcribed with primer LV76 (5′-TCTAGGAATTCTAGACGATCG(T) 40 -3′ (SEQ ID NO: 1); antisense; nucleotide (nt) 15088). The region flanking the introduced mutations was amplified by PCR using primers LV9 (5′-CTGCCGCCCGGGCAAGTGCC-3′ (SEQ ID NO: 2); sense; nt 11746) and LV22 (5′-CATAATAACCCTCAAGTTG-3′ (SEQ ID NO: 3); antisense; nt 12715) for vABV688. Nt numbers are based on the sequence of the LV isolate as deposited in GenBank, Accession number M96262 (SEQ ID NO: 4).  
         [0037]    The amplified fragments were analyzed in 2% agarose gels and the PCR fragments were excised from the gel and purified with SpinX columns (Costar). Sequence analysis of the fragments was performed using the antisense primer of the PCR, except for vABV688, for which primer LV24 (5′-AATCGGATCCTCAGGAAGCGTGCACACTGATGA-3′ (SEQ ID NO: 5); antisense; nt 12419) was used. The PRISM Ready Dye Deoxy Terminator cycle sequencing kit and the ABI PRISM 310 Genetic Analyzer (Perkin Elmer) were used to determine the sequences.  
         [0038]    Virus Isolation.  
         [0039]    10 5  PAMs/96 mm 2  were seeded in 96-wells plates and after one day, 50 μl of a 10-fold and 100-fold serial dilution of each serum sample was used to infect PAMs. After 48 hours, 25 μl of the culture supernatant was transferred to new PAMs seeded 16-24 hours prior to incubation. After 24 hours, the medium was discarded, the cells were washed with 0.05 M NaCl, dried and frozen for IPMA. (Wensvoort, 1986).  
         [0040]    Virus Titration.  
         [0041]    Virus titers were determined by end-point dilution on PAMs. (Wensvoort et al., 1986). Samples containing virus titers below the detection level (1,8) were considered negative. From the area under the curve of titer against time, the integrated virus titers after vaccination were determined. Statistical analysis was performed by the one-sided paired student&#39;s t-test. Results were considered statistically significant when the P-value was ≦0.05.  
         [0042]    Immunoperoxidase Monolayer Assay (IPMA).  
         [0043]    Immunostaining of PAMs was performed according to the method described. (Wensvoort et al, 1986). The expression of the EU- and US-PRRSV N protein was detected with monoclonal antibody (MAb) 122.  
         [0044]    Detection of Antibodies.  
         [0045]    The presence of antibodies against PRRSV in pig sera was determined by ELISA (IDEXX, Westbrook, Minn., US).  
       EXAMPLE 1  
       [0046]    Identification of Adaptation Sites.  
         [0047]    To identify important genomic regions for adaptation, the field isolate Lelystad virus was passaged 6 times on CL2621 cells followed by plaque purification for 3 times on the CL2621 cells and the virus was designated LV4.2.1. The entire sequence of the genome of the cell line-adapted strain LV4.2.1 was determined. RNA isolation, RT-PCR and PCR was performed yielding overlapping cDNA fragments of about 1.5 kb in length. All fragments were cloned into pGEM-T vector of PROMEGA using the TA-cloning strategy. When sequencing the whole sequence, every nucleotide was determined at least twice. To exclude mutations introduced by PCR-mismatches, a third or even a fourth cDNA-construct was made. When compared to the field strain Lelystad virus, 27 nucleotide differences in the coding region were identified and no nucleotide differences were identified in the 5′ and 3′ non-translated region. After translation, 8 amino acid differences resulted (Table 1).  
                                                                                                                 TABLE 1                           Amino acid differences between LV4.2.1, the field virus Lelystad virus       strain Ter Huurne, the infectious cDNA clone pABV437 and the       US prototype field virus strain ATCC VR2332.                ORFIA polyprotein   GP2A   GP2B   GP5                aa 663   aa 1084   aa 2343   aa 29   aa 88   aa 95   aa 27   aa 136                        LV4.2.1   Lys   Leu   Ala   Ser   Phe   Leu   Val   Tyr       Ter Huurne   Glu   Pro   Val   Pro   Val   Phe   Ala   Cys       pABV437   Glu   Pro   Val   Ser   Val   Phe   Val   Cys       ATCC-VR2332   Gln   Thr   Val   Leu   Thr   Leu   Ile   Trp                  
 
         [0048]    It was concluded that the observed difference in growth properties between LV4.2.1 and the field virus strain Lelystad virus was due to a different amino acid sequence in a non-structural protein, a structural protein or in a combination of two or more amino acid changes.  
         [0049]    Construction of full length cDNA with non-structural or structural gene mutations.  
         [0050]    A set of full length constructs (pABV647, 688, 689, and 690) was constructed based on pABV437 in which mutations were introduced. In vitro transcribed full length RNAs were transfected into the non-permissive BHK-21 cells using lipofectin as a cationic reagent. After 24 h, the supernatants (p0) were transferred to MARC-145 cells and after 24 h to 48 h, immunostaining was performed using MAb 122.17 directed against the N protein to detect the effect of the introduced mutations. Recombinant viruses containing substitutions responsible for adaptation showed cell line adapted phenotypes on MARC-145 cells similar to that of the positive control LV4.2.1. For the positive control, PAMs were infected with the same amount of p0 to show infection on the primary target cell of PRRSV. The supernatant p0 of pABV437 and LV4.2.1 virus served as positive controls in infection experiments.  
         [0051]    Two full length cDNA clones were constructed in which either mutations in the non-structural or in the structural region were introduced and designated as pABV647 and pABV690, respectively (FIG. 1B). In vitro analysis of these two constructs revealed that the RNA-transcript of pABV690 resulted in a phenotype similar to LV4.2.1. It was concluded that the mutations responsible for adaptation are located in the structural part of the genome.  
         [0052]    The next step was to determine whether the mutations in GP2a or in GP5 had any effect on the observed cell line-adaptation. pABV688 contains two amino acid differences in GP2a, while pABV689 contains only the amino acid change in GP5 (FIG. 1B). After performing the screening assay, pABV688 with the two mutations in GP2a resulted in a cell line-adapted phenotype similar to LV4.2.1 and the GP5 recombinant pABV689 did not. It was concluded that adaptation to the cell line is in majority caused by the two mutations in GP2a.  
         [0053]    Full Length Genome Constructs with Mutations in GP2a.  
         [0054]    Two amino acid differences in GP2a were studied. Two new constructs were generated. pABV772 contains a phenylalanine at amino acid 88 and pABV773 contains a leucine at position 95 in GP2a (FIG. 1B). In vitro, both substitutions were better adapted for growth in MARC-145 cells than the positive control pABV437, but the substitutions caused less cell pathologic effects (CPE) than vABV688.  
         [0055]    A multi-step and a one-step growth curve were performed to investigate the effect of the introduced amino acid residues on the infectivity process. The pO-medium of the recombinant viruses derived from pABV437/688/772/773 were harvested, transferred to PAMs and passaged three times (p3) in parallel with LV4.2.1 to increase the amount of virus. For further growth characterization, the presence of the introduced mutations was confirmed by sequence analysis and the TCID50/ml was determined (data not shown). Multi-step growth curves on both PAMs and MARC-145 were performed using a multiplicity of infection (m.o.i) of 0.05 with vABV437/688/772/773 and LV4.2.1. 10 6  cells were used in growth curves per 2 cm 2 . At different time points, virus was harvested and stored until the virus titer in TCID50/ml was determined.  
         [0056]    Introduction of a phenylalanine and a leucine in GP2a at amino acid 88 and 95, respectively, had a positive effect on the growth characteristics of a European strain of PRRSV on MARC-145 cells (FIG. 2A). No differences were observed when performing multi-step growth curves on PAMs (FIG. 2B). To secure the observed results, a total amount of 10 6  MARC-145 cells per 2 cm 2  were infected with a m.o.i. of 0.1 with supernatant p3 of vABV437/688/772/773 and LV4.2.1. After 12 h, an immunostaining was performed using a PRRSV-specific MAb (122.17) against the nucleocapsid protein and the total amount of positive cells were counted per 2 cm 2  (FIG. 2C). Based on the results, it was concluded that both amino acid residues at position 88 and 95 in the minor glycoprotein GP2a of PRRSV are important for adaptation to MARC-145 cells.  
         [0057]    The arteriviral reproduction process in permissive cells can be defined in seven distinct steps. The first two processes are called the viral entry and include (A) attachment of the virus to the cell surface of PAMs or MA-104 (or derivatives thereof) and (B) penetration of the virus through the cell membrane. Next, (C) the arterivirus needs to be uncoated in the cytoplasm before (D) replication, transcription and translation can occur. The next step is (E) RNA encapsidation and assembly, followed by (F) release of mature virions into the extracellular space which occurs before (G) spread of the virus can take place. Using a 12 h-time point in the one-step growth curves and knowing that the viral reproduction cycle of PRRSV takes about 10 hours, the observed difference in the amount of positive stained cells is caused by an increased number of cells being successfully infected (A-D). The observed difference is not due to a difference in encapsidation of the RNA, release of the newly produced virions or spread of the virus through a monolayer of MARC-145 cells (E-G). Since a role of glycoprotein GP2a in release of-viral RNA into the cytoplasm during uncoating (C) or in transcription and translation (D) is most unlikely, it was concluded that both residues, or a domain in which both residues are present, are important for the entry process, i.e., attachment and/or penetration of the virus (A and B).  
       EXAMPLE 2  
       [0058]    Animal Experiments with Cell Line-Adapted Recombinant PRRSV vABV688.  
         [0059]    In recombinant vABV688, amino acids 88 and 95 of the minor envelope glycoprotein GP2a were mutated and resulted in improved growth on MARC-145 cells. This characteristic facilitates the production of virus. In cell culture, these recombinant viruses were shown to be genetically stable and able to grow to virus titers sufficient to perform animal experiments. The properties of these PRRSV recombinants were studied with regard to safety and protective efficacy in animal experiments. The properties of the recombinant viruses were compared with those of virus derived from an infectious cDNA copy, vABV437. This virus is identical to wild type virus except for a PacI-restriction site and is, therefore, assumed to have similar properties as wild type virus. First, the in vivo genetic stability of vABV688 was determined in 8-week-old pigs. Subsequently, the immunogenicity, attenuation and efficacy of these viruses were tested in a homologous and heterologous immunization-challenge experiment in young pigs.  
         [0060]    Genetic Stability of the PRRSV Recombinant vABV688 in 8-Week-Old Pigs.  
         [0061]    In the first experiment, the genetic stability of the PRRSV recombinants was determined in vivo. Sequence analysis of viral RNA isolated from serum of all pigs inoculated from 14 days post inoculation (DPI) with the recombinant viruses at the last virus-positive day was performed. Sequence analysis of the fragments obtained by RT-PCR showed that the introduced mutations were present and that no additional changes were introduced in the domain of GP2a indicating that the recombinant viruses were genetically stable in vivo.  
         [0062]    Seroconversion of Virus inoculated Pigs and Sentinel Pigs.  
         [0063]    In the second experiment, it was determined whether pigs inoculated with the PRRSV recombinants and, subsequently, the sentinel pigs introduced into the inoculated groups had seroconverted. The presence of PRRSV antibodies was measured in IDEXX ELISA. Antibodies were detected in pigs inoculated with recombinant virus (i.e., ELISA sample-to-positive (S/P) ratio&gt;0.4) which indicated proper exposure of the viruses to the animals. Virus transmission is one of the characteristics of virulence. The sentinels had seroconverted at day 14 (vABV688) indicating that transmission of virus from the inoculated animals to these sentinels and, thus, conservation of the virulence.  
         [0064]    Duration and Height of Viremia after Inoculation.  
         [0065]    To determine the presence of virus in the serum of inoculated animals, virus isolation was performed for all sera collected. From the virus positive sera, virus titers were determined (FIG. 3A). All viruses induced viremia in the pigs and ranged from 2 dpi to 25 dpi.  
         [0066]    Viremia after Transmission of Virus to Sentinel Pigs.  
         [0067]    To determine spread of the recombinant virus to non-inoculated pigs, one sentinel animal was introduced into each inoculated group at 24 hours after inoculation. The virus titration of all virus positive sera indicated that the maximum virus titers did not differ between inoculated pigs and sentinels (data not shown).  
         [0068]    Challenge of virus-inoculated animals with homologous and heterologous PRRSV and transmission of challenge virus.  
         [0069]    To test whether inoculation with the recombinant virus protects pigs against wild type PRRSV, the inoculated pigs were challenged with EU- and US-PRRSV. Pigs inoculated with vABV437 remained virus negative after challenge with LV-Ter Huurne. Only one animal of the vABV688-inoculated pigs became virus positive for one day. After challenge with SDSU#73, all inoculated animals became viremic. All inoculated, unchallenged animals remained virus negative after they were united with their group members that were challenged with LV-Ter Huurne. In pigs inoculated with vABV688, viremia was induced after the inoculated pigs were united with SDSU#73-challenged pigs. In pigs used as challenge control, viremia was induced in all LV-Ter Huurne and SDSU#78-challenged pigs as well as in all sentinel animals (data not shown).  
         [0070]    Clinical Observations.  
         [0071]    After inoculation with the virus, no severe clinical signs of the disease were monitored. Only moderate rectal temperature increases were noted in all inoculated groups (data not shown). After challenge with LV-Ter Huurne in vABV688-inoculated animals and all SDSU#73-challenged animals, no temperature increases were measured.  
       EXAMPLE 3  
       [0072]    Attenuation of PRRS mutant virus strains with mutations in GP5 and/or ORFlab in addition to the mutations in GP2a (LV4.2.1). Comparison of virulence of 3 PRRS virus strains in an animal model.  
         [0073]    The clinical signs of PRRSV may differ among strains after exposure of pigs to PRRSV. The US PRRSV strains often cause respiratory and reproductive problems, whereas EU PRRSV strains predominantly cause reproductive problems. (Steverink et.al. 1999). Respiratory signs and the extent of lung involvement may vary per strain and are not consistently reproduced under experimental conditions. An infection model should include parameters to study and quantify the virulence of the virus based on frequency and severity of clinical signs, viral parameters (viremia, virus excretion and virus transmission) and seroconversion. Furthermore, duration, height and frequency of viremia and viral excretion after vaccination could also be important parameters to study vaccine safety. The same parameters after PRRSV challenge of vaccinated animals are important for vaccination efficacy since reducing virus shedding and virus transmission among pigs is an important target aiming at the control of PRRSV. Therefore pigs of 6 to 8 weeks old and 6 months old were infected with 3 different PRRSV strains, i.e., LV-Ter Huurne, SDSU#73 and LV4.2.1.  
         [0074]    The animals were conventional Landrace pigs obtained from a PRRSV-free farm in Denmark and born from PRRSV-unvaccinated sows. The pigs were free from antibodies against PRRSV as measured by an ELISA (IDEXX). Viremia was first detected in all pigs at 3 dpi except for the 6 month-old group infected with LV4.2.1 where viremia was first detected at 7 dpi and in one animal of this group, no viremia was detected. In 6-8 week-old pigs, viremia was detected until 42 dpi with a maximum frequency of positive animals of 88% and a virus titer ranging from 3, 4, 7, 10, 11 or 14 dpi and from 2.1-2.6.  
         [0075]    In 6 month-old pigs, viremia was detected until 28 dpi with a maximum frequency of 65% positive animals per time-point and virus titer ranging from 0.7-1. After 24 dpi, both LV-Ter Huurne and SDSU#73 were intermittently detected in the serum of pigs. LV4.2.1-infected pigs showed a shorter duration of viremia which was last detected at 21 dpi. The frequency of positive pigs at 7 and 10 dpi was significantly lower, had a maximum of 41% (p&gt;0.05) and a lower  10 log virus titer as compared to LV-Ter Huurne and SDSU#73. Kinetics of viremia was significantly different at 14 dpi for LV-Ter Huurne-infected pigs and SDSU#73-infected pigs.  
         [0076]    More pigs of 6-8 weeks excrete virus (maximum of positive animals per time-point was 50%) than pigs of 6 months (maximum of 37%). Furthermore, 6-8 week-old pigs showed a higher virus  10 log titer (between 0.2 and 1.9) in tonsillar swabs than 6 month-old pigs (ranging from 0.0 to 0.3) at day 3, 4, 7, 10 or 11 and 14. Per time-point, more pigs infected with LV-Ter Huurne excrete virus (maximum 100%) as compared to LV4.2.1 (maximum 17%) and SDSU#73 (maximum 50%). In addition, all pigs infected with LV-Ter Huurne excreted virus, but not all pigs infected with LV4.2.1 or SDSU#73 excreted virus. The  10 log virus titer in tonsillar swabs was higher for LV-Ter Huurne-infected pigs ranging from 0.0 to 3.0, while the  10 log titer of the LV4.2.1 infected group was 0.0 and the  10 log titer of the SDSU#73 infected pigs ranged from 0.0 to 0.5. At 14 dpi, pigs were still excreting virus.  
         [0077]    LV-Ter Huurne and SDSU#73 appear to be the most virulent due to the occurrence of a high level of virus-positive pigs, a long duration of viremia of both virus strains, the instigation of most severe clinical signs of SDSU#73 and a high level of virus excretion for LV-Ter Huurne. LV4.2.1 was less virulent since it showed less clinical signs and a low viremia and virus excretion. The reduced virulence of LV 4.2.1 was confirmed by impairment in its ability to cause reproductive problems for gestation sows as compared to LV-Ter Huurne. (Steverink et al. 1999). Sows infected with LV-Ter Huurne and LV4.2.1 resulted in the same number of piglets, but the piglets of the sows infected with the higher virulent strain were weaker. In addition, the group infected with the higher virulent virus LV-Ter Huurne showed a higher frequency of virus positive piglets until 28 days post farrowing. (Steverink et al 1999). Apparently, the lower virulence of LV4.2.1 is not related to macrophage infection since the growth curve of LV4.2.1 in vitro on macrophages is comparable to the growth curve of LV-Ter Huurne.  
       REFERENCES  
       [0078]    Bautista, E. M., Goyal, S. M., Yoon, I. J., Joo, H. S. &amp; Collins, J. E. (1993). Comparison of porcine alveolar macrophages and CL 2621 for the detection of porcine reproductive and respiratory syndrome (PRRS) virus and anti-PRRS antibody.  J Vet Diagn Invest  5, 163-5.  
         [0079]    Collins, J. E., Benfield, D. A., Christianson, W. T., Harris, L., Hennings, J. C., Shaw, D. P., Goyal, S. M., McCullough, S., Morrison, R. B., Joo, H. S. &amp; et al. (1992). Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs.  J Vet Diagn Invest  4, 117-26.  
         [0080]    Duan, X., Nauwynck, H. J., Favoreel, H. W. &amp; Pensaert, M. B. (1998). Identification of a putative receptor for porcine reproductive and respiratory syndrome virus on porcine alveolar macrophages.  J Virol  72, 4520-3.  
         [0081]    Jusa, E. R., Inaba, Y., Kouno, M. &amp; Hirose, O. (1997). Effect of heparin on infection of cells by porcine reproductive and respiratory syndrome virus.  Am J Vet Res  58, 488-91.  
         [0082]    Kreutz, L. C. (1998). Cellular membrane factors are the major determinants of porcine reproductive and respiratory syndrome virus tropism. Virus Res 53, 121-8.  
         [0083]    Meulenberg, J. J., Bos de Ruijter, J. N., van de Graaf, R., Wensvoort, G. &amp; Moormann, R. J. (1998). Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. J Virol 72, 380-7.  
         [0084]    Reed L J, Muench H. A simple method of estimating fifty percent end points. Am J Hyg 1938;27: 709-16.  
         [0085]    Snijder, E. J. &amp; Meulenberg, J. J. (1998). The molecular biology of arteriviruses.  J Gen Virol  79, 961-79.  
         [0086]    Wensvoort G, Terpstra C, Boonstra J, Bloemraad M, Van Zaane D. Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Vet Microbiol 1986;12: 101-8.  
         [0087]    Wensvoort, G., de Kluyver, E. P., Luijtze, E. A., den Besten, A., Harris, L., Collins, J. E., Christianson, W. T. &amp; Chladek, D. (1992). Antigenic comparison of Lelystad virus and swine infertility and respiratory syndrome (SIRS) virus.  J Vet Diagn Invest  4, 134-8.  
         [0088]    Wensvoort, G., Terpstra, C., Pol, J. M., ter Laak, E. A., Bloemraad, M., de Kluyver, E. P., Kragten, C., van Buiten, L., den Besten, A., Wagenaar, F. &amp; et al. (1991). Mystery swine disease in The Netherlands:the isolation of Lelystad virus.  Vet Q  13, 121-30.  
         [0089]    Steverink, P. J. G. M., Pol, J. M. A., Bos-De Ruijter, J. N. A. &amp; Meulenberg, J. J. M. Virulence of vABV414, the virus derived from the infectious cDNA clone of Lelystad virus, for third trimester pregnant gilts.  Proc. PRRS  &amp;  Aujeszky&#39;s disease  1999, 119.  
         [0090]    [0090] 
     
       
       
         0 
         
               
             
           
               
                   
               
               
                   
               
               
                   
               
               
                                                SEQUENCE LISTING  
               
               
                   
               
               
                   
               
               
                 &lt;160&gt; NUMBER OF SEQ ID NOS: 8  
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 1  
               
               
                 &lt;211&gt; LENGTH: 22  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;223&gt; OTHER INFORMATION: Synthesized sequence, primer  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 1  
               
               
                   
               
               
                 tctaggaatt ctagacgatc gt                                              22  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 2  
               
               
                 &lt;211&gt; LENGTH: 20  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;223&gt; OTHER INFORMATION: Synthesized sequence, primer  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 2  
               
               
                   
               
               
                 ctgccgcccg ggcaagtgcc                                                 20  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 3  
               
               
                 &lt;211&gt; LENGTH: 19  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;223&gt; OTHER INFORMATION: Synthesized sequence, primer  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 3  
               
               
                   
               
               
                 cataataacc ctcaagttg                                                  19  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 4  
               
               
                 &lt;211&gt; LENGTH: 15111  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Lelystad virus  
               
               
                 &lt;300&gt; PUBLICATION INFORMATION:  
               
               
                 &lt;308&gt; DATABASE ACCESSION NUMBER: Genbank/M96262  
               
               
                 &lt;309&gt; DATABASE ENTRY DATE: 2000-11-08  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 4  
               
               
                   
               
               
                 atgatgtgta gggtattccc cctacataca cgacacttct agtgtttgtg taccttggag     60  
               
               
                   
               
               
                 gcgtgggtac agccccgccc caccccttgg cccctgttct agcccaacag gtatccttct    120  
               
               
                   
               
               
                 ctctcggggc gagtgcgccg cctgctgctc ccttgcagcg ggaaggacct cccgagtatt    180  
               
               
                   
               
               
                 tccggagagc acctgcttta cgggatctcc accctttaac catgtctggg acgttctccc    240  
               
               
                   
               
               
                 ggtgcatgtg caccccggct gcccgggtat tttggaacgc cggccaagtc ttttgcacac    300  
               
               
                   
               
               
                 ggtgtctcag tgcgcggtct cttctctctc cagagcttca ggacactgac ctcggtgcag    360  
               
               
                   
               
               
                 ttggcttgtt ttacaagcct agggacaagc ttcactggaa agtccctatc ggcatccctc    420  
               
               
                   
               
               
                 aggtggaatg tactccatcc gggtgctgtt ggctctcagc tgttttccct ttggcgcgta    480  
               
               
                   
               
               
                 tgacctccgg caatcacaac ttcctccaac gacttgtgaa ggttgctgat gttttgtacc    540  
               
               
                   
               
               
                 gtgacggttg cttggcacct cgacaccttc gtgaactcca agtttacgag cgcggctgca    600  
               
               
                   
               
               
                 actggtaccc gatcacgggg cccgtgcccg ggatgggttt gtttgcgaac tccatgcacg    660  
               
               
                   
               
               
                 tatccgacca gccgttccct ggtgccaccc atgtgttgac taactcgcct ttgcctcaac    720  
               
               
                   
               
               
                 aggcttgtcg gcagccgttc tgtccatttg aggaggctca ttctagcgtg tacaggtgga    780  
               
               
                   
               
               
                 agaaatttgt ggttttcacg gactcctccc tcaacggtcg atctcgcatg atgtggacgc    840  
               
               
                   
               
               
                 cggaatccga tgattcagcc gccctggagg tactaccgcc tgagttagaa cgtcaggtcg    900  
               
               
                   
               
               
                 aaatcctcat tcggagtttt cctgctcatc accctgtcga cctggccgac tgggagctca    960  
               
               
                   
               
               
                 ctgagtcccc tgagaacggt ttttccttca acacgtctca ttcttgcggt caccttgtcc   1020  
               
               
                   
               
               
                 agaaccccga cgtgtttgat ggcaagtgct ggctctcctg ctttttgggc cagtcggtcg   1080  
               
               
                   
               
               
                 aagtgcgctg ccatgaggaa catctagctg acgccttcgg ttaccaaacc aagtggggcg   1140  
               
               
                   
               
               
                 tgcatggtaa gtacctccag cgcaggcttc aagttcgcgg cattcgtgct gtagtcgatc   1200  
               
               
                   
               
               
                 ctgatggtcc cattcacgtt gaagcgctgt cttgccccca gtcttggatc aggcacctga   1260  
               
               
                   
               
               
                 ctctggatga tgatgtcacc ccaggattcg ttcgcctgac atcccttcgc attgtgccga   1320  
               
               
                   
               
               
                 acacagagcc taccacttcc cggatctttc ggtttggagc gcataagtgg tatggcgctg   1380  
               
               
                   
               
               
                 ccggcaaacg ggctcgtgct aagcgtgccg ctaaaagtga gaaggattcg gctcccaccc   1440  
               
               
                   
               
               
                 ccaaggttgc cctgccggtc cccacctgtg gaattaccac ctactctcca ccgacagacg   1500  
               
               
                   
               
               
                 ggtcttgtgg ttggcatgtc cttgccgcca taatgaaccg gatgataaat ggtgacttca   1560  
               
               
                   
               
               
                 cgtcccctct gactcagtac aacagaccag aggatgattg ggcttctgat tatgatcttg   1620  
               
               
                   
               
               
                 ttcaggcgat tcaatgtcta cgactgcctg ctaccgtggt tcggaatcgc gcctgtccta   1680  
               
               
                   
               
               
                 acgccaagta ccttataaaa cttaacggag ttcactggga ggtagaggtg aggtctggaa   1740  
               
               
                   
               
               
                 tggctcctcg ctccctttct cgtgaatgtg tggttggcgt ttgctctgaa ggctgtgtcg   1800  
               
               
                   
               
               
                 caccgcctta tccagcagac gggctaccta aacgtgcact cgaggccttg gcgtctgctt   1860  
               
               
                   
               
               
                 acagactacc ctccgattgt gttagctctg gtattgctga ctttcttgct aatccacctc   1920  
               
               
                   
               
               
                 ctcaggaatt ctggaccctc gacaaaatgt tgacctcccc gtcaccagag cggtccggct   1980  
               
               
                   
               
               
                 tctctagttt gtataaatta ctattagagg ttgttccgca aaaatgcggt gccacggaag   2040  
               
               
                   
               
               
                 gggctttcat ctatgctgtt gagaggatgt tgaaggattg tccgagctcc aaacaggcca   2100  
               
               
                   
               
               
                 tggcccttct ggcaaaaatt aaagttccat cctcaaaggc cccgtctgtg tccctggacg   2160  
               
               
                   
               
               
                 agtgtttccc tacggatgtt ttagccgact tcgagccagc atctcaggaa aggccccaaa   2220  
               
               
                   
               
               
                 gttccggcgc tgctgttgtc ctgtgttcac cggatgcaaa agagttcgag gaagcagccc   2280  
               
               
                   
               
               
                 cggaagaagt tcaagagagt ggccacaagg ccgtccactc tgcactcctt gccgagggtc   2340  
               
               
                   
               
               
                 ctaacaatga gcaggtacag gtggttgccg gtgagcaact gaagctcggc ggttgtggtt   2400  
               
               
                   
               
               
                 tggcagtcgg gaatgctcat gaaggtgctc tggtctcagc tggtctaatt aacctggtag   2460  
               
               
                   
               
               
                 gcgggaattt gtccccctca gaccccatga aagaaaacat gctcaatagc cgggaagacg   2520  
               
               
                   
               
               
                 aaccactgga tttgtcccaa ccagcaccag cttccacaac gacccttgtg agagagcaaa   2580  
               
               
                   
               
               
                 cacccgacaa cccaggttct gatgccggtg ccctccccgt caccgttcga gaatttgtcc   2640  
               
               
                   
               
               
                 cgacggggcc tatactctgt catgttgagc actgcggcac ggagtcgggc gacagcagtt   2700  
               
               
                   
               
               
                 cgcctttgga tctatctgat gcgcaaaccc tggaccagcc tttaaatcta tccctggccg   2760  
               
               
                   
               
               
                 cttggccagt gagggccacc gcgtctgacc ctggctgggt ccacggtagg cgcgagcctg   2820  
               
               
                   
               
               
                 tctttgtaaa gcctcgaaat gctttctctg atggcgattc agcccttcag ttcggggagc   2880  
               
               
                   
               
               
                 tttctgaatc cagctctgtc atcgagtttg accggacaaa agatgctccg gtggttgacg   2940  
               
               
                   
               
               
                 cccctgtcga cttgacgact tcgaacgagg ccctctctgt agtcgatcct ttcgaatttg   3000  
               
               
                   
               
               
                 ccgaactcaa gcgcccgcgt ttctccgcac aagccttaat tgaccgaggc ggtccacttg   3060  
               
               
                   
               
               
                 ccgatgtcca tgcaaaaata aagaaccggg tatatgaaca gtgcctccaa gcttgtgagc   3120  
               
               
                   
               
               
                 ccggtagtcg tgcaacccca gccaccaggg agtggctcga caaaatgtgg gatagggtgg   3180  
               
               
                   
               
               
                 acatgaaaac ttggcgctgc acctcgcagt tccaagctgg tcgcattctt gcgtccctca   3240  
               
               
                   
               
               
                 aattcctccc tgacatgatt caagacacac cgcctcctgt tcccaggaag aaccgagcta   3300  
               
               
                   
               
               
                 gtgacaatgc cggcctgaag caactggtgg cacagtggga taggaaattg agtgtgaccc   3360  
               
               
                   
               
               
                 cccccccaaa accggttggg ccagtgcttg accagatcgt ccctccgcct acggatatcc   3420  
               
               
                   
               
               
                 agcaagaaga tgtcaccccc tccgatgggc caccccatgc gccggatttt cctagtcgag   3480  
               
               
                   
               
               
                 tgagcacggg cgggagttgg aaaggcctta tgctttccgg cacccgtctc gcggggtcta   3540  
               
               
                   
               
               
                 tcagccagcg ccttatgaca tgggtttttg aagttttctc ccacctccca gcttttatgc   3600  
               
               
                   
               
               
                 tcacactttt ctcgccgcgg ggctctatgg ctccaggtga ttggttgttt gcaggtgtcg   3660  
               
               
                   
               
               
                 ttttacttgc tctcttgctc tgtcgttctt acccgatact cggatgcctt cccttattgg   3720  
               
               
                   
               
               
                 gtgtcttttc tggttctttg cggcgtgttc gtctgggtgt ttttggttct tggatggctt   3780  
               
               
                   
               
               
                 ttgctgtatt tttattctcg actccatcca acccagtcgg ttcttcttgt gaccacgatt   3840  
               
               
                   
               
               
                 cgccggagtg tcatgctgag cttttggctc ttgagcagcg ccaactttgg gaacctgtgc   3900  
               
               
                   
               
               
                 gcggccttgt ggtcggcccc tcaggcctct tatgtgtcat tcttggcaag ttactcggtg   3960  
               
               
                   
               
               
                 ggtcacgtta tctctggcat gttctcctac gtttatgcat gcttgcagat ttggcccttt   4020  
               
               
                   
               
               
                 ctcttgttta tgtggtgtcc caggggcgtt gtcacaagtg ttggggaaag tgtataagga   4080  
               
               
                   
               
               
                 cagctcctgc ggaggtggct cttaatgtat ttcctttctc gcgcgccacc cgtgtctctc   4140  
               
               
                   
               
               
                 ttgtatcctt gtgtgatcga ttccaaacgc caaaaggggt tgatcctgtg cacttggcaa   4200  
               
               
                   
               
               
                 cgggttggcg cgggtgctgg cgtggtgaga gccccatcca tcaaccacac caaaagccca   4260  
               
               
                   
               
               
                 tagcttatgc caatttggat gaaaagaaaa tgtctgccca aacggtggtt gctgtcccat   4320  
               
               
                   
               
               
                 acgatcccag tcaggctatc aaatgcctga aagttctgca ggcgggaggg gccatcgtgg   4380  
               
               
                   
               
               
                 accagcctac acctgaggtc gttcgtgtgt ccgagatccc cttctcagcc ccatttttcc   4440  
               
               
                   
               
               
                 caaaagttcc agtcaaccca gattgcaggg ttgtggtaga ttcggacact tttgtggctg   4500  
               
               
                   
               
               
                 cggttcgctg cggttactcg acagcacaac tggttctggg ccggggcaac tttgccaagt   4560  
               
               
                   
               
               
                 taaatcagac cccccccagg aactctatct ccaccaaaac gactggtggg gcctcttaca   4620  
               
               
                   
               
               
                 cccttgctgt ggctcaagtg tctgcgtgga ctcttgttca tttcatcctc ggtctttggt   4680  
               
               
                   
               
               
                 tcacatcacc tcaagtgtgt ggccgaggaa ccgctgaccc atggtgttca aatccttttt   4740  
               
               
                   
               
               
                 catatcctac ctatggcccc ggagttgtgt gctcctctcg actttgtgtg tctgccgacg   4800  
               
               
                   
               
               
                 gggtcaccct gccattgttc tcagccgtgg cacaactctc cggtagagag gtggggattt   4860  
               
               
                   
               
               
                 ttattttggt gctcgtctcc ttgactgctt tggcccaccg catggctctt aaggcagaca   4920  
               
               
                   
               
               
                 tgttagtggt cttttcggct ttttgtgctt acgcctggcc catgagctcc tggttaatct   4980  
               
               
                   
               
               
                 gcttctttcc tatactcttg aagtgggtta cccttcaccc tcttactatg ctttgggtgc   5040  
               
               
                   
               
               
                 actcattctt ggtgttttgt ctgccagcag ccggcatcct ctcactaggg ataactggcc   5100  
               
               
                   
               
               
                 ttctttgggc aattggccgc tttacccagg ttgccggaat tattacacct tatgacatcc   5160  
               
               
                   
               
               
                 accagtacac ctctgggcca cgtggtgcag ctgctgtggc cacagcccca gaaggcactt   5220  
               
               
                   
               
               
                 atatggccgc cgtccggaga gctgctttaa ctgggcgaac tttaatcttc accccgtctg   5280  
               
               
                   
               
               
                 cagttggatc ccttctcgaa ggtgctttca ggactcataa accctgcctt aacaccgtga   5340  
               
               
                   
               
               
                 atgttgtagg ctcttccctt ggttccggag gggttttcac cattgatggc agaagaactg   5400  
               
               
                   
               
               
                 tcgtcactgc tgcccatgtg ttgaacggcg acacagctag agtcaccggc gactcctaca   5460  
               
               
                   
               
               
                 accgcatgca cactttcaag accaatggtg attatgcctg gtcccatgct gatgactggc   5520  
               
               
                   
               
               
                 agggcgttgc ccctgtggtc aaggttgcga aggggtaccg cggtcgtgcc tactggcaaa   5580  
               
               
                   
               
               
                 catcaactgg tgtcgaaccc ggtatcattg gggaagggtt cgccttctgt tttactaact   5640  
               
               
                   
               
               
                 gcggcgattc ggggtcaccc gtcatctcag aatctggtga tcttattgga atccacaccg   5700  
               
               
                   
               
               
                 gttcaaacaa acttggttct ggtcttgtga caacccctga aggggagacc tgcaccatca   5760  
               
               
                   
               
               
                 aagaaaccaa gctctctgac ctttccagac attttgcagg cccaagcgtt cctcttgggg   5820  
               
               
                   
               
               
                 acattaaatt gagtccggcc atcatccctg atgtaacatc cattccgagt gacttggcat   5880  
               
               
                   
               
               
                 cgctcctagc ctccgtccct gtagtggaag gcggcctctc gaccgttcaa cttttgtgtg   5940  
               
               
                   
               
               
                 tctttttcct tctctggcgc atgatgggcc atgcctggac acccattgtt gccgtgggct   6000  
               
               
                   
               
               
                 tctttttgct gaatgaaatt cttccagcag ttttggtccg agccgtgttt tcttttgcac   6060  
               
               
                   
               
               
                 tctttgtgct tgcatgggcc accccctggt ctgcacaggt gttgatgatt agactcctca   6120  
               
               
                   
               
               
                 cggcatctct caaccgcaac aagctttctc tggcgttcta cgcactcggg ggtgtcgtcg   6180  
               
               
                   
               
               
                 gtttggcagc tgaaatcggg acttttgctg gcagattgtc tgaattgtct caagctcttt   6240  
               
               
                   
               
               
                 cgacatactg cttcttacct agggtccttg ctatgaccag ttgtgttccc accatcatca   6300  
               
               
                   
               
               
                 ttggtggact ccataccctc ggtgtgattc tgtggttatt caaataccgg tgcctccaca   6360  
               
               
                   
               
               
                 acatgctggt tggtgatggg agtttttcaa gcgccttctt cctacggtat tttgcagagg   6420  
               
               
                   
               
               
                 gtaatctcag aaaaggtgtt tcacagtcct gtggcatgaa taacgagtcc ctaacggctg   6480  
               
               
                   
               
               
                 ctttagcttg caagttgtca caggctgacc ttgatttttt gtccagctta acgaacttca   6540  
               
               
                   
               
               
                 agtgctttgt atctgcttca aacatgaaaa atgctgccgg ccagtacatt gaagcagcgt   6600  
               
               
                   
               
               
                 atgccaaggc cctgcgccaa gagttggcct ctctagttca gattgacaaa atgaaaggag   6660  
               
               
                   
               
               
                 ttttgtccaa gctcgaggcc tttgctgaaa cagccacccc gtcccttgac ataggtgacg   6720  
               
               
                   
               
               
                 tgattgttct gcttgggcaa catcctcacg gatccatcct cgatattaat gtggggactg   6780  
               
               
                   
               
               
                 aaaggaaaac tgtgtccgtg caagagaccc ggagcctagg cggctccaaa ttcagtgttt   6840  
               
               
                   
               
               
                 gtactgtcgt gtccaacaca cccgtggacg ccttgaccgg catcccactc cagacaccaa   6900  
               
               
                   
               
               
                 cccctctttt tgagaatggt ccgcgtcatc gcagcgagga agacgatctt aaagtcgaga   6960  
               
               
                   
               
               
                 ggatgaagaa acactgtgta tccctcggct tccacaacat caatggcaaa gtttactgca   7020  
               
               
                   
               
               
                 aaatttggga caagtctacc ggtgacacct tttacacgga tgattcccgg tacacccaag   7080  
               
               
                   
               
               
                 accatgcttt tcaggacagg tcagccgact acagagacag ggactatgag ggtgtgcaaa   7140  
               
               
                   
               
               
                 ccacccccca acagggattt gatccaaagt ctgaaacccc tgttggcact gttgtgatcg   7200  
               
               
                   
               
               
                 gcggtattac gtataacagg tatctgatca aaggtaagga ggttctggtc cccaagcctg   7260  
               
               
                   
               
               
                 acaactgcct tgaagctgcc aagctgtccc ttgagcaagc tctcgctggg atgggccaaa   7320  
               
               
                   
               
               
                 cttgcgacct tacagctgcc gaggtggaaa agctaaagcg catcattagt caactccaag   7380  
               
               
                   
               
               
                 gtttgaccac tgaacaggct ttaaactgtt agccgccagc ggcttgaccc gctgtggccg   7440  
               
               
                   
               
               
                 cggcggccta gttgtgactg aaacggcggt aaaaattata aaataccaca gcagaacttt   7500  
               
               
                   
               
               
                 caccttaggc cctttagacc taaaagtcac ttccgaggtg gaggtaaaga aatcaactga   7560  
               
               
                   
               
               
                 gcagggccac gctgttgtgg caaacttatg ttccggtgtc atcttgatga gacctcaccc   7620  
               
               
                   
               
               
                 accgtccctt gtcgacgttc ttctgaaacc cggacttgac acaatacccg gcattcaacc   7680  
               
               
                   
               
               
                 agggcatggg gccgggaata tgggcgtgga cggttctatt tgggattttg aaaccgcacc   7740  
               
               
                   
               
               
                 cacaaaggca gaactcgagt tatccaagca aataatccaa gcatgtgaag ttaggcgcgg   7800  
               
               
                   
               
               
                 ggacgccccg aacctccaac tcccttacaa gctctatcct gttagggggg atcctgagcg   7860  
               
               
                   
               
               
                 gcataaaggc cgccttatca ataccaggtt tggagattta ccttacaaaa ctcctcaaga   7920  
               
               
                   
               
               
                 caccaagtcc gcaatccacg cggcttgttg cctgcacccc aacggggccc ccgtgtctga   7980  
               
               
                   
               
               
                 tggtaaatcc acactaggta ccactcttca acatggtttc gagctttatg tccctactgt   8040  
               
               
                   
               
               
                 gccctatagt gtcatggagt accttgattc acgccctgac acccctttta tgtgtactaa   8100  
               
               
                   
               
               
                 acatggcact tccaaggctg ctgcagagga cctccaaaaa tacgacctat ccacccaagg   8160  
               
               
                   
               
               
                 atttgtcctg cctggggtcc tacgcctagt acgcagattc atctttggcc atattggtaa   8220  
               
               
                   
               
               
                 ggcgccgcca ttgttcctcc catcaaccta tcccgccaag aactctatgg cagggatcaa   8280  
               
               
                   
               
               
                 tggccagagg ttcccaacaa aggacgttca gagcatacct gaaattgatg aaatgtgtgc   8340  
               
               
                   
               
               
                 ccgcgctgtc aaggagaatt ggcaaactgt gacaccttgc accctcaaga aacagtactg   8400  
               
               
                   
               
               
                 ttccaagccc aaaaccagga ccatcctggg caccaacaac tttattgcct tggctcacag   8460  
               
               
                   
               
               
                 atcggcgctc agtggtgtca cccaggcatt catgaagaag gcttggaagt ccccaattgc   8520  
               
               
                   
               
               
                 cttggggaaa aacaaattca aggagctgca ttgcactgtc gccggcaggt gtcttgaggc   8580  
               
               
                   
               
               
                 cgacttggcc tcctgtgacc gcagcacccc cgccattgta agatggtttg ttgccaacct   8640  
               
               
                   
               
               
                 cctgtatgaa cttgcaggat gtgaagagta cttgcctagc tatgtgctta attgctgcca   8700  
               
               
                   
               
               
                 tgacctcgtg gcaacacagg atggtgcctt cacaaaacgc ggtggcctgt cgtccgggga   8760  
               
               
                   
               
               
                 ccccgtcacc agtgtgtcca acaccgtata ttcactggta atttatgccc agcacatggt   8820  
               
               
                   
               
               
                 attgtcggcc ttgaaaatgg gtcatgaaat tggtcttaag ttcctcgagg aacagctcaa   8880  
               
               
                   
               
               
                 gttcgaggac ctccttgaaa ttcagcctat gttggtatac tctgatgatc ttgtcttgta   8940  
               
               
                   
               
               
                 cgctgaaaga cccacatttc ccaattacca ctggtgggtc gagcaccttg acctgatgct   9000  
               
               
                   
               
               
                 gggtttcaga acggacccaa agaaaaccgt cataactgat aaacccagct tcctcggctg   9060  
               
               
                   
               
               
                 cagaattgag gcagggcgac agctagtccc caatcgcgac cgcatcctgg ctgctcttgc   9120  
               
               
                   
               
               
                 atatcacatg aaggcgcaga acgcctcaga gtattatgcg tctgctgccg caatcctgat   9180  
               
               
                   
               
               
                 ggattcatgt gcttgcattg accatgaccc tgagtggtat gaggacctca tctgcggtat   9240  
               
               
                   
               
               
                 tgcccggtgc gcccgccagg atggttatag cttcccaggt ccggcatttt tcatgtccat   9300  
               
               
                   
               
               
                 gtgggagaag ctgagaagtc ataatgaagg gaagaaattc cgccactgcg gcatctgcga   9360  
               
               
                   
               
               
                 cgccaaagcc gactatgcgt ccgcctgtgg gcttgatttg tgtttgttcc attcgcactt   9420  
               
               
                   
               
               
                 tcatcaacac tgccctgtca ctctgagctg cggtcaccat gccggttcaa aggaatgttc   9480  
               
               
                   
               
               
                 gcagtgtcag tcacctgttg gggctggcag atcccctctt gatgccgtgc taaaacaaat   9540  
               
               
                   
               
               
                 tccatacaaa cctcctcgta ctgtcatcat gaaggtgggt aataaaacaa cggccctcga   9600  
               
               
                   
               
               
                 tccggggagg taccagtccc gtcgaggtct cgttgcagtc aagaggggta ttgcaggcaa   9660  
               
               
                   
               
               
                 tgaagttgat ctttctgatg gggactacca agtggtgcct cttttgccga cttgcaaaga   9720  
               
               
                   
               
               
                 cataaacatg gtgaaggtgg cttgcaatgt actactcagc aagttcatag tagggccacc   9780  
               
               
                   
               
               
                 aggttccgga aagaccacct ggctactgag tcaagtccag gacgatgatg tcatttacac   9840  
               
               
                   
               
               
                 acccacccat cagactatgt ttgatatagt cagtgctctc aaagtttgca ggtattccat   9900  
               
               
                   
               
               
                 tccaggagcc tcaggactcc ctttcccacc acctgccagg tccgggccgt gggttaggct   9960  
               
               
                   
               
               
                 tattgccagc gggcacgtcc ctggccgagt atcatacctc gatgaggctg gatattgtaa  10020  
               
               
                   
               
               
                 tcatctggac attcttagac tgctttccaa aacacccctt gtgtgtttgg gtgaccttca  10080  
               
               
                   
               
               
                 gcaacttcac cctgtcggct ttgattccta ctgttatgtg ttcgatcaga tgcctcagaa  10140  
               
               
                   
               
               
                 gcagctgacc actatttaca gatttggccc taacatctgc gcagccatcc agccttgtta  10200  
               
               
                   
               
               
                 cagggagaaa cttgaatcta aggctaggaa cactagggtg gtttttacca cccggcctgt  10260  
               
               
                   
               
               
                 ggcctttggt caggtgctga caccatacca taaagatcgc atcggctctg cgataaccat  10320  
               
               
                   
               
               
                 agattcatcc cagggggcca cctttgatat tgtgacattg catctaccat cgccaaagtc  10380  
               
               
                   
               
               
                 cctaaataaa tcccgagcac ttgtagccat cactcgggca agacacgggt tgttcattta  10440  
               
               
                   
               
               
                 tgaccctcat aaccagctcc aggagttttt caacttaacc cctgagcgca ctgattgtaa  10500  
               
               
                   
               
               
                 ccttgtgttc agccgtgggg atgagctggt agttctgaat gcggataatg cagtcacaac  10560  
               
               
                   
               
               
                 tgtagcgaag gcccttgaga caggtccatc tcgatttcga gtatcagacc cgaggtgcaa  10620  
               
               
                   
               
               
                 gtctctctta gccgcttgtt cggccagtct ggaagggagc tgtatgccac taccgcaagt  10680  
               
               
                   
               
               
                 ggcacataac ctggggtttt acttttcccc ggacagtcca acatttgcac ctctgccaaa  10740  
               
               
                   
               
               
                 agagttggcg ccacattggc cagtggttac ccaccagaat aatcgggcgt ggcctgatcg  10800  
               
               
                   
               
               
                 acttgtcgct agtatgcgcc caattgatgc ccgctacagc aagccaatgg tcggtgcagg  10860  
               
               
                   
               
               
                 gtatgtggtc gggccgtcca cctttcttgg tactcctggt gtggtgtcat actatctcac  10920  
               
               
                   
               
               
                 actatacatc aggggtgagc cccaggcctt gccagaaaca ctcgtttcaa cagggcgtat  10980  
               
               
                   
               
               
                 agccacagat tgtcgggagt atctcgacgc ggctgaggaa gaggcagcaa aagaactccc  11040  
               
               
                   
               
               
                 ccacgcattc attggcgatg tcaaaggtac cacggttggg gggtgtcatc acattacatc  11100  
               
               
                   
               
               
                 aaaataccta cctaggtccc tgcctaagga ctctgttgcc gtagttggag taagttcgcc  11160  
               
               
                   
               
               
                 cggcagggct gctaaagccg tgtgcactct caccgatgtg tacctccccg aactccggcc  11220  
               
               
                   
               
               
                 atatctgcaa cctgagacgg catcaaaatg ctggaaactc aaattagact tcagggacgt  11280  
               
               
                   
               
               
                 ccgactaatg gtctggaaag gagccaccgc ctatttccag ttggaagggc ttacatggtc  11340  
               
               
                   
               
               
                 ggcgctgccc gactatgcca ggtttattca gctgcccaag gatgccgttg tatacattga  11400  
               
               
                   
               
               
                 tccgtgtata ggaccggcaa cagccaaccg taaggtcgtg cgaaccacag actggcgggc  11460  
               
               
                   
               
               
                 cgacctggca gtgacaccgt atgattacgg tgcccagaac attttgacaa cagcctggtt  11520  
               
               
                   
               
               
                 cgaggacctc gggccgcagt ggaagatttt ggggttgcag ccctttaggc gagcatttgg  11580  
               
               
                   
               
               
                 ctttgaaaac actgaggatt gggcaatcct tgcacgccgt atgaatgacg gcaaggacta  11640  
               
               
                   
               
               
                 cactgactat aactggaact gtgttcgaga acgcccacac gccatctacg ggcgtgctcg  11700  
               
               
                   
               
               
                 tgaccatacg tatcattttg cccctggcac agaattgcag gtagagctag gtaaaccccg  11760  
               
               
                   
               
               
                 gctgccgcct gggcaagtgc cgtgaattcg gggtgatgca atggggtcac tgtggagtaa  11820  
               
               
                   
               
               
                 aatcagccag ctgttcgtgg acgccttcac tgagttcctt gttagtgtgg ttgatattgc  11880  
               
               
                   
               
               
                 cattttcctt gccatactgt ttgggttcac cgtcgcagga tggttactgg tctttcttct  11940  
               
               
                   
               
               
                 cagagtggtt tgctccgcgc ttctccgttc gcgctctgcc attcactctc ccgaactatc  12000  
               
               
                   
               
               
                 gaaggtccta tgaaggcttg ttgcccaact gcagaccgga tgtcccacaa tttgcagtca  12060  
               
               
                   
               
               
                 agcacccatt gggtatgttt tggcacatgc gagtttccca cttgattgat gagatggtct  12120  
               
               
                   
               
               
                 ctcgtcgcat ttaccagacc atggaacatt caggtcaagc ggcctggaag caggtggttg  12180  
               
               
                   
               
               
                 gtgaggccac tctcacgaag ctgtcagggc tcgatatagt tactcatttc caacacctgg  12240  
               
               
                   
               
               
                 ccgcagtgga ggcggattct tgccgctttc tcagctcacg actcgtgatg ctaaaaaatc  12300  
               
               
                   
               
               
                 ttgccgttgg caatgtgagc ctacagtaca acaccacgtt ggaccgcgtt gagctcatct  12360  
               
               
                   
               
               
                 tccccacgcc aggtacgagg cccaagttga ccgatttcag acaatggctc atcagtgtgc  12420  
               
               
                   
               
               
                 acgcttccat tttttcctct gtggcttcat ctgttacctt gttcatagtg ctttggcttc  12480  
               
               
                   
               
               
                 gaattccagc tctacgctat gtttttggtt tccattggcc cacggcaaca catcattcga  12540  
               
               
                   
               
               
                 gctgaccatc aactacacca tatgcatgcc ctgttctacc agtcaagcgg ctcgccaaag  12600  
               
               
                   
               
               
                 gctcgagccc ggtcgtaaca tgtggtgcaa aatagggcat gacaggtgtg aggagcgtga  12660  
               
               
                   
               
               
                 ccatgatgag ttgttaatgt ccatcccgtc cgggtacgac aacctcaaac ttgagggtta  12720  
               
               
                   
               
               
                 ttatgcttgg ctggcttttt tgtccttttc ctacgcggcc caattccatc cggagttgtt  12780  
               
               
                   
               
               
                 cgggataggg aatgtgtcgc gcgtcttcgt ggacaagcga caccagttca tttgtgccga  12840  
               
               
                   
               
               
                 gcatgatgga cacaattcaa ccgtatctac cggacacaac atctccgcat tatatgcggc  12900  
               
               
                   
               
               
                 atattaccac caccaaatag acgggggcaa ttggttccat ttggaatggc tgcggccact  12960  
               
               
                   
               
               
                 cttttcttcc tggctggtgc tcaacatatc atggtttctg aggcgttcgc ctgtaagccc  13020  
               
               
                   
               
               
                 tgtttctcga cgcatctatc agatattgag accaacacga ccgcggctgc cggtttcatg  13080  
               
               
                   
               
               
                 gtccttcagg acatcaattg tttccgacct cacggggtct cagcagcgca agagaaaatt  13140  
               
               
                   
               
               
                 tccttcggaa agtcgtccca atgtcgtgaa gccgtcggta ctccccagta catcacgata  13200  
               
               
                   
               
               
                 acggctaacg tgaccgacga atcatacttg tacaacgcgg acctgctgat gctttctgcg  13260  
               
               
                   
               
               
                 tgccttttct acgcctcaga aatgagcgag aaaggcttca aagtcatctt tgggaatgtc  13320  
               
               
                   
               
               
                 tctggcgttg tttctgcttg tgtcaatttc acagattatg tggcccatgt gacccaacat  13380  
               
               
                   
               
               
                 acccagcagc atcatctggt aattgatcac attcggttgc tgcatttcct gacaccatct  13440  
               
               
                   
               
               
                 gcaatgaggt gggctacaac cattgcttgt ttgttcgcca ttctcttggc aatatgagat  13500  
               
               
                   
               
               
                 gttctcacaa attggggcgt ttcttgactc cgcactcttg cttctggtgg ctttttttgc  13560  
               
               
                   
               
               
                 tgtgtaccgg cttgtcctgg tcctttgccg atggcaacgg cgacagctcg acataccaat  13620  
               
               
                   
               
               
                 acatatataa cttgacgata tgcgagctga atgggaccga ctggttgtcc agccattttg  13680  
               
               
                   
               
               
                 gttgggcagt cgagaccttt gtgctttacc cggttgccac tcatatcctc tcactgggtt  13740  
               
               
                   
               
               
                 ttctcacaac aagccatttt tttgacgcgc tcggtctcgg cgctgtatcc actgcaggat  13800  
               
               
                   
               
               
                 ttgttggcgg gcggtacgta ctctgcagcg tctacggcgc ttgtgctttc gcagcgttcg  13860  
               
               
                   
               
               
                 tatgttttgt catccgtgct gctaaaaatt gcatggcctg ccgctatgcc cgtacccggt  13920  
               
               
                   
               
               
                 ttaccaactt cattgtggac gaccggggga gagttcatcg atggaagtct ccaatagtgg  13980  
               
               
                   
               
               
                 tagaaaaatt gggcaaagcc gaagtcgatg gcaacctcgt caccatcaaa catgtcgtcc  14040  
               
               
                   
               
               
                 tcgaaggggt taaagctcaa cccttgacga ggacttcggc tgagcaatgg gaggcctaga  14100  
               
               
                   
               
               
                 cgatttttgc aacgatccta tcgccgcaca aaagctcgtg ctagccttta gcatcacata  14160  
               
               
                   
               
               
                 cacacctata atgatatacg cccttaaggt gtcacgcggc cgactcctgg ggctgttgca  14220  
               
               
                   
               
               
                 catcctaata tttctgaact gttcctttac attcggatac atgacatatg tgcattttca  14280  
               
               
                   
               
               
                 atccaccaac cgtgtcgcac ttaccctggg ggctgttgtc gcccttctgt ggggtgttta  14340  
               
               
                   
               
               
                 cagcttcaca gagtcatgga agtttatcac ttccagatgc agattgtgtt gccttggccg  14400  
               
               
                   
               
               
                 gcgatacatt ctggcccctg cccatcacgt agaaagtgct gcaggtctcc attcaatctc  14460  
               
               
                   
               
               
                 agcgtctggt aaccgagcat acgctgtgag aaagcccgga ctaacatcag tgaacggcac  14520  
               
               
                   
               
               
                 tctagtacca ggacttcgga gcctcgtgct gggcggcaaa cgagctgtta aacgaggagt  14580  
               
               
                   
               
               
                 ggttaacctc gtcaagtatg gccggtaaaa accagagcca gaagaaaaag aaaagtacag  14640  
               
               
                   
               
               
                 ctccgatggg gaatggccag ccagtcaatc aactgtgcca gttgctgggt gcaatgataa  14700  
               
               
                   
               
               
                 agtcccagcg ccagcaacct aggggaggac aggccaaaaa gaaaaagcct gagaagccac  14760  
               
               
                   
               
               
                 attttcccct ggctgctgaa gatgacatcc ggcaccacct cacccagact gaacgctccc  14820  
               
               
                   
               
               
                 tctgcttgca atcgatccag acggctttca atcaaggcgc aggaactgcg tcgctttcat  14880  
               
               
                   
               
               
                 ccagcgggaa ggtcagtttt caggttgagt ttatgctgcc ggttgctcat acagtgcgcc  14940  
               
               
                   
               
               
                 tgattcgcgt gacttctaca tccgccagtc agggtgcaag ttaatttgac agtcaggtga  15000  
               
               
                   
               
               
                 atggccgcga ttggcgtgtg gcctctgagt cacctattca attagggcga tcacatgggg  15060  
               
               
                   
               
               
                 gtcatactta atcaggcagg aaccatgtga ccgaaattaa aaaaaaaaaa a           15111  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 5  
               
               
                 &lt;211&gt; LENGTH: 33  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Artificial sequence  
               
               
                 &lt;220&gt; FEATURE:  
               
               
                 &lt;223&gt; OTHER INFORMATION: Synthesized sequence, primer  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 5  
               
               
                   
               
               
                 aatcggatcc tcaggaagcg tgcacactga tga                                  33  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 6  
               
               
                 &lt;211&gt; LENGTH: 249  
               
               
                 &lt;212&gt; TYPE: PRT  
               
               
                 &lt;213&gt; ORGANISM: Lelystad virus  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 6  
               
               
                   
               
               
                 Met Gln Trp Gly His Cys Gly Val Lys Ser Ala Ser Cys Ser Trp Thr  
               
               
                 1               5                   10                  15  
               
               
                   
               
               
                 Pro Ser Leu Ser Ser Leu Leu Val Trp Leu Ile Leu Pro Phe Ser Leu  
               
               
                             20                  25                  30  
               
               
                   
               
               
                 Pro Tyr Cys Leu Gly Ser Pro Ser Gln Asp Gly Tyr Trp Ser Phe Phe  
               
               
                         35                  40                  45  
               
               
                   
               
               
                 Ser Glu Trp Phe Ala Pro Arg Phe Ser Val Arg Ala Leu Pro Phe Thr  
               
               
                     50                  55                  60  
               
               
                   
               
               
                 Leu Pro Asn Tyr Arg Arg Ser Tyr Glu Gly Leu Leu Pro Asn Cys Arg  
               
               
                 65                  70                  75                  80  
               
               
                   
               
               
                 Pro Asp Val Pro Gln Phe Ala Val Lys His Pro Leu Gly Met Phe Trp  
               
               
                                 85                  90                  95  
               
               
                   
               
               
                 His Met Arg Val Ser His Leu Ile Asp Glu Met Val Ser Arg Arg Ile  
               
               
                             100                 105                 110  
               
               
                   
               
               
                 Tyr Gln Thr Met Glu His Ser Gly Gln Ala Ala Trp Lys Gln Val Val  
               
               
                         115                 120                 125  
               
               
                   
               
               
                 Gly Glu Ala Thr Leu Thr Lys Leu Ser Gly Leu Asp Ile Val Thr His  
               
               
                     130                 135                 140  
               
               
                   
               
               
                 Phe Gln His Leu Ala Ala Val Glu Ala Asp Ser Cys Arg Phe Leu Ser  
               
               
                 145                 150                 155                 160  
               
               
                   
               
               
                 Ser Arg Leu Val Met Leu Lys Asn Leu Ala Val Gly Asn Val Ser Leu  
               
               
                                 165                 170                 175  
               
               
                   
               
               
                 Gln Tyr Asn Thr Thr Leu Asp Arg Val Glu Leu Ile Phe Pro Thr Pro  
               
               
                             180                 185                 190  
               
               
                   
               
               
                 Gly Thr Arg Pro Lys Leu Thr Asp Phe Arg Gln Trp Leu Ile Ser Val  
               
               
                         195                 200                 205  
               
               
                   
               
               
                 His Ala Ser Ile Phe Ser Ser Val Ala Ser Ser Val Thr Leu Phe Ile  
               
               
                     210                 215                 220  
               
               
                   
               
               
                 Val Leu Trp Leu Arg Ile Pro Ala Leu Arg Tyr Val Phe Gly Phe His  
               
               
                 225                 230                 235                 240  
               
               
                   
               
               
                 Trp Pro Thr Ala Thr His His Ser Ser  
               
               
                                 245  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 7  
               
               
                 &lt;211&gt; LENGTH: 201  
               
               
                 &lt;212&gt; TYPE: PRT  
               
               
                 &lt;213&gt; ORGANISM: Lelystad virus  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 7  
               
               
                   
               
               
                 Met Arg Cys Ser His Lys Leu Gly Arg Phe Leu Thr Pro His Ser Cys  
               
               
                 1               5                   10                  15  
               
               
                   
               
               
                 Phe Trp Trp Leu Phe Leu Leu Cys Thr Gly Leu Ser Trp Ser Phe Ala  
               
               
                             20                  25                  30  
               
               
                   
               
               
                 Asp Gly Asn Gly Asp Ser Ser Thr Tyr Gln Tyr Ile Tyr Asn Leu Thr  
               
               
                         35                  40                  45  
               
               
                   
               
               
                 Ile Cys Glu Leu Asn Gly Thr Asp Trp Leu Ser Ser His Phe Gly Trp  
               
               
                     50                  55                  60  
               
               
                   
               
               
                 Ala Val Glu Thr Phe Val Leu Tyr Pro Val Ala Thr His Ile Leu Ser  
               
               
                 65                  70                  75                  80  
               
               
                   
               
               
                 Leu Gly Phe Leu Thr Thr Ser His Phe Phe Asp Ala Leu Gly Leu Gly  
               
               
                                 85                  90                  95  
               
               
                   
               
               
                 Ala Val Ser Thr Ala Gly Phe Val Gly Gly Arg Tyr Val Leu Cys Ser  
               
               
                             100                 105                 110  
               
               
                   
               
               
                 Val Tyr Gly Ala Cys Ala Phe Ala Ala Phe Val Cys Phe Val Ile Arg  
               
               
                         115                 120                 125  
               
               
                   
               
               
                 Ala Ala Lys Asn Cys Met Ala Cys Arg Tyr Ala Arg Thr Arg Phe Thr  
               
               
                     130                 135                 140  
               
               
                   
               
               
                 Asn Phe Ile Val Asp Asp Arg Gly Arg Val His Arg Trp Lys Ser Pro  
               
               
                 145                 150                 155                 160  
               
               
                   
               
               
                 Ile Val Val Glu Lys Leu Gly Lys Ala Glu Val Asp Gly Asn Leu Val  
               
               
                                 165                 170                 175  
               
               
                   
               
               
                 Thr Ile Lys His Val Val Leu Glu Gly Val Lys Ala Gln Pro Leu Thr  
               
               
                             180                 185                 190  
               
               
                   
               
               
                 Arg Thr Ser Ala Glu Gln Trp Glu Ala  
               
               
                         195                 200  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 8  
               
               
                 &lt;211&gt; LENGTH: 3859  
               
               
                 &lt;212&gt; TYPE: PRT  
               
               
                 &lt;213&gt; ORGANISM: Lelystad virus  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 8  
               
               
                   
               
               
                 Met Ser Gly Thr Phe Ser Arg Cys Met Cys Thr Pro Ala Ala Arg Val  
               
               
                 1               5                   10                  15  
               
               
                   
               
               
                 Phe Trp Asn Ala Gly Gln Val Phe Cys Thr Arg Cys Leu Ser Ala Arg  
               
               
                             20                  25                  30  
               
               
                   
               
               
                 Ser Leu Leu Ser Pro Glu Leu Gln Asp Thr Asp Leu Gly Ala Val Gly  
               
               
                         35                  40                  45  
               
               
                   
               
               
                 Leu Phe Tyr Lys Pro Arg Asp Lys Leu His Trp Lys Val Pro Ile Gly  
               
               
                     50                  55                  60  
               
               
                   
               
               
                 Ile Pro Gln Val Glu Cys Thr Pro Ser Gly Cys Cys Trp Leu Ser Ala  
               
               
                 65                  70                  75                  80  
               
               
                   
               
               
                 Val Phe Pro Leu Ala Arg Met Thr Ser Gly Asn His Asn Phe Leu Gln  
               
               
                                 85                  90                  95  
               
               
                   
               
               
                 Arg Leu Val Lys Val Ala Asp Val Leu Tyr Arg Asp Gly Cys Leu Ala  
               
               
                             100                 105                 110  
               
               
                   
               
               
                 Pro Arg His Leu Arg Glu Leu Gln Val Tyr Glu Arg Gly Cys Asn Trp  
               
               
                         115                 120                 125  
               
               
                   
               
               
                 Tyr Pro Ile Thr Gly Pro Val Pro Gly Met Gly Leu Phe Ala Asn Ser  
               
               
                     130                 135                 140  
               
               
                   
               
               
                 Met His Val Ser Asp Gln Pro Phe Pro Gly Ala Thr His Val Leu Thr  
               
               
                 145                 150                 155                 160  
               
               
                   
               
               
                 Asn Ser Pro Leu Pro Gln Gln Ala Cys Arg Gln Pro Phe Cys Pro Phe  
               
               
                                 165                 170                 175  
               
               
                   
               
               
                 Glu Glu Ala His Ser Ser Val Tyr Arg Trp Lys Lys Phe Val Val Phe  
               
               
                             180                 185                 190  
               
               
                   
               
               
                 Thr Asp Ser Ser Leu Asn Gly Arg Ser Arg Met Met Trp Thr Pro Glu  
               
               
                         195                 200                 205  
               
               
                   
               
               
                 Ser Asp Asp Ser Ala Ala Leu Glu Val Leu Pro Pro Glu Leu Glu Arg  
               
               
                     210                 215                 220  
               
               
                   
               
               
                 Gln Val Glu Ile Leu Ile Arg Ser Phe Pro Ala His His Pro Val Asp  
               
               
                 225                 230                 235                 240  
               
               
                   
               
               
                 Leu Ala Asp Trp Glu Leu Thr Glu Ser Pro Glu Asn Gly Phe Ser Phe  
               
               
                                 245                 250                 255  
               
               
                   
               
               
                 Asn Thr Ser His Ser Cys Gly His Leu Val Gln Asn Pro Asp Val Phe  
               
               
                             260                 265                 270  
               
               
                   
               
               
                 Asp Gly Lys Cys Trp Leu Ser Cys Phe Leu Gly Gln Ser Val Glu Val  
               
               
                         275                 280                 285  
               
               
                   
               
               
                 Arg Cys His Glu Glu His Leu Ala Asp Ala Phe Gly Tyr Gln Thr Lys  
               
               
                     290                 295                 300  
               
               
                   
               
               
                 Trp Gly Val His Gly Lys Tyr Leu Gln Arg Arg Leu Gln Val Arg Gly  
               
               
                 305                 310                 315                 320  
               
               
                   
               
               
                 Ile Arg Ala Val Val Asp Pro Asp Gly Pro Ile His Val Glu Ala Leu  
               
               
                                 325                 330                 335  
               
               
                   
               
               
                 Ser Cys Pro Gln Ser Trp Ile Arg His Leu Thr Leu Asp Asp Asp Val  
               
               
                             340                 345                 350  
               
               
                   
               
               
                 Thr Pro Gly Phe Val Arg Leu Thr Ser Leu Arg Ile Val Pro Asn Thr  
               
               
                         355                 360                 365  
               
               
                   
               
               
                 Glu Pro Thr Thr Ser Arg Ile Phe Arg Phe Gly Ala His Lys Trp Tyr  
               
               
                     370                 375                 380  
               
               
                   
               
               
                 Gly Ala Ala Gly Lys Arg Ala Arg Ala Lys Arg Ala Ala Lys Ser Glu  
               
               
                 385                 390                 395                 400  
               
               
                   
               
               
                 Lys Asp Ser Ala Pro Thr Pro Lys Val Ala Leu Pro Val Pro Thr Cys  
               
               
                                 405                 410                 415  
               
               
                   
               
               
                 Gly Ile Thr Thr Tyr Ser Pro Pro Thr Asp Gly Ser Cys Gly Trp His  
               
               
                             420                 425                 430  
               
               
                   
               
               
                 Val Leu Ala Ala Ile Met Asn Arg Met Ile Asn Gly Asp Phe Thr Ser  
               
               
                         435                 440                 445  
               
               
                   
               
               
                 Pro Leu Thr Gln Tyr Asn Arg Pro Glu Asp Asp Trp Ala Ser Asp Tyr  
               
               
                     450                 455                 460  
               
               
                   
               
               
                 Asp Leu Val Gln Ala Ile Gln Cys Leu Arg Leu Pro Ala Thr Val Val  
               
               
                 465                 470                 475                 480  
               
               
                   
               
               
                 Arg Asn Arg Ala Cys Pro Asn Ala Lys Tyr Leu Ile Lys Leu Asn Gly  
               
               
                                 485                 490                 495  
               
               
                   
               
               
                 Val His Trp Glu Val Glu Val Arg Ser Gly Met Ala Pro Arg Ser Leu  
               
               
                             500                 505                 510  
               
               
                   
               
               
                 Ser Arg Glu Cys Val Val Gly Val Cys Ser Glu Gly Cys Val Ala Pro  
               
               
                         515                 520                 525  
               
               
                   
               
               
                 Pro Tyr Pro Ala Asp Gly Leu Pro Lys Arg Ala Leu Glu Ala Leu Ala  
               
               
                     530                 535                 540  
               
               
                   
               
               
                 Ser Ala Tyr Arg Leu Pro Ser Asp Cys Val Ser Ser Gly Ile Ala Asp  
               
               
                 545                 550                 555                 560  
               
               
                   
               
               
                 Phe Leu Ala Asn Pro Pro Pro Gln Glu Phe Trp Thr Leu Asp Lys Met  
               
               
                                 565                 570                 575  
               
               
                   
               
               
                 Leu Thr Ser Pro Ser Pro Glu Arg Ser Gly Phe Ser Ser Leu Tyr Lys  
               
               
                             580                 585                 590  
               
               
                   
               
               
                 Leu Leu Leu Glu Val Val Pro Gln Lys Cys Gly Ala Thr Glu Gly Ala  
               
               
                         595                 600                 605  
               
               
                   
               
               
                 Phe Ile Tyr Ala Val Glu Arg Met Leu Lys Asp Cys Pro Ser Ser Lys  
               
               
                     610                 615                 620  
               
               
                   
               
               
                 Gln Ala Met Ala Leu Leu Ala Lys Ile Lys Val Pro Ser Ser Lys Ala  
               
               
                 625                 630                 635                 640  
               
               
                   
               
               
                 Pro Ser Val Ser Leu Asp Glu Cys Phe Pro Thr Asp Val Leu Ala Asp  
               
               
                                 645                 650                 655  
               
               
                   
               
               
                 Phe Glu Pro Ala Ser Gln Glu Arg Pro Gln Ser Ser Gly Ala Ala Val  
               
               
                             660                 665                 670  
               
               
                   
               
               
                 Val Leu Cys Ser Pro Asp Ala Lys Glu Phe Glu Glu Ala Ala Pro Glu  
               
               
                         675                 680                 685  
               
               
                   
               
               
                 Glu Val Gln Glu Ser Gly His Lys Ala Val His Ser Ala Leu Leu Ala  
               
               
                     690                 695                 700  
               
               
                   
               
               
                 Glu Gly Pro Asn Asn Glu Gln Val Gln Val Val Ala Gly Glu Gln Leu  
               
               
                 705                 710                 715                 720  
               
               
                   
               
               
                 Lys Leu Gly Gly Cys Gly Leu Ala Val Gly Asn Ala His Glu Gly Ala  
               
               
                                 725                 730                 735  
               
               
                   
               
               
                 Leu Val Ser Ala Gly Leu Ile Asn Leu Val Gly Gly Asn Leu Ser Pro  
               
               
                             740                 745                 750  
               
               
                   
               
               
                 Ser Asp Pro Met Lys Glu Asn Met Leu Asn Ser Arg Glu Asp Glu Pro  
               
               
                         755                 760                 765  
               
               
                   
               
               
                 Leu Asp Leu Ser Gln Pro Ala Pro Ala Ser Thr Thr Thr Leu Val Arg  
               
               
                     770                 775                 780  
               
               
                   
               
               
                 Glu Gln Thr Pro Asp Asn Pro Gly Ser Asp Ala Gly Ala Leu Pro Val  
               
               
                 785                 790                 795                 800  
               
               
                   
               
               
                 Thr Val Arg Glu Phe Val Pro Thr Gly Pro Ile Leu Cys His Val Glu  
               
               
                                 805                 810                 815  
               
               
                   
               
               
                 His Cys Gly Thr Glu Ser Gly Asp Ser Ser Ser Pro Leu Asp Leu Ser  
               
               
                             820                 825                 830  
               
               
                   
               
               
                 Asp Ala Gln Thr Leu Asp Gln Pro Leu Asn Leu Ser Leu Ala Ala Trp  
               
               
                         835                 840                 845  
               
               
                   
               
               
                 Pro Val Arg Ala Thr Ala Ser Asp Pro Gly Trp Val His Gly Arg Arg  
               
               
                     850                 855                 860  
               
               
                   
               
               
                 Glu Pro Val Phe Val Lys Pro Arg Asn Ala Phe Ser Asp Gly Asp Ser  
               
               
                 865                 870                 875                 880  
               
               
                   
               
               
                 Ala Leu Gln Phe Gly Glu Leu Ser Glu Ser Ser Ser Val Ile Glu Phe  
               
               
                                 885                 890                 895  
               
               
                   
               
               
                 Asp Arg Thr Lys Asp Ala Pro Val Val Asp Ala Pro Val Asp Leu Thr  
               
               
                             900                 905                 910  
               
               
                   
               
               
                 Thr Ser Asn Glu Ala Leu Ser Val Val Asp Pro Phe Glu Phe Ala Glu  
               
               
                         915                 920                 925  
               
               
                   
               
               
                 Leu Lys Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly  
               
               
                     930                 935                 940  
               
               
                   
               
               
                 Pro Leu Ala Asp Val His Ala Lys Ile Lys Asn Arg Val Tyr Glu Gln  
               
               
                 945                 950                 955                 960  
               
               
                   
               
               
                 Cys Leu Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr Arg  
               
               
                                 965                 970                 975  
               
               
                   
               
               
                 Glu Trp Leu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg  
               
               
                             980                 985                 990  
               
               
                   
               
               
                 Cys Thr Ser Gln Phe Gln Ala Gly  Arg Ile Leu Ala Ser  Leu Lys Phe  
               
               
                         995                 1000                 1005  
               
               
                   
               
               
                 Leu Pro  Asp Met Ile Gln Asp  Thr Pro Pro Pro Val  Pro Arg Lys  
               
               
                     1010                 1015                 1020  
               
               
                   
               
               
                 Asn Arg  Ala Ser Asp Asn Ala  Gly Leu Lys Gln Leu  Val Ala Gln  
               
               
                     1025                 1030                 1035  
               
               
                   
               
               
                 Trp Asp  Arg Lys Leu Ser Val  Thr Pro Pro Pro Lys  Pro Val Gly  
               
               
                     1040                 1045                 1050  
               
               
                   
               
               
                 Pro Val  Leu Asp Gln Ile Val  Pro Pro Pro Thr Asp  Ile Gln Gln  
               
               
                     1055                 1060                 1065  
               
               
                   
               
               
                 Glu Asp  Val Thr Pro Ser Asp  Gly Pro Pro His Ala  Pro Asp Phe  
               
               
                     1070                 1075                 1080  
               
               
                   
               
               
                 Pro Ser  Arg Val Ser Thr Gly  Gly Ser Trp Lys Gly  Leu Met Leu  
               
               
                     1085                 1090                 1095  
               
               
                   
               
               
                 Ser Gly  Thr Arg Leu Ala Gly  Ser Ile Ser Gln Arg  Leu Met Thr  
               
               
                     1100                 1105                 1110  
               
               
                   
               
               
                 Trp Val  Phe Glu Val Phe Ser  His Leu Pro Ala Phe  Met Leu Thr  
               
               
                     1115                 1120                 1125  
               
               
                   
               
               
                 Leu Phe  Ser Pro Arg Gly Ser  Met Ala Pro Gly Asp  Trp Leu Phe  
               
               
                     1130                 1135                 1140  
               
               
                   
               
               
                 Ala Gly  Val Val Leu Leu Ala  Leu Leu Leu Cys Arg  Ser Tyr Pro  
               
               
                     1145                 1150                 1155  
               
               
                   
               
               
                 Ile Leu  Gly Cys Leu Pro Leu  Leu Gly Val Phe Ser  Gly Ser Leu  
               
               
                     1160                 1165                 1170  
               
               
                   
               
               
                 Arg Arg  Val Arg Leu Gly Val  Phe Gly Ser Trp Met  Ala Phe Ala  
               
               
                     1175                 1180                 1185  
               
               
                   
               
               
                 Val Phe  Leu Phe Ser Thr Pro  Ser Asn Pro Val Gly  Ser Ser Cys  
               
               
                     1190                 1195                 1200  
               
               
                   
               
               
                 Asp His  Asp Ser Pro Glu Cys  His Ala Glu Leu Leu  Ala Leu Glu  
               
               
                     1205                 1210                 1215  
               
               
                   
               
               
                 Gln Arg  Gln Leu Trp Glu Pro  Val Arg Gly Leu Val  Val Gly Pro  
               
               
                     1220                 1225                 1230  
               
               
                   
               
               
                 Ser Gly  Leu Leu Cys Val Ile  Leu Gly Lys Leu Leu  Gly Gly Ser  
               
               
                     1235                 1240                 1245  
               
               
                   
               
               
                 Arg Tyr  Leu Trp His Val Leu  Leu Arg Leu Cys Met  Leu Ala Asp  
               
               
                     1250                 1255                 1260  
               
               
                   
               
               
                 Leu Ala  Leu Ser Leu Val Tyr  Val Val Ser Gln Gly  Arg Cys His  
               
               
                     1265                 1270                 1275  
               
               
                   
               
               
                 Lys Cys  Trp Gly Lys Cys Ile  Arg Thr Ala Pro Ala  Glu Val Ala  
               
               
                     1280                 1285                 1290  
               
               
                   
               
               
                 Leu Asn  Val Phe Pro Phe Ser  Arg Ala Thr Arg Val  Ser Leu Val  
               
               
                     1295                 1300                 1305  
               
               
                   
               
               
                 Ser Leu  Cys Asp Arg Phe Gln  Thr Pro Lys Gly Val  Asp Pro Val  
               
               
                     1310                 1315                 1320  
               
               
                   
               
               
                 His Leu  Ala Thr Gly Trp Arg  Gly Cys Trp Arg Gly  Glu Ser Pro  
               
               
                     1325                 1330                 1335  
               
               
                   
               
               
                 Ile His  Gln Pro His Gln Lys  Pro Ile Ala Tyr Ala  Asn Leu Asp  
               
               
                     1340                 1345                 1350  
               
               
                   
               
               
                 Glu Lys  Lys Met Ser Ala Gln  Thr Val Val Ala Val  Pro Tyr Asp  
               
               
                     1355                 1360                 1365  
               
               
                   
               
               
                 Pro Ser  Gln Ala Ile Lys Cys  Leu Lys Val Leu Gln  Ala Gly Gly  
               
               
                     1370                 1375                 1380  
               
               
                   
               
               
                 Ala Ile  Val Asp Gln Pro Thr  Pro Glu Val Val Arg  Val Ser Glu  
               
               
                     1385                 1390                 1395  
               
               
                   
               
               
                 Ile Pro  Phe Ser Ala Pro Phe  Phe Pro Lys Val Pro  Val Asn Pro  
               
               
                     1400                 1405                 1410  
               
               
                   
               
               
                 Asp Cys  Arg Val Val Val Asp  Ser Asp Thr Phe Val  Ala Ala Val  
               
               
                     1415                 1420                 1425  
               
               
                   
               
               
                 Arg Cys  Gly Tyr Ser Thr Ala  Gln Leu Val Leu Gly  Arg Gly Asn  
               
               
                     1430                 1435                 1440  
               
               
                   
               
               
                 Phe Ala  Lys Leu Asn Gln Thr  Pro Pro Arg Asn Ser  Ile Ser Thr  
               
               
                     1445                 1450                 1455  
               
               
                   
               
               
                 Lys Thr  Thr Gly Gly Ala Ser  Tyr Thr Leu Ala Val  Ala Gln Val  
               
               
                     1460                 1465                 1470  
               
               
                   
               
               
                 Ser Ala  Trp Thr Leu Val His  Phe Ile Leu Gly Leu  Trp Phe Thr  
               
               
                     1475                 1480                 1485  
               
               
                   
               
               
                 Ser Pro  Gln Val Cys Gly Arg  Gly Thr Ala Asp Pro  Trp Cys Ser  
               
               
                     1490                 1495                 1500  
               
               
                   
               
               
                 Asn Pro  Phe Ser Tyr Pro Thr  Tyr Gly Pro Gly Val  Val Cys Ser  
               
               
                     1505                 1510                 1515  
               
               
                   
               
               
                 Ser Arg  Leu Cys Val Ser Ala  Asp Gly Val Thr Leu  Pro Leu Phe  
               
               
                     1520                 1525                 1530  
               
               
                   
               
               
                 Ser Ala  Val Ala Gln Leu Ser  Gly Arg Glu Val Gly  Ile Phe Ile  
               
               
                     1535                 1540                 1545  
               
               
                   
               
               
                 Leu Val  Leu Val Ser Leu Thr  Ala Leu Ala His Arg  Met Ala Leu  
               
               
                     1550                 1555                 1560  
               
               
                   
               
               
                 Lys Ala  Asp Met Leu Val Val  Phe Ser Ala Phe Cys  Ala Tyr Ala  
               
               
                     1565                 1570                 1575  
               
               
                   
               
               
                 Trp Pro  Met Ser Ser Trp Leu  Ile Cys Phe Phe Pro  Ile Leu Leu  
               
               
                     1580                 1585                 1590  
               
               
                   
               
               
                 Lys Trp  Val Thr Leu His Pro  Leu Thr Met Leu Trp  Val His Ser  
               
               
                     1595                 1600                 1605  
               
               
                   
               
               
                 Phe Leu  Val Phe Cys Leu Pro  Ala Ala Gly Ile Leu  Ser Leu Gly  
               
               
                     1610                 1615                 1620  
               
               
                   
               
               
                 Ile Thr  Gly Leu Leu Trp Ala  Ile Gly Arg Phe Thr  Gln Val Ala  
               
               
                     1625                 1630                 1635  
               
               
                   
               
               
                 Gly Ile  Ile Thr Pro Tyr Asp  Ile His Gln Tyr Thr  Ser Gly Pro  
               
               
                     1640                 1645                 1650  
               
               
                   
               
               
                 Arg Gly  Ala Ala Ala Val Ala  Thr Ala Pro Glu Gly  Thr Tyr Met  
               
               
                     1655                 1660                 1665  
               
               
                   
               
               
                 Ala Ala  Val Arg Arg Ala Ala  Leu Thr Gly Arg Thr  Leu Ile Phe  
               
               
                     1670                 1675                 1680  
               
               
                   
               
               
                 Thr Pro  Ser Ala Val Gly Ser  Leu Leu Glu Gly Ala  Phe Arg Thr  
               
               
                     1685                 1690                 1695  
               
               
                   
               
               
                 His Lys  Pro Cys Leu Asn Thr  Val Asn Val Val Gly  Ser Ser Leu  
               
               
                     1700                 1705                 1710  
               
               
                   
               
               
                 Gly Ser  Gly Gly Val Phe Thr  Ile Asp Gly Arg Arg  Thr Val Val  
               
               
                     1715                 1720                 1725  
               
               
                   
               
               
                 Thr Ala  Ala His Val Leu Asn  Gly Asp Thr Ala Arg  Val Thr Gly  
               
               
                     1730                 1735                 1740  
               
               
                   
               
               
                 Asp Ser  Tyr Asn Arg Met His  Thr Phe Lys Thr Asn  Gly Asp Tyr  
               
               
                     1745                 1750                 1755  
               
               
                   
               
               
                 Ala Trp  Ser His Ala Asp Asp  Trp Gln Gly Val Ala  Pro Val Val  
               
               
                     1760                 1765                 1770  
               
               
                   
               
               
                 Lys Val  Ala Lys Gly Tyr Arg  Gly Arg Ala Tyr Trp  Gln Thr Ser  
               
               
                     1775                 1780                 1785  
               
               
                   
               
               
                 Thr Gly  Val Glu Pro Gly Ile  Ile Gly Glu Gly Phe  Ala Phe Cys  
               
               
                     1790                 1795                 1800  
               
               
                   
               
               
                 Phe Thr  Asn Cys Gly Asp Ser  Gly Ser Pro Val Ile  Ser Glu Ser  
               
               
                     1805                 1810                 1815  
               
               
                   
               
               
                 Gly Asp  Leu Ile Gly Ile His  Thr Gly Ser Asn Lys  Leu Gly Ser  
               
               
                     1820                 1825                 1830  
               
               
                   
               
               
                 Gly Leu  Val Thr Thr Pro Glu  Gly Glu Thr Cys Thr  Ile Lys Glu  
               
               
                     1835                 1840                 1845  
               
               
                   
               
               
                 Thr Lys  Leu Ser Asp Leu Ser  Arg His Phe Ala Gly  Pro Ser Val  
               
               
                     1850                 1855                 1860  
               
               
                   
               
               
                 Pro Leu  Gly Asp Ile Lys Leu  Ser Pro Ala Ile Ile  Pro Asp Val  
               
               
                     1865                 1870                 1875  
               
               
                   
               
               
                 Thr Ser  Ile Pro Ser Asp Leu  Ala Ser Leu Leu Ala  Ser Val Pro  
               
               
                     1880                 1885                 1890  
               
               
                   
               
               
                 Val Val  Glu Gly Gly Leu Ser  Thr Val Gln Leu Leu  Cys Val Phe  
               
               
                     1895                 1900                 1905  
               
               
                   
               
               
                 Phe Leu  Leu Trp Arg Met Met  Gly His Ala Trp Thr  Pro Ile Val  
               
               
                     1910                 1915                 1920  
               
               
                   
               
               
                 Ala Val  Gly Phe Phe Leu Leu  Asn Glu Ile Leu Pro  Ala Val Leu  
               
               
                     1925                 1930                 1935  
               
               
                   
               
               
                 Val Arg  Ala Val Phe Ser Phe  Ala Leu Phe Val Leu  Ala Trp Ala  
               
               
                     1940                 1945                 1950  
               
               
                   
               
               
                 Thr Pro  Trp Ser Ala Gln Val  Leu Met Ile Arg Leu  Leu Thr Ala  
               
               
                     1955                 1960                 1965  
               
               
                   
               
               
                 Ser Leu  Asn Arg Asn Lys Leu  Ser Leu Ala Phe Tyr  Ala Leu Gly  
               
               
                     1970                 1975                 1980  
               
               
                   
               
               
                 Gly Val  Val Gly Leu Ala Ala  Glu Ile Gly Thr Phe  Ala Gly Arg  
               
               
                     1985                 1990                 1995  
               
               
                   
               
               
                 Leu Ser  Glu Leu Ser Gln Ala  Leu Ser Thr Tyr Cys  Phe Leu Pro  
               
               
                     2000                 2005                 2010  
               
               
                   
               
               
                 Arg Val  Leu Ala Met Thr Ser  Cys Val Pro Thr Ile  Ile Ile Gly  
               
               
                     2015                 2020                 2025  
               
               
                   
               
               
                 Gly Leu  His Thr Leu Gly Val  Ile Leu Trp Leu Phe  Lys Tyr Arg  
               
               
                     2030                 2035                 2040  
               
               
                   
               
               
                 Cys Leu  His Asn Met Leu Val  Gly Asp Gly Ser Phe  Ser Ser Ala  
               
               
                     2045                 2050                 2055  
               
               
                   
               
               
                 Phe Phe  Leu Arg Tyr Phe Ala  Glu Gly Asn Leu Arg  Lys Gly Val  
               
               
                     2060                 2065                 2070  
               
               
                   
               
               
                 Ser Gln  Ser Cys Gly Met Asn  Asn Glu Ser Leu Thr  Ala Ala Leu  
               
               
                     2075                 2080                 2085  
               
               
                   
               
               
                 Ala Cys  Lys Leu Ser Gln Ala  Asp Leu Asp Phe Leu  Ser Ser Leu  
               
               
                     2090                 2095                 2100  
               
               
                   
               
               
                 Thr Asn  Phe Lys Cys Phe Val  Ser Ala Ser Asn Met  Lys Asn Ala  
               
               
                     2105                 2110                 2115  
               
               
                   
               
               
                 Ala Gly  Gln Tyr Ile Glu Ala  Ala Tyr Ala Lys Ala  Leu Arg Gln  
               
               
                     2120                 2125                 2130  
               
               
                   
               
               
                 Glu Leu  Ala Ser Leu Val Gln  Ile Asp Lys Met Lys  Gly Val Leu  
               
               
                     2135                 2140                 2145  
               
               
                   
               
               
                 Ser Lys  Leu Glu Ala Phe Ala  Glu Thr Ala Thr Pro  Ser Leu Asp  
               
               
                     2150                 2155                 2160  
               
               
                   
               
               
                 Ile Gly  Asp Val Ile Val Leu  Leu Gly Gln His Pro  His Gly Ser  
               
               
                     2165                 2170                 2175  
               
               
                   
               
               
                 Ile Leu  Asp Ile Asn Val Gly  Thr Glu Arg Lys Thr  Val Ser Val  
               
               
                     2180                 2185                 2190  
               
               
                   
               
               
                 Gln Glu  Thr Arg Ser Leu Gly  Gly Ser Lys Phe Ser  Val Cys Thr  
               
               
                     2195                 2200                 2205  
               
               
                   
               
               
                 Val Val  Ser Asn Thr Pro Val  Asp Ala Leu Thr Gly  Ile Pro Leu  
               
               
                     2210                 2215                 2220  
               
               
                   
               
               
                 Gln Thr  Pro Thr Pro Leu Phe  Glu Asn Gly Pro Arg  His Arg Ser  
               
               
                     2225                 2230                 2235  
               
               
                   
               
               
                 Glu Glu  Asp Asp Leu Lys Val  Glu Arg Met Lys Lys  His Cys Val  
               
               
                     2240                 2245                 2250  
               
               
                   
               
               
                 Ser Leu  Gly Phe His Asn Ile  Asn Gly Lys Val Tyr  Cys Lys Ile  
               
               
                     2255                 2260                 2265  
               
               
                   
               
               
                 Trp Asp  Lys Ser Thr Gly Asp  Thr Phe Tyr Thr Asp  Asp Ser Arg  
               
               
                     2270                 2275                 2280  
               
               
                   
               
               
                 Tyr Thr  Gln Asp His Ala Phe  Gln Asp Arg Ser Ala  Asp Tyr Arg  
               
               
                     2285                 2290                 2295  
               
               
                   
               
               
                 Asp Arg  Asp Tyr Glu Gly Val  Gln Thr Thr Pro Gln  Gln Gly Phe  
               
               
                     2300                 2305                 2310  
               
               
                   
               
               
                 Asp Pro  Lys Ser Glu Thr Pro  Val Gly Thr Val Val  Ile Gly Gly  
               
               
                     2315                 2320                 2325  
               
               
                   
               
               
                 Ile Thr  Tyr Asn Arg Tyr Leu  Ile Lys Gly Lys Glu  Val Leu Val  
               
               
                     2330                 2335                 2340  
               
               
                   
               
               
                 Pro Lys  Pro Asp Asn Cys Leu  Glu Ala Ala Lys Leu  Ser Leu Glu  
               
               
                     2345                 2350                 2355  
               
               
                   
               
               
                 Gln Ala  Leu Ala Gly Met Gly  Gln Thr Cys Asp Leu  Thr Ala Ala  
               
               
                     2360                 2365                 2370  
               
               
                   
               
               
                 Glu Val  Glu Lys Leu Lys Arg  Ile Ile Ser Gln Leu  Gln Gly Leu  
               
               
                     2375                 2380                 2385  
               
               
                   
               
               
                 Thr Thr  Glu Gln Ala Leu Asn  Cys Thr Gly Phe Lys  Leu Leu Ala  
               
               
                     2390                 2395                 2400  
               
               
                   
               
               
                 Ala Ser  Gly Leu Thr Arg Cys  Gly Arg Gly Gly Leu  Val Val Thr  
               
               
                     2405                 2410                 2415  
               
               
                   
               
               
                 Glu Thr  Ala Val Lys Ile Ile  Lys Tyr His Ser Arg  Thr Phe Thr  
               
               
                     2420                 2425                 2430  
               
               
                   
               
               
                 Leu Gly  Pro Leu Asp Leu Lys  Val Thr Ser Glu Val  Glu Val Lys  
               
               
                     2435                 2440                 2445  
               
               
                   
               
               
                 Lys Ser  Thr Glu Gln Gly His  Ala Val Val Ala Asn  Leu Cys Ser  
               
               
                     2450                 2455                 2460  
               
               
                   
               
               
                 Gly Val  Ile Leu Met Arg Pro  His Pro Pro Ser Leu  Val Asp Val  
               
               
                     2465                 2470                 2475  
               
               
                   
               
               
                 Leu Leu  Lys Pro Gly Leu Asp  Thr Ile Pro Gly Ile  Gln Pro Gly  
               
               
                     2480                 2485                 2490  
               
               
                   
               
               
                 His Gly  Ala Gly Asn Met Gly  Val Asp Gly Ser Ile  Trp Asp Phe  
               
               
                     2495                 2500                 2505  
               
               
                   
               
               
                 Glu Thr  Ala Pro Thr Lys Ala  Glu Leu Glu Leu Ser  Lys Gln Ile  
               
               
                     2510                 2515                 2520  
               
               
                   
               
               
                 Ile Gln  Ala Cys Glu Val Arg  Arg Gly Asp Ala Pro  Asn Leu Gln  
               
               
                     2525                 2530                 2535  
               
               
                   
               
               
                 Leu Pro  Tyr Lys Leu Tyr Pro  Val Arg Gly Asp Pro  Glu Arg His  
               
               
                     2540                 2545                 2550  
               
               
                   
               
               
                 Lys Gly  Arg Leu Ile Asn Thr  Arg Phe Gly Asp Leu  Pro Tyr Lys  
               
               
                     2555                 2560                 2565  
               
               
                   
               
               
                 Thr Pro  Gln Asp Thr Lys Ser  Ala Ile His Ala Ala  Cys Cys Leu  
               
               
                     2570                 2575                 2580  
               
               
                   
               
               
                 His Pro  Asn Gly Ala Pro Val  Ser Asp Gly Lys Ser  Thr Leu Gly  
               
               
                     2585                 2590                 2595  
               
               
                   
               
               
                 Thr Thr  Leu Gln His Gly Phe  Glu Leu Tyr Val Pro  Thr Val Pro  
               
               
                     2600                 2605                 2610  
               
               
                   
               
               
                 Tyr Ser  Val Met Glu Tyr Leu  Asp Ser Arg Pro Asp  Thr Pro Phe  
               
               
                     2615                 2620                 2625  
               
               
                   
               
               
                 Met Cys  Thr Lys His Gly Thr  Ser Lys Ala Ala Ala  Glu Asp Leu  
               
               
                     2630                 2635                 2640  
               
               
                   
               
               
                 Gln Lys  Tyr Asp Leu Ser Thr  Gln Gly Phe Val Leu  Pro Gly Val  
               
               
                     2645                 2650                 2655  
               
               
                   
               
               
                 Leu Arg  Leu Val Arg Arg Phe  Ile Phe Gly His Ile  Gly Lys Ala  
               
               
                     2660                 2665                 2670  
               
               
                   
               
               
                 Pro Pro  Leu Phe Leu Pro Ser  Thr Tyr Pro Ala Lys  Asn Ser Met  
               
               
                     2675                 2680                 2685  
               
               
                   
               
               
                 Ala Gly  Ile Asn Gly Gln Arg  Phe Pro Thr Lys Asp  Val Gln Ser  
               
               
                     2690                 2695                 2700  
               
               
                   
               
               
                 Ile Pro  Glu Ile Asp Glu Met  Cys Ala Arg Ala Val  Lys Glu Asn  
               
               
                     2705                 2710                 2715  
               
               
                   
               
               
                 Trp Gln  Thr Val Thr Pro Cys  Thr Leu Lys Lys Gln  Tyr Cys Ser  
               
               
                     2720                 2725                 2730  
               
               
                   
               
               
                 Lys Pro  Lys Thr Arg Thr Ile  Leu Gly Thr Asn Asn  Phe Ile Ala  
               
               
                     2735                 2740                 2745  
               
               
                   
               
               
                 Leu Ala  His Arg Ser Ala Leu  Ser Gly Val Thr Gln  Ala Phe Met  
               
               
                     2750                 2755                 2760  
               
               
                   
               
               
                 Lys Lys  Ala Trp Lys Ser Pro  Ile Ala Leu Gly Lys  Asn Lys Phe  
               
               
                     2765                 2770                 2775  
               
               
                   
               
               
                 Lys Glu  Leu His Cys Thr Val  Ala Gly Arg Cys Leu  Glu Ala Asp  
               
               
                     2780                 2785                 2790  
               
               
                   
               
               
                 Leu Ala  Ser Cys Asp Arg Ser  Thr Pro Ala Ile Val  Arg Trp Phe  
               
               
                     2795                 2800                 2805  
               
               
                   
               
               
                 Val Ala  Asn Leu Leu Tyr Glu  Leu Ala Gly Cys Glu  Glu Tyr Leu  
               
               
                     2810                 2815                 2820  
               
               
                   
               
               
                 Pro Ser  Tyr Val Leu Asn Cys  Cys His Asp Leu Val  Ala Thr Gln  
               
               
                     2825                 2830                 2835  
               
               
                   
               
               
                 Asp Gly  Ala Phe Thr Lys Arg  Gly Gly Leu Ser Ser  Gly Asp Pro  
               
               
                     2840                 2845                 2850  
               
               
                   
               
               
                 Val Thr  Ser Val Ser Asn Thr  Val Tyr Ser Leu Val  Ile Tyr Ala  
               
               
                     2855                 2860                 2865  
               
               
                   
               
               
                 Gln His  Met Val Leu Ser Ala  Leu Lys Met Gly His  Glu Ile Gly  
               
               
                     2870                 2875                 2880  
               
               
                   
               
               
                 Leu Lys  Phe Leu Glu Glu Gln  Leu Lys Phe Glu Asp  Leu Leu Glu  
               
               
                     2885                 2890                 2895  
               
               
                   
               
               
                 Ile Gln  Pro Met Leu Val Tyr  Ser Asp Asp Leu Val  Leu Tyr Ala  
               
               
                     2900                 2905                 2910  
               
               
                   
               
               
                 Glu Arg  Pro Thr Phe Pro Asn  Tyr His Trp Trp Val  Glu His Leu  
               
               
                     2915                 2920                 2925  
               
               
                   
               
               
                 Asp Leu  Met Leu Gly Phe Arg  Thr Asp Pro Lys Lys  Thr Val Ile  
               
               
                     2930                 2935                 2940  
               
               
                   
               
               
                 Thr Asp  Lys Pro Ser Phe Leu  Gly Cys Arg Ile Glu  Ala Gly Arg  
               
               
                     2945                 2950                 2955  
               
               
                   
               
               
                 Gln Leu  Val Pro Asn Arg Asp  Arg Ile Leu Ala Ala  Leu Ala Tyr  
               
               
                     2960                 2965                 2970  
               
               
                   
               
               
                 His Met  Lys Ala Gln Asn Ala  Ser Glu Tyr Tyr Ala  Ser Ala Ala  
               
               
                     2975                 2980                 2985  
               
               
                   
               
               
                 Ala Ile  Leu Met Asp Ser Cys  Ala Cys Ile Asp His  Asp Pro Glu  
               
               
                     2990                 2995                 3000  
               
               
                   
               
               
                 Trp Tyr  Glu Asp Leu Ile Cys  Gly Ile Ala Arg Cys  Ala Arg Gln  
               
               
                     3005                 3010                 3015  
               
               
                   
               
               
                 Asp Gly  Tyr Ser Phe Pro Gly  Pro Ala Phe Phe Met  Ser Met Trp  
               
               
                     3020                 3025                 3030  
               
               
                   
               
               
                 Glu Lys  Leu Arg Ser His Asn  Glu Gly Lys Lys Phe  Arg His Cys  
               
               
                     3035                 3040                 3045  
               
               
                   
               
               
                 Gly Ile  Cys Asp Ala Lys Ala  Asp Tyr Ala Ser Ala  Cys Gly Leu  
               
               
                     3050                 3055                 3060  
               
               
                   
               
               
                 Asp Leu  Cys Leu Phe His Ser  His Phe His Gln His  Cys Pro Val  
               
               
                     3065                 3070                 3075  
               
               
                   
               
               
                 Thr Leu  Ser Cys Gly His His  Ala Gly Ser Lys Glu  Cys Ser Gln  
               
               
                     3080                 3085                 3090  
               
               
                   
               
               
                 Cys Gln  Ser Pro Val Gly Ala  Gly Arg Ser Pro Leu  Asp Ala Val  
               
               
                     3095                 3100                 3105  
               
               
                   
               
               
                 Leu Lys  Gln Ile Pro Tyr Lys  Pro Pro Arg Thr Val  Ile Met Lys  
               
               
                     3110                 3115                 3120  
               
               
                   
               
               
                 Val Gly  Asn Lys Thr Thr Ala  Leu Asp Pro Gly Arg  Tyr Gln Ser  
               
               
                     3125                 3130                 3135  
               
               
                   
               
               
                 Arg Arg  Gly Leu Val Ala Val  Lys Arg Gly Ile Ala  Gly Asn Glu  
               
               
                     3140                 3145                 3150  
               
               
                   
               
               
                 Val Asp  Leu Ser Asp Gly Asp  Tyr Gln Val Val Pro  Leu Leu Pro  
               
               
                     3155                 3160                 3165  
               
               
                   
               
               
                 Thr Cys  Lys Asp Ile Asn Met  Val Lys Val Ala Cys  Asn Val Leu  
               
               
                     3170                 3175                 3180  
               
               
                   
               
               
                 Leu Ser  Lys Phe Ile Val Gly  Pro Pro Gly Ser Gly  Lys Thr Thr  
               
               
                     3185                 3190                 3195  
               
               
                   
               
               
                 Trp Leu  Leu Ser Gln Val Gln  Asp Asp Asp Val Ile  Tyr Thr Pro  
               
               
                     3200                 3205                 3210  
               
               
                   
               
               
                 Thr His  Gln Thr Met Phe Asp  Ile Val Ser Ala Leu  Lys Val Cys  
               
               
                     3215                 3220                 3225  
               
               
                   
               
               
                 Arg Tyr  Ser Ile Pro Gly Ala  Ser Gly Leu Pro Phe  Pro Pro Pro  
               
               
                     3230                 3235                 3240  
               
               
                   
               
               
                 Ala Arg  Ser Gly Pro Trp Val  Arg Leu Ile Ala Ser  Gly His Val  
               
               
                     3245                 3250                 3255  
               
               
                   
               
               
                 Pro Gly  Arg Val Ser Tyr Leu  Asp Glu Ala Gly Tyr  Cys Asn His  
               
               
                     3260                 3265                 3270  
               
               
                   
               
               
                 Leu Asp  Ile Leu Arg Leu Leu  Ser Lys Thr Pro Leu  Val Cys Leu  
               
               
                     3275                 3280                 3285  
               
               
                   
               
               
                 Gly Asp  Leu Gln Gln Leu His  Pro Val Gly Phe Asp  Ser Tyr Cys  
               
               
                     3290                 3295                 3300  
               
               
                   
               
               
                 Tyr Val  Phe Asp Gln Met Pro  Gln Lys Gln Leu Thr  Thr Ile Tyr  
               
               
                     3305                 3310                 3315  
               
               
                   
               
               
                 Arg Phe  Gly Pro Asn Ile Cys  Ala Ala Ile Gln Pro  Cys Tyr Arg  
               
               
                     3320                 3325                 3330  
               
               
                   
               
               
                 Glu Lys  Leu Glu Ser Lys Ala  Arg Asn Thr Arg Val  Val Phe Thr  
               
               
                     3335                 3340                 3345  
               
               
                   
               
               
                 Thr Arg  Pro Val Ala Phe Gly  Gln Val Leu Thr Pro  Tyr His Lys  
               
               
                     3350                 3355                 3360  
               
               
                   
               
               
                 Asp Arg  Ile Gly Ser Ala Ile  Thr Ile Asp Ser Ser  Gln Gly Ala  
               
               
                     3365                 3370                 3375  
               
               
                   
               
               
                 Thr Phe  Asp Ile Val Thr Leu  His Leu Pro Ser Pro  Lys Ser Leu  
               
               
                     3380                 3385                 3390  
               
               
                   
               
               
                 Asn Lys  Ser Arg Ala Leu Val  Ala Ile Thr Arg Ala  Arg His Gly  
               
               
                     3395                 3400                 3405  
               
               
                   
               
               
                 Leu Phe  Ile Tyr Asp Pro His  Asn Gln Leu Gln Glu  Phe Phe Asn  
               
               
                     3410                 3415                 3420  
               
               
                   
               
               
                 Leu Thr  Pro Glu Arg Thr Asp  Cys Asn Leu Val Phe  Ser Arg Gly  
               
               
                     3425                 3430                 3435  
               
               
                   
               
               
                 Asp Glu  Leu Val Val Leu Asn  Ala Asp Asn Ala Val  Thr Thr Val  
               
               
                     3440                 3445                 3450  
               
               
                   
               
               
                 Ala Lys  Ala Leu Glu Thr Gly  Pro Ser Arg Phe Arg  Val Ser Asp  
               
               
                     3455                 3460                 3465  
               
               
                   
               
               
                 Pro Arg  Cys Lys Ser Leu Leu  Ala Ala Cys Ser Ala  Ser Leu Glu  
               
               
                     3470                 3475                 3480  
               
               
                   
               
               
                 Gly Ser  Cys Met Pro Leu Pro  Gln Val Ala His Asn  Leu Gly Phe  
               
               
                     3485                 3490                 3495  
               
               
                   
               
               
                 Tyr Phe  Ser Pro Asp Ser Pro  Thr Phe Ala Pro Leu  Pro Lys Glu  
               
               
                     3500                 3505                 3510  
               
               
                   
               
               
                 Leu Ala  Pro His Trp Pro Val  Val Thr His Gln Asn  Asn Arg Ala  
               
               
                     3515                 3520                 3525  
               
               
                   
               
               
                 Trp Pro  Asp Arg Leu Val Ala  Ser Met Arg Pro Ile  Asp Ala Arg  
               
               
                     3530                 3535                 3540  
               
               
                   
               
               
                 Tyr Ser  Lys Pro Met Val Gly  Ala Gly Tyr Val Val  Gly Pro Ser  
               
               
                     3545                 3550                 3555  
               
               
                   
               
               
                 Thr Phe  Leu Gly Thr Pro Gly  Val Val Ser Tyr Tyr  Leu Thr Leu  
               
               
                     3560                 3565                 3570  
               
               
                   
               
               
                 Tyr Ile  Arg Gly Glu Pro Gln  Ala Leu Pro Glu Thr  Leu Val Ser  
               
               
                     3575                 3580                 3585  
               
               
                   
               
               
                 Thr Gly  Arg Ile Ala Thr Asp  Cys Arg Glu Tyr Leu  Asp Ala Ala  
               
               
                     3590                 3595                 3600  
               
               
                   
               
               
                 Glu Glu  Glu Ala Ala Lys Glu  Leu Pro His Ala Phe  Ile Gly Asp  
               
               
                     3605                 3610                 3615  
               
               
                   
               
               
                 Val Lys  Gly Thr Thr Val Gly  Gly Cys His His Ile  Thr Ser Lys  
               
               
                     3620                 3625                 3630  
               
               
                   
               
               
                 Tyr Leu  Pro Arg Ser Leu Pro  Lys Asp Ser Val Ala  Val Val Gly  
               
               
                     3635                 3640                 3645  
               
               
                   
               
               
                 Val Ser  Ser Pro Gly Arg Ala  Ala Lys Ala Val Cys  Thr Leu Thr  
               
               
                     3650                 3655                 3660  
               
               
                   
               
               
                 Asp Val  Tyr Leu Pro Glu Leu  Arg Pro Tyr Leu Gln  Pro Glu Thr  
               
               
                     3665                 3670                 3675  
               
               
                   
               
               
                 Ala Ser  Lys Cys Trp Lys Leu  Lys Leu Asp Phe Arg  Asp Val Arg  
               
               
                     3680                 3685                 3690  
               
               
                   
               
               
                 Leu Met  Val Trp Lys Gly Ala  Thr Ala Tyr Phe Gln  Leu Glu Gly  
               
               
                     3695                 3700                 3705  
               
               
                   
               
               
                 Leu Thr  Trp Ser Ala Leu Pro  Asp Tyr Ala Arg Phe  Ile Gln Leu  
               
               
                     3710                 3715                 3720  
               
               
                   
               
               
                 Pro Lys  Asp Ala Val Val Tyr  Ile Asp Pro Cys Ile  Gly Pro Ala  
               
               
                     3725                 3730                 3735  
               
               
                   
               
               
                 Thr Ala  Asn Arg Lys Val Val  Arg Thr Thr Asp Trp  Arg Ala Asp  
               
               
                     3740                 3745                 3750  
               
               
                   
               
               
                 Leu Ala  Val Thr Pro Tyr Asp  Tyr Gly Ala Gln Asn  Ile Leu Thr  
               
               
                     3755                 3760                 3765  
               
               
                   
               
               
                 Thr Ala  Trp Phe Glu Asp Leu  Gly Pro Gln Trp Lys  Ile Leu Gly  
               
               
                     3770                 3775                 3780  
               
               
                   
               
               
                 Leu Gln  Pro Phe Arg Arg Ala  Phe Gly Phe Glu Asn  Thr Glu Asp  
               
               
                     3785                 3790                 3795  
               
               
                   
               
               
                 Trp Ala  Ile Leu Ala Arg Arg  Met Asn Asp Gly Lys  Asp Tyr Thr  
               
               
                     3800                 3805                 3810  
               
               
                   
               
               
                 Asp Tyr  Asn Trp Asn Cys Val  Arg Glu Arg Pro His  Ala Ile Tyr  
               
               
                     3815                 3820                 3825  
               
               
                   
               
               
                 Gly Arg  Ala Arg Asp His Thr  Tyr His Phe Ala Pro  Gly Thr Glu  
               
               
                     3830                 3835                 3840  
               
               
                   
               
               
                 Leu Gln  Val Glu Leu Gly Lys  Pro Arg Leu Pro Pro  Gly Gln Val  
               
               
                     3845                 3850                 3855  
               
               
                   
               
               
                 Pro