Patent Document

CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application is a divisional application of co-pending U.S. application Ser. No. 09/565,864, filed May 5, 2000, now U.S. Patent ______, issued ______, which itself is a divisional application of co-pending U.S. application Ser. No. 08/747,863, filed Nov. 13, 1996, now U.S. Pat. No. 6,197,310 , issued Mar. 6, 2001, which itself is a divisional of U.S. patent application Ser. No. 08/157,005, filed Nov. 26, 1993, now U.S. Pat. No. 5,620,691, which is a U.S. National Stage under 35 U.S.C. § 371 of International Patent Application PCT/NL92/00096, filed Jun. 5, 1992, the contents of all of which are incorporated by this reference. 
     
    
     
       TECHNICAL FIELD  
         [0002]    The invention relates to the isolation, characterization and utilization of the causative agent of the Mystery Swine Disease (MSD). The invention utilizes the discovery of the agent causing the disease and the determination of its genome organization, the genomic nucleotide sequence and the proteins encoded by the genome, for providing protection against and diagnosis of infections, in particular, protection against and diagnosis of MSD infections, and for providing vaccine compositions and diagnostic kits, either for use with MSD or with other pathogen-caused diseases.  
         BACKGROUND  
         [0003]    In the winter and early spring of 1991, the Dutch pig industry was struck by a sudden outbreak of a new disease among breeding sows. Most sows showed anorexia, some aborted late in gestation (around day 110), showed stillbirths or gave birth to mummified fetuses and some had fever. Occasionally, sows with bluish ears were found, therefore, the disease was commonly named “Abortus Blauw”. The disease in the sows was often accompanied by respiratory distress and death of their young piglets and often by respiratory disease and growth retardation of older piglets and fattening pigs.  
           [0004]    The cause of this epizootic was not known, but the symptoms resembled those of a similar disease occurring in Germany since late 1990, and resembled those of the so-called “Mystery Swine Disease” as seen since 1987 in the mid-west of the United States of America and in Canada (Hill, 1990). Various other names have been used for the disease; in Germany it is known as “Seuchenhafter Spätabort der Schweine” and in North America it is also known as “Mystery Pig Disease”, “Mysterious Reproductive Syndrome”, and “Swine Infertility and Respiratory Syndrome”. In North America, Loula (1990) described the general clinical signs as:  
           [0005]    1) off feed, sick animals of all ages;  
           [0006]    2) abortions, stillbirths, weak pigs, mummies;  
           [0007]    3) post-farrowing respiratory problems; and  
           [0008]    4) breeding problems.  
           [0009]    No causative agent has as yet been identified, but encephalomyocarditis virus (“EMCV”), porcine parvo virus (“PPV”), pseudorabies virus (“PRV”), swine influenza virus (“SIV”), bovine viral diarrhea virus (“BVDV”), hog cholera virus (“HCV”), porcine entero viruses (“PEV”), an influenza-like virus, chlamidiae, leptospirae, have all been named as a possible cause (Loula, 1990; Mengeling and Lager, 1990; among others).  
         SUMMARY OF THE INVENTION  
         [0010]    The invention provides a composition of matter comprising isolated Lelystad Agent which is the causative agent of Mystery Swine Disease, the Lelystad Agent essentially corresponding to the isolate Lelystad Agent (CDI-NL-2.91) deposited Jun. 5, 1991 with the Institut Pasteur,  Collection Nationale de Cultures De Microorganismes  (C.N.C.M.) 25, rue du Docteur Roux, 75724 -Paris Cedex 15, France, deposit number I-1102. The words “essentially corresponding” refer to variations that occur in nature and to artificial variations of Lelystad Agent, particularly those which still allow detection by techniques like hybridization, PCR and ELISA, using Lelystad Agent-specific materials, such as Lelystad Agent-specific DNA or antibodies.  
           [0011]    The composition of matter may comprise live, killed, or attenuated isolated Lelystad Agent; a recombinant vector derived from Lelystad Agent; an isolated part or component of Lelystad Agent; isolated or synthetic protein (poly)peptide, or nucleic acid derived from Lelystad Agent; recombinant nucleic acid which comprises a nucleotide sequence derived from the genome of Lelystad Agent; a (poly)peptide having an amino acid sequence derived from a protein of Lelystad Agent, the (poly)peptide being produced by a cell capable of producing it due to genetic engineering with appropriate recombinant DNA; an isolated or synthetic antibody which specifically recognizes a part or component of Lelystad Agent; or a recombinant vector which contains nucleic acid comprising a nucleotide sequence coding for a protein or antigenic peptide derived from Lelystad Agent.  
           [0012]    On the DNA level, the invention specifically provides a recombinant nucleic acid, more specifically recombinant DNA, which comprises a Lelystad Agent-specific nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) which includes FIGS. 1 a  ; through  1   q . Preferably, the Lelystad Agent-specific nucleotide sequence is selected from any one of the ORFs (Open Reading Frames) shown in FIG. 1 (SEQ ID NO: 1).  
           [0013]    On the peptide/protein level, the invention specifically provides a peptide comprising a Lelystad Agent-specific amino acid sequence shown in FIG. 1 (SEQ ID NO: 1).  
           [0014]    The invention further provides a vaccine composition for vaccinating animals, in particular mammals, more in particular pigs or swine, to protect them against Mystery Swine Disease, comprising Lelystad Agent, either live, killed, or attenuated; or a recombinant vector which contains nucleic acid comprising a nucleotide sequence coding for a protein or antigenic peptide derived from Lelystad Agent; an antigenic part or component of Lelystad Agent; a protein or antigenic polypeptide derived from, or a peptide mimicking an antigenic component of, Lelystad Agent; and a suitable carrier or adjuvant.  
           [0015]    The invention also provides a vaccine composition for vaccinating animals, in particular mammals, more in particular pigs or swine, to protect them against a disease caused by a pathogen, comprising a recombinant vector derived from Lelystad Agent, the nucleic acid of the recombinant vector comprising a nucleotide sequence coding for a protein or antigenic peptide derived from the pathogen, and a suitable carrier or adjuvant.  
           [0016]    The invention further provides a diagnostic kit for detecting nucleic acid from Lelystad Agent in a sample, in particular a biological sample such as blood or blood serum, sputum, saliva, or tissue, derived from an animal, in particular a mammal, more in particular a pig or swine, comprising a nucleic acid probe or primer which comprises a nucleotide sequence derived from the genome of Lelystad Agent, and suitable detection means of a nucleic acid detection assay.  
           [0017]    The invention also provides a diagnostic kit for detecting antigen from Lelystad Agent in a sample, in particular a biological sample such as blood or blood serum, sputum, saliva, or tissue, derived from an animal, in particular a mammal, more in particular a pig or swine, comprising an antibody which specifically recognizes a part or component of Lelystad Agent, and suitable detection means of an antigen detection assay.  
           [0018]    The invention also provides a diagnostic kit for detecting an antibody which specifically recognizes Lelystad Agent in a sample, in particular a biological sample such as blood or blood serum, sputum, saliva, or tissue, derived from an animal, in particular a mammal, more in particular a pig or swine, comprising Lelystad Agent; an antigenic part or component of Lelystad Agent; a protein or antigenic polypeptide derived from Lelystad Agent; or a peptide mimicking an antigenic component of Lelystad Agent; and suitable detection means of an antibody detection assay.  
           [0019]    The invention also relates to a process for diagnosing whether an animal, in particular a mammal, more in particular a pig or swine, is contaminated with the causative agent of Mystery Swine Disease, comprising preparing a sample, in particular a biological sample such as blood or blood serum, sputum, saliva, or tissue, derived from the animal, and examining whether it contains Lelystad Agent nucleic acid, Lelystad Agent antigen, or antibody specifically recognizing Lelystad Agent, the Lelystad Agent being the causative agent of Mystery Swine Disease and essentially corresponding to the isolate Lelystad Agent (CDI-NL-2.91) deposited Jun. 5, 1991 with the Institut Pasteur, Paris, France, deposit number I-1102.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0020]    The invention is a result of combined efforts of the Central Veterinary Institute (CVI) and the Regional Animal Health Services (RAHS) in the Netherlands in trying to find the cause of the new disease MSD. Farms with pigs affected by the new disease were visited by field veterinarians of the RAHS. Sick pigs, specimens of sick pigs, and sow sera taken at the time of the acute and convalescent phase of the disease were sent for virus isolation to the RAHS and the CVI. Paired sera of affected sows were tested for antibodies against ten known pig-viruses. Three different viruses, encephalomyocarditis virus, porcine entero virus type 2, porcine entero virus type 7, and an unknown agent, Lelystad Agent (LA), were isolated. Sows which had reportedly been struck with the disease mainly seroconverted to LA, and rarely to any of the other virus isolates or the known viral pathogens. In order to reproduce MSD experimentally, eight pregnant sows were inoculated intranasally with LA at day 84 of gestation. One sow gave birth to seven dead and four live but very weak piglets at day 109 of gestation; the four live piglets died one day after birth. Another sow gave birth at day 116 to three mummified fetuses, six dead piglets and three live piglets; two of the live piglets died within one day. A third sow gave birth at day 117 to two mummified fetuses, eight dead and seven live piglets. The other sows farrowed around day 115 and had less severe reproductive losses. The mean number of live piglets from all eight sows at birth was 7.3 and the mean number of dead piglets at birth was 4.6. Antibodies directed against LA were detected in 10 out of 42 serum samples collected before the pigs had sucked. LA was isolated from three piglets that died shortly after birth. These results justify the conclusion that LA is the causal agent of mystery swine disease.  
           [0021]    LA grows with a cytopathic affect in pig lung macrophages and can be identified by staining in an immuno-peroxidase-monolayer assay (IPMA) with post-infection sera of pigs c 829 and b 822, or with any of the other post-infection sera of the SPF pigs listed in table 5. Antibodies to LA can be identified by indirect staining procedures in IPMA. LA did not grow in any other cell system tested. LA was not neutralized by homologous sera, or by sera directed against a set of known viruses (Table 3). LA did not haemagglutinate with the red blood cells tested. LA is smaller then 200 nm since it passes through a filter with pores of this size. LA is sensitive to chloroform. The above results show that Lelystad Agent is not yet identified as belonging to a certain virus group or other microbiological species. It has been deposited Jun. 5, 1991 under number I-1102 at Institute Pasteur, France.  
           [0022]    The genome organization, nucleotide sequences, and polypeptides derived therefrom, of LA have now been found. These data together with those of others (see below) justify classification of LA (hereafter also called Lelystad Virus or LV) as a member of a new virus family, the Arteriviridae. As prototype virus of this new family we propose Equine Arteritis Virus (EAV), the first member of the new family of which data regarding the replication strategy of the genome and genome organization became available (de Vries et al., 1990, and references therein). On the basis of a comparison of our sequence data with those available for Lactate Dehydrogenase-Elevating Virus (LDV; Godeny et al., 1990), we propose that LDV is also a member of the Arteriviridae.  
           [0023]    Given the genome organization and translation strategy of Arteriviridae, it seems appropriate to place this new virus family into the superfamily of coronaviruses (Snijder et al., 1990a).  
           [0024]    Arteriviruses have in common that their primary target cells in respective hosts are macrophages. Replication of LDV has been shown to be restricted to macrophages in its host, the mouse; whereas this strict propensity for macrophages has not been resolved yet for EAV and LV.  
           [0025]    Arteriviruses are spherical enveloped particles having a diameter of 45-60 nm and containing an icosahedral nucleocapsid (Brinton-Darnell and Plagemann, 1975; Horzinek et al., 1971; Hyllseth, 1973).  
           [0026]    The genome of Arteriviridae consists of a positive stranded polyadenylated RNA molecule with a size of about 12-13 kilobases (kb) (Brinton-Darnell and Plageman, 1975; van der Zeijst et al., 1975). EAV replicates via a 3′ nested set of six subgenomic mRNAs, ranging in size from 0.8 to 3.6 kb, which are composed of a leader sequence, derived from the 5′ end of the genomic RNA, which is joined to the 3′ terminal body sequences (de Vries et al., 1990).  
           [0027]    Here we show that the genome organization and replication strategy of LV is similar to that of EAV, coronaviruses and toroviruses, whereas the genome sizes of the latter viruses are completely different from those of LV and EAV.  
           [0028]    The genome of LV consists of a genomic RNA molecule of about 14.5 to 15.5 kb in length (estimated on a neutral agarose gel), which replicates via a 3′ nested set of subgenomic RNAs. The subgenomic RNAs consist of a leader sequence, the length of which is yet unknown, which is derived from the 5′ end of the genomic RNA and which is fused to the body sequences derived from the 3′ end of the genomic RNA (FIG. 2).  
           [0029]    The nucleotide sequence of the genomic RNA of LV was determined from overlapping cDNA clones. A consecutive sequence of 15,088 bp was obtained covering nearly the complete genome of LV (FIG. 1, SEQ ID NO: 1). In this sequence 8 open reading frames (ORFs) were identified: ORF 1A, ORF 1B, and ORFs 2 to 7.  
           [0030]    ORF 1A and ORF 1B are predicted to encode the viral replicase or polymerase (SEQ ID NO: 2 and SEQ ID NO: 3), whereas ORFs 2 to 6 are predicted to encode structural viral membrane (envelope) associated proteins (SEQ ID NOS: 4-8). ORF 7 is predicted to encode the structural viral nucleocapsid protein (SEQ ID NO: 9).  
           [0031]    Because the products of ORF 6 and ORF 7 of LV (SEQ ID NO: 8 and SEQ ID NO: 9) show a significant similarity with VpX and Vp1 of LDV, respectively, it is predicted that the sequences of ORFs 6 and 7 will also be highly conserved among antigenic variants of LV.  
           [0032]    The complete nucleotide sequence of FIG. 1 (SEQ ID NO: 1) and all the sequences and protein products encoded by ORFs 1 to 7 (SEQ ID NOS: 1-9) and possible other ORFs located in the sequence of FIG. 1 (SEQ ID NO: 1) are especially suited for vaccine development, in whatever sense, and for the development of diagnostic tools, in whatever sense. All possible modes are well known to persons skilled in the art.  
           [0033]    Since it is now possible to unambiguously identify LA, the causal agent of MSD, it can now be tested whether pigs are infected with LA or not. Such diagnostic tests have, until now, been unavailable.  
           [0034]    The test can be performed by virus isolation in macrophages, or other cell culture systems in which LA might grow, and staining the infected cultures with antibodies directed against LA (such as post-infection sera c 829 or b 822), but it is also feasible to develop and employ other types of diagnostic tests.  
           [0035]    For instance, it is possible to use direct or indirect immunohistological staining techniques, i.e., with antibodies directed to LA that are labeled with fluorescent compounds such as isothiocyanate, or labeled with enzymes such as horseradish peroxidase. These techniques can be used to detect LA antigen in tissue sections or other samples from pigs suspected to have MSD. The antibodies needed for these tests can be c 829 or b 822 or other polyclonal antibodies directed against LA, but monoclonal antibodies directed against LA can also be used.  
           [0036]    Furthermore, since the nature and organization of the genome of LA and the nucleotide sequence of this genome have been determined, LA-specific nucleotide sequences can be identified and used to develop oligonucleotide sequences that can be used as probes or primers in diagnostic techniques such as hybridization, polymerase chain reaction, or any other techniques that are developed to specifically detect nucleotide acid sequences.  
           [0037]    It is also possible to test for antibodies directed against LA. Table 5 shows that experimentally infected pigs rapidly develop antibodies against LA, and table 4 shows that pigs in the field also have strong antibody responses against LA. Thus, it can now also be determined whether pigs have been infected with LA in the past. Such testing is of utmost importance in determining whether pigs or pig herds or pig populations or pigs in whole regions or countries are free of LA. The test can be done by using the IPMA as described, but it is also feasible to develop and employ other types of diagnostic tests for the detection of antibodies directed against LA.  
           [0038]    LA-specific proteins, polypeptides, and peptides, or peptide sequences mimicking antigenic components of LA, can be used in such tests. Such proteins can be derived from the LA itself, but it is also possible to make such proteins by recombinant DNA or peptide synthesis techniques. These tests can use specific polyclonal and/or monoclonal antibodies directed against LA or specific components of LA, and/or use cell systems infected with LA or cell systems expressing LA antigen. The antibodies can be used, for example, as a means for immobilizing the LA antigen (a solid surface is coated with the antibody whereafter the LA antigen is bound by the antibody) which leads to a higher specificity of the test, or can be used in a competitive assay (labeled antibody and unknown antibody in the sample compete for available LA antigen).  
           [0039]    Furthermore, the above described diagnostic possibilities can be applied to test whether other animals, such as mammals, birds, insects or fish, or plants, or other living creatures, can be, or are, or have been infected with LA or related agents.  
           [0040]    Since LA has now been identified as the causal agent of MSD, it is possible to make a vaccine to protect pigs against this disease. Such a vaccine can simply be made by growing LA in pig lung macrophage cultures, or in other cell systems in which LA grows. LA can then be purified or not, and killed by established techniques, such as inactivation with formaline or ultra-violet light. The inactivated LA can then be combined with adjuvantia, such as Freund&#39;s adjuvans or aluminum hydroxide or others, and this composition can then be injected in pigs.  
           [0041]    Dead vaccines can also be made with LA protein preparations derived from LA infected cultures, or derived from cell systems expressing specifically LA protein through DNA recombinant techniques. Such subunits of LA would then be treated as above, and this would result in a subunit vaccine.  
           [0042]    Vaccines using even smaller components of LA, such as polypeptides, peptides, or peptides mimicking antigenic components of LA, are also feasible for use as dead vaccine.  
           [0043]    Dead vaccines against MSD can also be made by recombinant DNA techniques through which the genome of LA, or parts thereof, is incorporated in vector systems such as vaccinia virus, herpesvirus, pseudorabies virus, adeno virus, baculo virus or other suitable vector systems that can so express LA antigen in appropriate cells systems. LA antigen from these systems can then be used to develop a vaccine as above, and pigs, vaccinated with such products would develop protective immune responses against LA.  
           [0044]    Vaccines against MSD can also be based on live preparations of LA. Since only young piglets and pregnant sows seem to be seriously affected by infection with LA, it is possible to use unattenuated LA, grown in pig lung macrophages, as vaccine for older piglets, or breeding gilts. In this way, sows can be protected against MSD before they get pregnant, which results in protection against abortions and stillbirth, and against congenital infections of piglets. Also the maternal antibody that these vaccinated sows give to their offspring would protect their offspring against the disease.  
           [0045]    Attenuated vaccines (modified-live-vaccines) against MSD can be made by serially passaging LA in pig lung macrophages, in lung macrophages of other species, or in other cell systems, or in other animals, such as rabbits, until it has lost its pathogenicity.  
           [0046]    Live vaccines against MSD can also be made by recombinant DNA techniques through which the genome of LA, or parts thereof, is incorporated in vector systems such as vaccinia virus, herpesvirus, pseudorabies virus, adeno virus or other suitable vector systems that can so express LA antigen. Pigs vaccinated with such live vector systems would then develop protective immune responses against LA.  
           [0047]    Lelystad Agent itself would be specifically suited to use as a live vector system. Foreign genes could be inserted in the genome of LA and could be expressing the corresponding protein during the infection of the macrophages. This cell, which is an antigen-presenting cell, would process the foreign antigen and present it to B-lymphocytes and T-lymphocytes which will respond with the appropriate immune response.  
           [0048]    Since LA seems to be very cell specific and possibly also very species specific, this vector system might be a very safe system, which does not harm other cells or species. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0049]    [0049]FIG. 1 (SEQ ID NO: 1) shows the nucleotide sequence of the LV genome. The deduced amino acid sequence of the identified ORFs (SEQ ID NOS: 2-9) are shown. The methionines encoded by the (putative) ATG start sites are indicated in bold and putative N-glycosylation sites are underlined. Differences in the nucleotide and amino acid sequence, as identified by sequencing different cDNA clones, are shown. The nucleotide sequence of primer 25, which has been used in hybridization experiments (see FIG. 2 and section “results”), is underlined.  
         [0050]    [0050]FIG. 2 shows the organization of the LV genome. The cDNA clones, which have been used for the determination of the nucleotide sequence, are indicated in the upper part of the figure. The parts of the clones, which were sequenced, are indicated in black. In the lower part of the FIG. the ORFs, identified in the nucleotide sequence, and the subgenomic set of mRNAs, encoding these ORFs are shown. The dashed lines in the ORFs represent alternative initiation sites (ATGs) of these ORFs. The leader sequence of the genomic and subgenomic RNAs is indicated by a solid box.  
         [0051]    [0051]FIG. 3 shows the growth characteristics of LA:  
         [0052]    empty squares—titre of cell-free virus;  
         [0053]    solid squares—titre of cell-associated virus;  
         [0054]    solid line—percentage cytopathic effect (CPE).  
     
    
     MATERIALS AND METHODS  
       [0055]    Sample Collection  
         [0056]    Samples and pigs were collected from farms where a herd epizootic of MSD seemed to occur. Important criteria for selecting the farm as being affected with MSD were: sows that were off feed, the occurrence of stillbirth and abortion, weak offspring, respiratory disease and death among young piglets. Samples from four groups of pigs have been investigated:  
         [0057]    (1) tissue samples and an oral swab from affected piglets from the field (Table 1A);  
         [0058]    (2) blood samples and oral swabs from affected sows in the field (Tables 1B and 4);  
         [0059]    (3) tissue samples, nasal swabs and blood samples collected from specific-pathogen-free (SPF) pigs experimentally infected by contact with affected sows from the field; or  
         [0060]    (4) tissue samples, nasal swabs and blood samples collected from specific-pathogen-free (SPF) pigs experimentally infected by inoculation with blood samples of affected sows from the field (Tables 2 and 5).  
         [0061]    Sample Preparation  
         [0062]    Samples for virus isolation were obtained from piglets and sows which on clinical grounds were suspected to have MSD, and from experimentally infected SPF pigs, sows and their piglets.  
         [0063]    Tissue samples were cut on a cryostat microtome and sections were submitted for direct immunofluorescence testing (IFT) with conjugates directed against various pig pathogens.  
         [0064]    10% Suspensions of tissues samples were prepared in Hank&#39;s BSS supplemented with antibiotics, and oral and nasal swabs were soaked in Hank&#39;s BSS supplemented with antibiotics. After one hour at room temperature, the suspensions were clarified for 10 min at 6000 g and the supernatant was stored at −70° C. for further use. Leucocyte fractions were isolated from EDTA or heparin blood as described earlier (Wensvoort and Terpstra, 1988) and stored at −70° C. Plasma and serum for virus isolation were stored at −70° C.  
         [0065]    Serum for serology was obtained from sows suspected to be in the acute phase of MSD, a paired serum was taken 3-9 weeks later. Furthermore, sera were taken from the experimentally infected SPF pigs at regular intervals and colostrum and serum was taken from experimentally infected sows and their piglets. Sera for serology were stored at −20° C.  
         [0066]    Cells  
         [0067]    Pig lung macrophages were obtained from lungs of 5-6 weeks old SPF pigs or from lungs of adult SPF sows from the Central Veterinary Institute&#39;s own herd. The lungs were washed five to eight times with phosphate buffered saline (PBS). Each aliquot of washing fluid was collected and centrifuged for 10 min at 300 g. The resulting cell pellet was washed again in PBS and resuspended in cell culture medium (160 ml medium 199, supplemented with 20 ml 2.95% tryptose phosphate, 20 ml fetal bovine serum (FBS), and 4.5 ml 1.4% sodium bicarbonate) to a concentration of 4×10 7 cells/ml. The cell suspension was then slowly mixed with an equal volume of DMSO mix (6.7 ml of above medium, 1.3 ml FBS, 2 ml dimethylsulfoxide 97%), aliquoted in 2 ml ampoules and stored in liquid nitrogen.  
         [0068]    Macrophages from one ampoule were prepared for cell culture by washing twice in Earle&#39;s MEM, and resuspended in 30 ml growth medium (Earle&#39;s MEM, supplemented with 10% FBS, 200 U/ml penicillin, 0.2 mg/ml streptomycine, 100 U/ml mycostatin, and 0.3 mg/ml glutamine). PK-15 cells (American Type Culture Collection, CCL33) and SK-6 cells (Kasza et al., 1972) were grown as described by Wensvoort et al. (1989). Secondary porcine kidney (PK2) cells were grown in Earle&#39;s MEM, supplemented with 10% FBS and the above antibiotics. All cells were grown in a cell culture cabinet at 37° C. and 5% CO 2 .  
         [0069]    Virus Isolation Procedures  
         [0070]    Virus isolation was performed according to established techniques using PK2, PK-15 and SK-6 cells, and pig lung macrophages. The former three cells were grown in 25 ml flasks (Greiner), and inoculated with the test sample when monolayers had reached 70-80% confluency. Macrophages were seeded in 100 μl aliquots in 96-well microtiter plates (Greiner) or in larger volumes in appropriate flasks, and inoculated with the test sample within one hour after seeding. The cultures were observed daily for cytopathic effects (CPE), and frozen at −70° C. when 50-70% CPE was reached or after five to ten days of culture. Further passages were made with freeze-thawed material of passage level 1 and 2 or higher. Some samples were also inoculated into nine to twelve day old embryonated hen eggs. Allantoic fluid was subinoculated two times using an incubation interval of three days and the harvest of the third passage was examined by haemagglutination at 4° C. using chicken red blood cells, and by an ELISA specifically detecting nucleoprotein of influenza A viruses (De Boer et al., 1990).  
         [0071]    Serology  
         [0072]    Sera were tested in haemagglutinating inhibition tests (HAI) to study the development of antibody against haemagglutinating encephalitis virus (HEV), and swine influenza viruses H1N1 and H3N2 according to the protocol of Masurel (1976). Starting dilutions of the sera in HAI were 1:9, after which the sera were diluted twofold.  
         [0073]    Sera were tested in established enzyme-linked immuno-sorbent assays (ELISA) for antibodies against the glycoprotein gI of pseudorabies virus (PRV; Van Oirschot et al., 1988), porcine parvo virus (PPV; Westenbrink et al., 1989), bovine viral diarrhea virus (BVDV; Westenbrink et al., 1986), and hog cholera virus (HCV; Wensvoort et al., 1988). Starting dilutions in the ELISA&#39;s were 1:5, after which the sera were diluted twofold.  
         [0074]    Sera were tested for neutralizing antibodies against 30-300 TCID 50  of encephalomyocarditis viruses (EMCV), porcine enteroviruses (PEV), and Lelystad Agent (LA) according to the protocol of Terpstra (1978). Starting dilutions of the sera in the serum neutralization tests (SNT) were 1:5, after which the sera were diluted twofold.  
         [0075]    Sera were tested for binding with LA in an immuno-peroxidase-monolayer assay (IPMA). Lelystad Agent (LA; code: CDI-NL-2.91) was seeded in microtiter plates by adding 50 ml growth medium containing 100 TCID 50  LA to the wells of a microtiter plate containing freshly seeded lung macrophages. The cells were grown for two days and then fixed as described (Wensvoort, 1986). The test sera were diluted 1:10 in 0.15 M NaCl, 0.05% Tween 80, 4% horse serum, or diluted further in fourfold steps, added to the wells and then incubated for one hour at 37° C. Sheep-anti-pig immunoglobulins (Ig) conjugated to horse radish peroxidase (HRPO, DAKO) were diluted in the same buffer and used in a second incubation for one hour at 37° C., after which the plates were stained as described (Wensvoort et al., 1986). An intense red staining of the cytoplasm of infected macrophages indicated binding of the sera to LA.  
         [0076]    Virus Identification Procedures  
         [0077]    The identity of cytopathic isolates was studied by determining the buoyant density in CsCl, by estimating particle. size in negatively stained preparations through electron microscopy, by determining the sensitivity of the isolate to chloroform and by neutralizing the CPE of the isolate with sera with known specificity (Table 3). Whenever an isolate was specifically neutralized by a serum directed against a known virus, the isolate was considered to be a representative of this known virus.  
         [0078]    Isolates that showed CPE on macrophage cultures were also studied by staining in IPMA with post-infection sera of pigs c 829 or b 822. The isolates were reinoculated on macrophage cultures and fixed at day 2 after inoculation before the isolate showed CPE. Whenever an isolate showed reactivity in IPMA with the post-infection sera of pigs c 829 or b 822, the isolate was considered to be a representative of the Lelystad Agent. Representatives of the other isolates grown in macrophages or uninfected macrophages were also stained with these sera to check the specificity of the sera.  
         [0079]    Further Identification of Lelystad Agent  
         [0080]    Lelystad Agent was further studied by haemagglutination at 4° C. and 37° C. with chicken, guinea pig, pig, sheep, or human O red blood cells. SIV, subtype H3N2, was used as positive control in the haemagglutination studies.  
         [0081]    The binding of pig antisera specifically directed against pseudorabies virus (PRV), transmissible gastroenteritis virus (TGE), porcine epidemic diarrhea virus (PED), haemagglutinating encephalitis virus (HEV), African swine fever virus (ASFV), hog cholera virus (HCV) and swine influenza virus (SIV) type H1N1 and H3N2, of bovine antisera specifically directed against bovine herpes viruses type 1 and 4 (BHV 1 and 4), malignant catarrhal fever (MCF), parainfluenza virus 3 (PI3), bovine respiratory syncitial virus (BRSV) and bovine leukemia virus (BLV), and of avian antisera specifically directed against avian leukemia virus (ALV) and infectious bronchitis virus (IBV) was studied with species-Ig-specific HRPO conjugates in an IPMA on LA infected and uninfected pig lung macrophages as described above.  
         [0082]    We also tested in IPMA antisera of various species directed against mumps virus, Sendai virus, canine distemper virus, rinderpest virus, measles virus, pneumonia virus of mice, bovine respiratory syncytial virus, rabies virus, foamy virus, maedi-visna virus, bovine and murine leukemia virus, human, feline and simian immunodeficiency virus, lymphocytic choriomeningitis virus, feline infectious peritonitis virus, mouse hepatitis virus, Breda virus, Hantaan virus, Nairobi sheep disease virus, Eastern, Western and Venezuelan equine encephalomyelitis virus, rubellavirus, equine arteritis virus, lactic dehydrogenase virus, yellow fever virus, tick-born encephalitis virus and hepatitis C virus.  
         [0083]    LA was blindly passaged in PK2, PK- 15, and SK-6 cells, and in embryonated hen eggs. After two passages, the material was inoculated again into pig lung macrophage cultures for reisolation of LA.  
         [0084]    LA was titrated in pig lung macrophages prior to and after passing through a 0.2 micron filter (Schleicher and Schuell). The LA was detected in IPMA and by its CPE. Titres were calculated according to Reed and Muench (1938).  
         [0085]    We further prepared pig antisera directed against LA. Two SPF pigs (21 and 23) were infected intranasally with 10 5 TCID 50  of a fifth cell culture passage of LA. Two other SPF pigs (25 and 29) were infected intranasally with a fresh suspension of the lungs of an LA-infected SPF piglet containing 10 5  TCID 50  LA. Blood samples were taken at 0, 14, 28, and 42 days post-infection (dpi).  
         [0086]    We further grew LA in porcine alveolar macrophages to determine its growth pattern over time. Porcine alveolar macrophages were seeded in F25 flasks (Greiner), infected with LA with a multiplicity of infection of 0.01 TCID 50  per cell. At 8, 16,24, 32,40,48, 56, and 64 h after infection, one flask was examined and the percentage of CPE in relation to a noninfected control culture was determined. The culture medium was then harvested and replaced with an equal volume of phosphate-buffered saline. The medium and the flask were stored at −70° C. After all cultures had been harvested, the LA titres were determined and expressed as log TCID 50  ml −1 .  
         [0087]    The morphology of LA was studied by electronmicroscopy. LA was cultured as above. After 48 h, the cultures were freeze-thawed and centrifuged for 10 min at 6000.times.g. An amount of 30 ml supernatant was then mixed with 0.3 ml LA-specific pig serum and incubated for 1.5 h at 37° C. After centrifugation for 30 min at 125,000× g, the resulting pellet was suspended in 1% Seakem agarose ME in phosphate-buffered saline at 40° C. After coagulation, the agarose block was immersed in 0.8% glutaraldehyde and 0.8% osmiumtetroxide (Hirsch et al., 1968) in veronal/acetate buffer, pH 7.4 (230 mOsm/kg H 2 O), and fixed by microwave irradiation. This procedure was repeated once with fresh fixative. The sample was washed with water, immersed in 1% uranyl acetate, and stained by microwave irradiation. Throughout all steps, the sample was kept at 0° C. and the microwave (Samsung RE211D) was set at defrost for 5 min. Thin sections were prepared with standard techniques, stained with lead citrate (Venable et al., 1965), and examined in a Philips CM 10 electron microscope.  
         [0088]    We further continued isolating LA from sera of pigs originating from cases of MSD. Serum samples originated from the Netherlands (field case the Netherlands 2), Germany (field cases Germany 1 and Germany 2; courtesy Drs. Berner, Müinchen and Nienhoff, Münster), and the United States [experimental case United States 1 (experiment performed with ATCC VR-2332; courtesy Drs. Collins, St. Paul and Chladek, St. Joseph), and field cases United States 2 and United States 3; courtesy Drs. van Alstine, West Lafayette and Slife, Galesburg]. All samples were sent to the “Centraal Diergeneeskundig Instituut, Lelystad” for LA diagnosis. All samples were used for virus isolation on porcine alveolar macrophages as described. Cytophatic isolates were passaged three times and identified as LA by specific immunostaining with anti-LA post infection sera b 822 and c 829.  
         [0089]    We also studied the antigenic relationships of isolates NL1 (the first LA isolate; code CDI-NL-2.91), NL2, GE1, GE2, US1, US2, and US3. The isolates were grown in macrophages as above and were tested in IPMA with a set of field sera and two sets of experimental sera. The sera were also tested in IPMA with uninfected macrophages.  
         [0090]    The field sera were: Two sera positive for LV (TH-187 and TO-36) were selected from a set of LA-positive Dutch field sera. Twenty-two sera were selected from field sera sent from abroad to Lelystad for serological diagnosis. The sera originated from Germany (BE-352, BE-392 and NI-f2; courtesy Dr. Bemer, München and Dr. Nienhoff, Münster), the United Kingdom (PA-141615, PA-141617 and PA-142440; courtesy Dr. Paton, Weybridge), Belgium (PE-1960; courtesy Prof. Pensaert, Gent), France (EA-2975 and EA-2985; courtesy Dr. Albina, Ploufragan), the United States (SL-441, SL-451, AL-RP9577, AL-P10814/33, AL-4994A, AL-7525, JC-MN41, JC-MN44 and JC-MN45; courtesy Dr. Slife, Galesburg, Dr. van Alstine, West Lafayette, and Dr. Collins, St. Paul), and Canada (RB-16, RB- 19, RB-22 and RB-23; courtesy Dr. Robinson, Quebec).  
         [0091]    The experimental sera were: The above described set of sera of pigs 21, 23, 25, and 29, taken at dpi 0, 14, 28, and 42. A set of experimental sera (obtained by courtesy of Drs. Chladek, St. Joseph, and Collins, St. Paul) that originated from four six-month-old gilts that were challenged intranasally with 10 5.1 TCID 50  of the isolate ATCC VR-2332. Blood samples were taken from gilt 2B at 0, 20, 36, and 63 dpi; from gilt 9G at 0, 30, 44, and 68 dpi; from gilt 16W at 0, 25, 40, and 64 dpi; and from gilt 16Y at 0, 36, and 64 dpi.  
         [0092]    To study by radio-immunoprecipitation assay (RIP; de Mazancourt et al., 1986) the proteins of LA in infected porcine alveolar macrophages, we grew LA-infected and uninfected macrophages for 16 hours in the presence of labeling medium containing  35 S-Cysteine. Then the labeled cells were precipitated according to standard methods with 42 dpi post-infection sera of pig b 822 and pig 23 and with serum MN 8 which was obtained 26 days after infecting a sow with the isolate ATCC VR-2332 (courtesy Dr. Collins, St. Paul). The precipitated proteins were analyzed by electrophoresis in a 12% SDS-PAGE gel and visualized by fluorography.  
         [0093]    To characterize the genome of LA, we extracted nuclear DNA and cytoplasmatic RNA from macrophage cultures that were infected with LA and grown for 24 h or were left uninfected. The cell culture medium was discarded, and the cells were washed twice with phosphate-buffered saline. DNA was extracted as described (Strauss, 1987). The cytoplasmic RNA was extracted as described (Favaloro et al., 1980), purified by centrifugation through a 5.7 M CsCl cushion (Setzer et al., 1980), treated with RNase-free DNase (Pharmacia), and analyzed in a 0.8% neutral agarose gel (Moormann and Hulst, 1988).  
         [0094]    Cloning and Sequencing  
         [0095]    To clone LV RNA, intracellular RNA of LV-infected porcine lung alveolar macrophages (10 μg) was incubated with 10 mM methylmercury hydroxide for 10 minutes at room temperature. The denatured RNA was incubated at 42° C. with 50 mM Tris-HCI, pH 7.8, 10 mM MgCl 2 , 70 mM KCl, 0.5 mM dATP, dCTP, dGTP and dTTP, 0.6 μg calf thymus oligonucleotide primers pd(N)6 (Pharmacia) and 300 units of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories) in a total volume of 100μl 20 mM EDTA was added after 1 hr; the reaction mixture was then extracted with phenol/chloroform, passed through a Sephadex G50 column and precipitated with ethanol.  
         [0096]    For synthesis of the second cDNA strand, DNA polymerase I (Boehringer) and RNase H (Pharmacia) were used (Gübler and Hoffinan, 1983). To generate blunt ends at the termini, double-stranded cDNA was incubated with T4 DNA polymerase (Pharmacia) in a reaction mixture which contained 0.05 mM deoxynucleotide-triphosphates. Subsequently, cDNA was fractionated in a 0.8% neutral agarose gel (Moormann and Hulst, 1988). Fragments of 1 to 4 kb were electroeluted, ligated into the Smal site of pGEM-4Z (Promega), and used for transformation of  Escherichia coli  strain DH5α (Hanahan, 1985). Colony filters were hybridized with a  32 P-labeled single-stranded cDNA probe. The probe was reverse transcribed from LV RNA which had been fractionated in a neutral agarose gel (Moormann and Hulst, 1988). Before use, the single stranded DNA probe was incubated with cytoplasmic RNA from mock-infected lung alveolar macrophages.  
         [0097]    The relationship between LV cDNA clones was determined by restriction enzyme analysis and by hybridization of Southern blots of the digested DNA with nick-translated cDNA probes (Sambrook et al., 1989).  
         [0098]    To obtain the 3′ end of the viral genome, we constructed a second cDNA library, using oligo (dT) 12-18  and a 3′ LV-specific oligonucleotide that was complementary to the minus-strand viral genome as a primer in the first-strand reaction. The reaction conditions for first- and second-strand synthesis were identical to those described above. This library was screened with virus-specific 3′ end oligonucleotide probes.  
         [0099]    Most (&gt;95%) of the cDNA sequences were determined with an Automated Laser Fluorescent A.L.F.™. DNA sequencer from Pharmacia LKB. Fluorescent oligonucleotide primer directed sequencing was performed on double-stranded DNA using the AutoRead™. Sequencing Kit (Pharmacia) essentially according to procedures C and D described in the Autoread™ Sequencing Kit protocol. Fluorescent primers were prepared with FluorePrime™. (Pharmacia). The remaining part of the sequence was determined via double-stranded DNA sequencing using oligonucleotide primers in conjunction with a T7 polymerase based sequencing kit (Pharmacia) and α- 32 S-dATP (Amersham). Sequence data were analyzed using the sequence analysis programs PCGENE (Intelligenetics, Inc, Mountain View, U.S.A.) and FASTA (Pearson and Lipman, 1988).  
         [0100]    Experimental Reproduction of MSD  
         [0101]    Fourteen conventionally reared pregnant sows that were pregnant for 10-11 weeks were tested for antibody against LA in the IPMA. All were negative. Then two groups of four sows were formed and brought to the CVI. At week 12 of gestation, these sows were inoculated intranasally with 2 ml LA (passage level 3, titre 10 4.8  TCID 50  /ml). Serum and EDTA blood samples were taken at day 10 after inoculation. Food intake, rectal temperature, and other clinical symptoms were observed daily. At farrowing, the date of birth and the number of dead and living piglets per sow were recorded, and samples were taken for virus isolation and serology.  
       Results  
       [0102]    Immunofluorescence  
         [0103]    Tissue sections of pigs with MSD were stained in an IFT with FITC-conjugates directed against African swine fever virus, hog cholera virus, pseudorabies virus, porcine parvo virus, porcine influenza virus, encephalomyocarditis virus and Chlamydia psittaci. The sections were stained, examined by fluorescent microscopy and all were found negative.  
         [0104]    Virus Isolation from Piglets from MSD Affected Farms  
         [0105]    Cytopathic isolates were detected in macrophage cultures inoculated with tissue samples of MSD affected, two-to-ten day old piglets. Sixteen out of 19 piglets originating from five different farms were positive (Table 1A). These isolates all reacted in IPMA with the post-infection serum of pig c 829, whereas non-inoculated control cultures did not react. The isolates, therefore, were representatives of LA. One time a cytopathic isolate was detected in an SK-6 cell culture inoculated with a suspension of an oral swab from a piglet from a sixth farm (farm VE) (Table 1A). This isolate showed characteristics of the picoma viridae and was neutralized by serum specific for PEV 2, therefore, the isolate was identified as PEV 2 (Table 3). PK2, PK-15 cells and hen eggs inoculated with samples from this group remained negative throughout.  
         [0106]    Virus Isolation from Sows from MSD Affected Farms  
         [0107]    Cytopathic isolates were detected in macrophage cultures inoculated with samples of MSD affected sows. 41 out of 63 sows originating from 11 farms were positive (Table 1B). These isolates all reacted in IPMA with the post-infection serum of pig b 822 and were, therefore, representatives of LA. On one occasion a cytopathic isolate was detected in a PK2 cell culture inoculated with a suspension of a leucocyte fraction of a sow from farm HU (Table 1B). This isolate showed characteristics of the picoma viridae and was neutralized by serum specific for EMCV, therefore, the isolate was identified as EMCV (Table 3). SK-6, PK-15 cells and hen eggs inoculated with samples from this group remained negative.  
         [0108]    Virus Isolation from SPF Pigs Kept in Contact with MSD Affected Sows  
         [0109]    Cytopathic isolates were detected in macrophage cultures inoculated with samples of SPF pigs kept in contact with MSD affected sows. Four of the 12 pigs were positive (Table 2). These isolates all reacted in IPMA with the post-infection serum of pig c 829 and of pig b 822 and were, therefore, representatives of LA. Cytopathic isolates were also detected in PK2, PK-15 and SK-6 cell cultures inoculated with samples of these SPF pigs. Seven of the 12 pigs were positive (Table 2), these isolates were all neutralized by serum directed against PEV 7. One of these seven isolates was studied further and other characteristics also identified the isolate as PEV 7 (Table 3).  
         [0110]    Virus Isolation from SPF Pigs Inoculated with Blood of MSD Affected Sows  
         [0111]    Cytopathic isolates were detected in macrophage cultures inoculated with samples of SPF pigs inoculated with blood of MSD affected sows. Two out of the eight pigs were positive (Table 2). These isolates all reacted in IPMA with the post-infection serum of pig c 829 and of pig b 822 and were, therefore, representatives of LA. PK2, SK-6 and PK-15 cells inoculated with samples from this group remained negative.  
         [0112]    Summarizing, four groups of pigs were tested for the presence of agents that could be associated with mystery swine disease (MSD).  
         [0113]    In group one, MSD affected piglets, the Lelystad Agent (LA) was isolated from 16 out of 20 piglets; one time PEV 2 was isolated.  
         [0114]    In group two, MSD affected sows, the Lelystad Agent was isolated from 41 out of 63 sows; one time EMCV was isolated. Furthermore, 123 out of 165 MSD affected sows seroconverted to the Lelystad Agent, as tested in the IPMA. Such massive seroconversion was not demonstrated against any of the other viral pathogens tested.  
         [0115]    In group three, SPF pigs kept in contact with MSD affected sows, LA was isolated from four of the 12 pigs; PEV 7 was isolated from seven pigs. All 12 pigs seroconverted to LA and PEV 7.  
         [0116]    In group four, SPF pigs inoculated with blood of MSD affected sows, the LA was isolated from two pigs. All eight pigs seroconverted to LA.  
         [0117]    Serology of Sows from MSD Affected Farms  
         [0118]    Paired sera from sows affected with MSD were tested against a variety of viral pathogens and against the isolates obtained during this study (Table 4). An overwhelming antibody response directed against LA was measured in the IPMA (75% of the sows seroconverted, in 23 out of the 26 farms seroconversion was found), whereas with none of the other viral pathogens a clear pattern of seroconversion was found. Neutralizing antibody directed against LA was not detected.  
         [0119]    Serology of SPF Pigs Kept in Contact with MSD Affected Sows  
         [0120]    All eight SPF pigs showed an antibody response in the IPMA against LA (Table 5). None of these sera were positive in the IPMA performed on uninfected macrophages. None of these sera were positive in the SNT for LA. The sera taken two weeks after contact had all high neutralizing antibody titres (&gt;1280) against PEV 7, whereas the pre-infection sera were negative (&lt;10), indicating that all pigs had also been infected with PEV 7.  
         [0121]    Serology of SPF Pigs Inoculated with Blood of MSD Affected Sows  
         [0122]    All eight SPF pigs showed an antibodyresponse in the IPMA against LA (Table 5). None of these sera were positive in the IPMA performed on uninfected macrophages. None of these sera were positive in the SNT for LA. The pre- and two weeks post-inoculation sera were negative (&lt;10) against PEV 7.  
         [0123]    Further Identification of Lelystad Agent  
         [0124]    LA did not haemagglutinate with chicken, guinea pig, pig, sheep, or human O red blood cells.  
         [0125]    LA did not react in IPMA with sera directed against PRV, TGE, PED, ASFV, etc.  
         [0126]    After two blind passages, LA did not grow in PK2, PK-15, or SK-6 cells, or in embryonated hen eggs, inoculated through the allantoic route.  
         [0127]    LA was still infectious after it was filtered through a 0.2 micron filter, titres before and after filitration were 10 5.05  and 10 5.3  TCID 50  as detected by IPMA.  
         [0128]    Growth curve of LA (see FIG. 3). Maximum titres of cell-free virus were approximately 10 5.5 TCID 50  ml −1  from 32-48 h after inoculation. After that time the macrophages he cytopathic effect of LA.  
         [0129]    Electronmicroscopy. Clusters of spherical LA particles were found. The particles measured 45-55 nm in diameter and contained a 30-35 nm nucleocapsid that was surrounded by a lipid bilayer membrane. LA particles were not found in infected cultures that were treated with negative serum or in negative control preparations.  
         [0130]    Isolates from the Netherlands, Germany, and the United States. All seven isolates were isolated in porcine alveolar macrophages and passaged three to five times. All isolates caused a cytopathic effect in macrophages and could be specifically immunostained with anti-LA sera b 822 and the 42 dpi serum 23. The isolates were named NL2, GE1, GE 2, US1, US2, and US3.  
         [0131]    Antigenic relationships ofisolates NL1, NL2, GE1, GE2, US 1, US2, and US3. None of the field sera reacted in IPMA with uninfected macrophages but all sera contained antibodies directed against one or more of the seven isolates (Table 7). None of the experimental sera reacted in IPMA with uninfected macrophages, and none of the 0 dpi experimental sera reacted with any of the seven isolates in IPMA (Table 8). All seven LA isolates reacted with all or most of the sera from the set of experimental sera of pigs 21, 23, 25, and 29, taken after 0 dpi. Only the isolates US1, US2, and US3 reacted with all or most of the sera from the set of experimental sera of gilts 2B, 9G, 16W, and 16Y, taken after 0 dpi.  
         [0132]    Radioimmunoprecipitation studies. Seven LA-specific proteins were detected in LA-infected macrophages but not in uninfected macrophages precipitated with the 42 dpi sera of pigs b 822 and 23. The proteins had estimated molecular weights of 65, 39, 35, 26, 19, 16, and 15 kilodalton. Only two of these LA-specific proteins, of 16 and 15 kilodalton, were also precipitated by the 26 dpi serum MN8.  
         [0133]    Sequence and Organization of the Genome of LV  
         [0134]    The nature of the genome of LV was determined by analyzing DNA and RNA from infected porcine lung alveolar macrophages. No LV-specific DNA was detected. However, we did detect LV-specific RNA. In a 0.8% neutral agarose gel, LV RNA migrated slightly slower than a preparation of hog cholera virus RNA of 12.3 kb (Moormann et al., 1990) did. Although no accurate size determination can be performed in neutral agarose gels, it was estimated that the LV-specific RNA is about 14.5 to 15.5 kb in length.  
         [0135]    To determine the complexity of the LV-specific RNAs in infected cells and to establish the nucleotide sequence of the genome of LV, we prepared cDNA from RNA of LV-infected porcine lung alveolar macrophages and selected and mapped LV-specific cDNA clones as described under Materials and Methods. The specificity of the cDNA clones was reconfirmed by hybridizing specific clones, located throughout the overlapping cDNA sequence, to Northern blots carrying RNA of LV-infected and uninfected macrophages. Remarkably, some of the cDNA clones hybridized with the 14.5 to 15.5 kb RNA detected in infected macrophages only, whereas others hybridized with the 14.5 to 15.5 kb RNA as well as with a panel of 4 or 5 RNAs of lower molecular weight (estimated size, 1 to 4 kb). The latter clones were all clustered at one end of the cDNA map and covered about 4 kb of DNA. These data suggested that the genome organization of LV may be similar to that of coronaviridae (Spaan et al., 1988), Berne virus (BEV; Snijder et al., 1990b), a torovirus, and EAV (de Vries et al., 1990), i.e., besides a genomic RNA there are subgenomic mRNAs which form a nested set which is located at the 3′ end of the genome. This assumption was confirmed when sequences of the cDNA clones became available and specific primers could be selected to probe the blots with. A compilation of the hybridization data obtained with cDNA clones and specific primers, which were hybridized to Northern blots carrying the RNA of LV-infected and uninfected macrophages, is shown in FIG. 2. Clones 12 and 20 which are located in the 5′ part and the centre of the sequence, respectively, hybridize to the 14.5 to 15.5 kb genomic RNA detected in LV-infected cells only. Clones 41 and 39, however, recognize the 14.5 to 15.5 kb genomic RNA and a set of 4 and 5 RNAs of lower molecular weight, respectively. The most instructive and conclusive hybridization pattern, however, was obtained with primer 25, which is located at the ultimate 5′ end in the LV sequence (compare FIG. 1). Primer 25 hybridized to a panel of 7 RNAs, with an estimated molecular weight ranging in size from 0.7 to 3.3 kb (subgenomic mRNAs), as well as the genomic RNA. The most likely explanation for the hybridization pattern of primer 25 is that 5′ end genomic sequences, the length of which is yet unknown, fuse with the body of the mRNAs which are transcribed from the 3′ end of the genome. In fact, the hybridization pattern obtained with primer 25 suggests that 5′ end genomic sequences function as a so called “leader sequence” in subgenomic mRNAs. Such a transcription pattern is a hallmark of replication of coronaviridae (Spaan et al., 1988), and of EAV (de Vries et al., 1990).  
         [0136]    The only remarkable discrepancy between LV and EAV which could be extracted from the above data is that the genome size of LV is about 2.5 kb larger than that of EAV.  
         [0137]    The consensus nucleotide sequence of overlapping cDNA clones is shown in FIG. 1 (SEQ ID NO: 1). The length of the sequence is 15,088 basepairs, which is in good agreement with the estimated size of the genomic LV RNA.  
         [0138]    Since the LV cDNA library was made by random priming of the reverse transcriptase reaction with calf thymus pd(N) 6 primers, no cDNA clones were obtained which started with a poly-A stretch at their 3′ end. To clone the 3′ end of the viral genome, we constructed a second cDNA library, using oligo (dT) and primer 39U183R in the reverse transcriptase reaction. Primer 39U183R is complementary to LV minus-strand RNA, which is likely present in a preparation of RNA isolated from LV-infected cells. This library was screened with virus-specific probes (nick-translated cDNA clone 119 and oligonucleotide 119R64R), resulting in the isolation of five additional cDNA clones (e.g., cDNA clone 151, FIG. 2). Sequencing of these cDNA clones revealed that LV contains a 3′ poly(A) tail. The length of the poly(A) tail varied between the various cDNA clones, but its maximum length was twenty nucleotides. Besides clone 25 and 155 (FIG. 2), four additional cDNA clones were isolated at the 5′ end of the genome, which were only two to three nucleotides shorter than the ultimate 5′ nucleotide shown in FIG. 1 (SEQ ID NO: 1). Given this finding and given the way cDNA was synthesized, we assume to be very close to the 5′ end of the sequence of LV genomic RNA.  
         [0139]    Nearly 75% of the genomic sequence of LV encodes ORF 1A and ORF 1B. ORF 1A probably initiates at the first AUG (nucleotide position 212, FIG. 1) encountered in the LV sequence. The C-terminus of ORF 1A overlaps the putative N-terminus of ORF 1 B over a small distance of 16 nucleotides. It thus seems that translation of ORF 1B proceeds via ribosomal frameshifling, a hallmark of the mode of translation of the polymerase or replicase gene of coronaviruses (Boursnell et al., 1987; Bredenbeek et al. 1990) and the torovirus BEV (Snijder et al., 1990a). The characteristic RNA pseudoknot structure which is predicted to be formed at the site of the ribosomal frameshifting is also found at this location in the sequence of LV (results not shown).  
         [0140]    ORF 1B encodes an amino acid sequence (SEQ ID NO: 3) of nearly 1400 residues which is much smaller than ORF 1B of the coronaviruses MHV and IBV (about 3,700 amino acid residues; Bredenbeek et al., 1990; Boursnell et al., 1987) and BEV (about 2,300 amino acid residues; Snijder et al., 1990a). Characteristic features of the ORF 1B product (SEQ ID NO: 3) of members of the superfamily of coronaviridae, like the replicase motif and the Zinc finger domain, can also be found in ORF 1B of LV (results not shown).  
         [0141]    Whereas ORF 1A and ORF 1B encode the viral polymerase (SEQ ID NO:2 and SEQ ID NO:3) and, therefore, are considered to encode a non-structural viral protein, ORFs 2 to 7 are believed to encode structural viral proteins (SEQ ID NOS:4-9).  
         [0142]    The products of ORFs 2 to 6 (SEQ ID NOS:4-8) all show features reminiscent of membrane (envelope) associated proteins. ORF 2 encodes a protein (SEQ ID NO:4) of 249 amino acids containing two predicted N-linked glycosylation sites (Table 9). At the N-terminus a hydrophobic sequence, which may function as a so-called signal sequence, is identified. The C-terminus also ends with a hydrophobic sequence, which in this case may function as a transmembrane region, which anchors the ORF 2 product (SEQ ID NO:4) in the viral envelope membrane.  
         [0143]    ORF 3 may initiate at the AUG starting at nucleotide position 12394 or at the AUG starting at nucleotide position 12556 and then encodes proteins (SEQ ID NO:5) of 265 and 211 amino acids, respectively. The protein of 265 residues contains seven putative N-linked glycosylation sites, whereas the protein of 211 residues contains four (Table 9). At the N-terminus of the protein (SEQ ID NO:5) of 265 residues a hydrophobic sequence is identified.  
         [0144]    Judged by hydrophobicity analysis, the topology of the protein encoded by ORF 4 (SEQ ID NO:6) is similar to that encoded by ORF 2 (SEQ ID NO:4) if the product of ORF 4 (SEQ ID NO:6) initiates at the AUG starting at nucleotide position 12936. However, ORF 4 may also initiate at two other AUG codons (compare FIGS. 1 and 2) starting at positions 12981 and 13068 in the sequence respectively. Up to now it is unclear which start codon is used. Depending on the start codon used, ORF 4 may encode proteins (SEQ ID NO:6) of 183 amino acids containing four putative N-linked glycosylation sites, of 168 amino acids containing four putative N-linked glycosylation sites, or of 139 amino acids containing three putative N-linked glycosylation sites (Table 9).  
         [0145]    ORF 5 is predicted to encode a protein (SEQ ID NO:7) of 201 amino acids having two putative N-linked glycosylation sites (Table 9). A characteristic feature of the ORF 5 product (SEQ ID NO:7) is the internal hydrophobic sequence between amino acid 108 to amino acid 132.  
         [0146]    Analysis for membrane spanning segments andhydrophilicity of the product of ORF 6 (SEQ ID NO:8) shows that it contains three transmembrane spanning segments in the N-terminal 90 amino acids of its sequence. This remarkable feature is also a characteristic of the small envelope glycoprotein M or E1 of several coronaviruses, e.g., Infectious Bronchitis Virus (IBV; Boursnell et al., 1984) and Mouse Hepatitis Virus (MHV: Rottier et al., 1986). It is, therefore, predicted that the protein encoded by ORF 6 (SEQ ID NO:8) was a membrane topology analogous to that of the M or E1 protein of coronaviruses (Rottier et al., 1986). A second characteristic of the M or E1 protein is a so-called surface helix which is located immediately adjacent to the presumed third transmembrane region. This sequence of about 25 amino acids which is very well conserved among coronaviruses is also recognized, although much more degenerate, in LV. Yet we predict the product of LV ORF 6 (SEQ ID NO:8) to have an analogous membrane associated function as the coronavirus M or E1 protein. Furthermore, the protein encoded by ORF 6 (SEQ ID NO:8) showed a strong similarity (53% identical amino acids) with VpX (Godeny et al., 1990) of LDV.  
         [0147]    The protein encoded by ORF 7 (SEQ ID NO:9) has a length of 128 amino acid residues (Table 9) which is 13 amino acids longer than Vp1 of LDV (Godeny et al., 1990). Yet a significant similarity (43% identical amino acids) was observed between the protein encoded by ORF 7 (SEQ ID NO:9) and Vp1. Another shared characteristic between the product of ORF 7 (SEQ ID NO:9) and Vp1 is the high concentration of basic residues (Arg, Lys and His) in the N-terminal half of the protein. Up to amino acid 55, the LV sequence contains 26% Arg, Lys and His. This finding is fully in line with the proposed function of the ORF 7 product (SEQ ID NO:9) or Vp1 (Godeny et al., 1990), namely encapsidation of the viral genomic RNA. On the basis of the above data, we propose the LV ORF 7 product (SEQ ID NO:9) to be the nucleocapsid protein N of the virus.  
         [0148]    A schematic representation of the organization of the LV genome is shown in FIG. 2. The map of overlapping clones used to determine the sequence of LV is shown in the top panel. A linear compilation of this map indicating the 5′ and 3′ end of the nucleotide sequence of LV, shown in FIG. 1 (SEQ ID NO:1), including a division in kilobases, is shown below the map of cDNA clones and allows the positioning of these clones in the sequence. The position of the ORFs identified in the LV genome is indicated below the linear map of the LV sequence. The bottom panel shows the nested set of subgenomic mRNAs, and the position of these RNAs relative to the LV sequence.  
         [0149]    In line with the translation strategy of coronavirus, torovirus and arterivirus subgenomic mRNAs, it is predicted that ORFs 1 to 6 are translated from the unique 5′ end of their genomic or mRNAs. This unique part of the mRNAs is considered to be that part of the RNA that is obtained when a lower molecular weight RNA is “subtracted” from the higher molecular weight RNA which is next in line. Although RNA 7 forms the 3′ end of all the other genomic and subgenomic RNAs, and thus does not have a unique region, it is believed that ORF 7 is only translated from this smallest sized mRNA. The “leader sequence” at the 5′ end of the subgenomic RNAs is indicated with a solid box. The length of this sequence is about 200 bases, but the precise site of fusion with the body of the genomic RNAs still has to be determined.  
         [0150]    Experimental Reproduction of MSD  
         [0151]    Eight pregnant sows were inoculated with LA and clinical signs of MSD such as inappetance and reproductive losses were reproduced in these sows. From day four to day 10-12 post-inoculation (p.i.), all sows showed a reluctance to eat. None of the sows had elevated body temperatures. Two sows had bluish ears at day 9 and 10 p.i. In Table 6 the day of birth and the number of living and dead piglets per sow is given. LA was isolated from 13 of the born piglets.  
                                                             TABLE 1                       Description and results of virus isolation of field samples.                   A Samples of piglets suspected of infection with MSD.                number   age               farm   of pigs   days   material used   results*               RB    5    2   lung, tonsil, and brains    5 × LA       DV    4    3   lung, brains,    3 × LA                   pools of kidney,                   spleen       TH    3   3-5   lung, pools of kidney, tonsil    3 × LA       DO    3   10   lung, tonsil    2 × LA       ZA    4    1   lung, tonsil    3 × LA       VE    1   ?   oral swab    1 × PEV 2       TOTAL   20           16 × LA,                        1 × PEV 2                    B Samples of sows suspected of infection with MSD.                number               farm   of sows   material used   results               TH   2   plasma and leucocytes    1 × LA       HU   5   plasma and leucocytes    2 × LA, 1 × EMCV       TS   10   plasma and leucocytes    6 × LA       HK   5   plasma and leucocytes    2 × LA       LA   6   plasma and leucocytes    2 × LA       VL   6   serum and leucocytes    5 × LA       TA   15   serum   11 × LA       LO   4   plasma and leucocytes    2 × LA       JA   8   plasma and leucocytes    8 × LA       VD   1   plasma and leucocytes    1 × LA       VW   1   serum    1 × LA       TOTAL   63       41 × LA, 1 × EMCV                                                  
 
         [0152]    [0152]                                                                                       TABLE 2                           Description and results of virus isolation of       samples of pigs with experimentally induced infections.                sow   pig@   material used   results*                       A (LO) #   c 835   lung, tonsil    2 × LA               c 836   nasal swabs    2 × PEV 7               c 837   nasal swabs           B (JA)   c 825   lung, tonsil               c 821   nasal swabs    1 × PEV 7               c 823   nasal swabs    4 × PEV 7           C (JA)   c 833   lung, tonsil    1 × LA,                        1 × PEV 7               c 832   nasal swabs    2 × PEV 7               c 829   nasal swabs,                   plasma and    3 × LA,                   leucocytes    2 × PEV 7           D (VD)   c 816   lung, tonsil               c 813   nasal swabs    1 × LA               c 815   nasal swabs    1 × PEV 7                TOTAL isolates from contact pigs    7 × LA,               13 × PEV 7                A   b 809   nasal swabs                   b 817   nasal swabs           B   b 818   nasal swabs, plasma   1 × LA                   and leucocytes               b 820   nasal swabs           C   b 822   nasal swabs               b 826   nasal swabs           D   b 830   nasal swabs   1 × LA               b 834   nasal swabs                TOTAL isolates from blood inoculated pigs   2 × LA                                   #a separate stable. EDTA blood for virus isolation from plasma and leucocytes was taken whenever a pig had fever.                                                             
         [0153]    [0153]                                     TABLE 3                           Identification of viral isolates                buoyant 1     particle 2         neutralized by 4         origin and   density   size in   sens 3  to   serum directed       cell culture   in CsCl   FM (nm)   chloroform   against (titre)               leucocytes   1.33 g/ml   28-30   not sens.   EMCV ( 1280)       sow farm HU       PK-15,       PK2, SK6       oral swab   ND   28-30   not sens.   PEV 2 (&gt;1280)       piglet farm VE       SK6       nasal swabs,   ND   28-30   not sens.   PEV 7 (&gt;1280)       tonsil       SPF pigs CVI       PK-15, PK2,       SK6       various   1.19 g/ml   pleomorf   sens.   none (all &lt;5)       samples       various farms       pig lung       macrophages                                               #entero viruses (PEV) 1 to 11 (courtesy Dr. Knowles, Pirbright, UK), against encephalomyocarditis virus (EMCV; courtesy Dr. Ahl, Tübingen, Germany), against porcine parvo virus, and against swine vesicular disease.                #leukemia virus from the SPF-pigs (see Table 5).             
         [0154]    [0154]                                                                                                                                                                       TABLE 4                           Results of serology of paired field sera taken from sows       suspected to have MSD. Sera were taken       in the acute phase of the disease and 3-9 weeks       later. Given is the number of sows which showed       a fourfold or higher rise in titre/number of sows tested.                Interval i                                     Farm   in weeks   HAI HEV   H1N1   H3N2   ELISA PPV   PPV   BVDV   HCV               TH   3   0/6   0/6   0/6   0/6   0/6   0/5   0/6       RB   5   0/13   1/13   0/13   1/9   0/7   0/6   0/9       HU   4   0/5   0/5   3/5   0/5   0/5   0/5   0/5       TS   3   1/10   0/10   0/10   0/10   0/10   0/4   0/10       VL   3   0/5   0/5   0/5   0/5   1/5   0/5   0/5       JA   3   0/11   1/11   3/11   0/11   2/11   0/11   0/11       WE   4   1/6   1/6   1/6   3/7   3/7   0/7   0/7       GI   4   0/4   1/4   0/4   0/4   0/4   0/4   0/4       SE   5   0/8   0/8   0/8   0/8   0/6   0/3   0/8       KA   5   0/1   0/1   0/1   0/1   0/1   ND   0/1       HO   3   1/6   0/5   1/6   0/6   0/6   0/6   0/6       NY   4   0/5   1/5   1/5   0/3   0/4   0/2   0/4       JN   3   0/10   5/10   0/10   0/10   1/10   0/10   0/10       KO f     3   1/10   0/10   0/10   0/10   2/10   0/10   0/10       OE   9   ND   ND   ND   0/6   0/6   0/6   0/6       LO   6   ND   ND   ND   0/3   0/3   0/2   0/3       WI   4   ND   ND   ND   0/1   1/1   0/1   0/3       RR   3   ND   ND   ND   1/8   0/8   0/8   0/8       RY   4   ND   ND   ND   0/3   0/4   0/3   0/4       BE   5   ND   ND   ND   0/10   0/10   0/10   0/10       BU   3   ND   ND   ND   1/6   0/6   0/6   0/6       KR   3   ND   ND   ND   1/4   0/4   0/4   0/4       KW   5   ND   ND   ND   0/10   0/10   0/10   0/10       VR   5   ND   ND   ND   1/6   0/6   0/6   0/6       HU   4   ND   ND   ND   1/4   0/3   0/3   0/4       ME   3   ND   ND   ND   0/5   1/5   0/5   0/5            total negative n      19   41    29    97    16   140   165       total positive p      77   48    62    55   131    1    0       total sero-converted s      4   10    9    9    11    0    0       total tested   100   99   100   161   158   141   165                        Interval   SNT                           IPMA       Farm   in weeks   EMCV   EMCVi   PEV2   PEV2i   PEV7   PEV7i   LA   LA               TH   3   0/6   0/6   0/5   0/5   0/6   0/5   0/6    6/6       RB   5   1/7   1/9   0/6   2/6   1/8   0/6   0/13    7/9       HU   4   ND   0/5   0/5   0/5   ND   0/5   0/5    5/5       TS   3   0/10   0/10   0/7   0/4   0/10   0/7   ND   10/10       VL   3   ND   ND   1/5   0/5   ND   0/5   ND    5/5       JA   3   0/11   0/11   0/11   0/11   1/11   2/11   0/5    8/11       WE   4   1/7   1/6   1/6   1/7   1/7   1/7   0/7    7/7       GI   4   0/4   0/4   0/4   0/4   0/4   0/4   0/4    4/4       SE   5   0/8   0/8   0/6   1/8   0/8   1/5   0/8    6/8       KA   5   0/1   0/1   0/1   0/1   0/1   0/1   0/1    0/1       HO   3   0/6   0/6   0/6   0/6   0/6   0/6   0/6    4/6       NY   4   0/4   0/4   0/2   0/2   0/4   0/3   0/4    4/4       JN   3   0/10   0/10   1/10   0/9   0/10   0/10   0/10    5/10       KO f     3   0/10   0/10   2/10   2/10   1/10   3/10   ND    8/10       OE   9   0/6   0/6   1/6   1/5   ND   1/6   ND    4/6       LO   6   0/3   0/3   0/3   0/3   0/3   0/3   ND    3/3       WI   4   ND   ND   0/1   0/1   ND   0/1   ND    0/3       RR   3   0/8   1/8   0/8   0/8   0/8   0/8   ND    8/8       RY   4   0/4   ND   0/4   0/1   ND   1/4   ND    1/4       BE   5   ND   ND   0/10   0/10   ND   1/10   ND    0/10       BU   3   ND   ND   0/6   0/6   ND   0/6   ND    6/6       KR   3   ND   ND   0/4   0/4   ND   0/4   ND    1/4       KW   5   ND   ND   0/10   0/10   ND   1/10   ND   10/10       VR   5   ND   ND   0/6   1/6   ND   0/6   ND    6/6       HU   4   ND   ND   0/3   0/4   ND   0/3   ND    3/4       ME   3   ND   ND   0/5   0/5   ND   0/5   ND    2/5            total neg. n      15    29    0    0    2    1   69    15       total pos. p      88    74   144   138   90   136    0    27       total sero-converted s      2    3    6    8    4    10    0   123       total tested   105   107   150   146   96   147   69   165                                                                                    
         [0155]    [0155]                                                                                                         TABLE 5                       Development of antibody directed against       Lelystad Agent as measured by IPMA.                   A contact pigs serum titres in IPMA                    Weeks post contact:                Pig   0   2   3   4   5                       c 836   0   10   640   640   640           c 837   0   10   640   640   640           c 821   0   640   640   640   640           c 823   0   160   2560   640   640           c 829   0   160   640   10240   10240           c 832   0   160   640   640   2560           c 813   0   640   2560   2560   2560           c 815   0   160   640   640   640                        B blood inoculated pigs serum titres in IPMA                Weeks post inoculation:                Pig   0   2   3   4   6                       b 809   0   640   2560   2560   2560           b 817   0   160   640   640   640           b 818   0   160   640   640   640           b 820   0   160   640   640   640           b 822   0   640   2560   2560   10240           b 826   0   640   640   640   10240           b 830   0   640   640   640   2560           b 834   0   160   640   2560   640                                    
         [0156]    [0156]                                                                                                                           TABLE 6                           Experimental reproduction of MSD.                    No. of piglets                   Length   at birth   No. of   LA 1  in piglets                of   alive   dead   deaths   born   died in            Sow   gestation   (Number Ab pos) 2     week 1   dead   week 1                    52   113   12 (5)    3 (2)   6   2   4       965   116    3 (0)    9 (3)   2   4       997   114    9 (0)    1 (0)   0       1305   116    7 (0)    2 (0)   1       134   109    4 (4)    7 (4)   4   3       941   117    7   10       1056   113    7 (1)    3 (0)   4       1065   115    9    2                                    
         [0157]    [0157]                                                     TABLE 7                           Reactivity in IPMA of a collection of field sera from       Europe and North America tested with LA       isolates from the Netherlands (NL1 and NL2),       Germany (GE1 and GE2), and the United States       (US1, US2 and US3).            Isolates:       NL1   NL2   GE1   GE2   US1   US2   US3               Sera from:                                       The Netherlands       TH-187       3.5 t      3.5   2.5   3.5   −   −   −       TO-36       3.5   3.0   2.5   3.0   −   1.0   −       Germany       BE-352       4.0   3.5   2.5   3.0   −   1.5   −       BE-392       3.5   3.5   2.5   2.5   1.5   1.5   0.5       NI-f2   2.5   1.5   2.0   2.5   −   −   −       United Kingdom       PA-141615       4.0   3.0   3.0   3.5   −   −   −       PA-141617       4.0   3.5   3.0   3.5   −   2.5   2.0       PA-142440       3.5   3.0   2.5   3.5   −   2.0   2.5       Belgium       PE-1960       4.5   4.5   3.0   4.0   1.5   −   −       France       EA-2975       4.0   3.5   3.0   3.0   2.0   −   −       EA-2985       3.5   3.0   3.0   2.5   −   −   −       United States       SL-441       3.5   1.5   2.5   2.5   3.5   3.5   3.0       SL-451       3.0   2.0   2.5   2.5   3.5   4.5   4.0       AL-RP9577       1.5   −   −   1.0   3.0   4.0   2.5       AL-P10814/33       0.5   2.5   −   −   2.5   3.5   3.0       AL-4094A       −   −   −   −   1.0   2.0   0.5       AL-7525       −   −   −   −   −   1.0   −       JC-MN41       −   −   −   −   1.0   3.5   1.0       JC-MN44       −   −   −   −   2.0   3.5   2.0       JC-MN45       −   −   −   −   2.0   3.5   2.5       Canada       RB-16       2.5   −   3.0   2.0   3.0   3.5   −       RB-19       1.0   −   1.0   −   2.5   1.5   −       RB-22       1.5   −   2.0   2.5   2.5   3.5   −       RB-23       −   −   −   −   −   3.0   −                                    
         [0158]    [0158]                                                                                       TABLE 8                           Reactivity in IPMA of a collection of experimental sera       raised against LA and SIRSV tested with       LA isolates from the Netherlands (NL1 and NL2),       Germany (GE1 and GE2), and the United States       (US1, US2 and US3).            Isolates:   NL1   NL2   GE1   GE2   US1   US2   US3               Sera:                                   anti-LA:            21   14 dpi   2.5 t     2.0   2.5   3.0   1.5   2.0   1.5           28 dpi   4.0   3.5   3.5   4.0   −   2.5   1.5           42 dpi   4.0   3.5   3.0   3.5   1.5   2.5   2.0       23   14 dpi   3.0   2.0   2.5   3.0   1.0   2.0   1.0           28 dpi   3.5   3.5   3.5   4.0   1.5   2.0   2.0           42 dpi   4.0   4.0   3.0   4.0   −   2.5   2.5       25   14 dpi   2.5   2.0   2.5   3.0   1.5   2.0   1.0           28 dpi   4.0   3.5   4.0   3.5   −   1.5   2.0           42 dpi   3.5   4.0   3.5   3.5   1.5   2.0   2.0       29   14 dpi   3.5   3.5   3.0   3.5   −   2.0   1.5           28 dpi   3.5   3.5   3.0   3.5   −   2.5   2.0           42 dpi   4.0   3.5   3.5   4.0   1.5   2.5   2.5       anti-       SIRSV:       2B   20 dpi   −   −   −   −   2.0   2.0   −           36 dpi   −   −   −   −   1.5   2.0   −           63 dpi   −   −   −   −   1.0   1.0   −       9G   30 dpi   −   −   −   −   2.5   3.0   −           44 dpi   −   −   −   −   2.5   3.5   −           68 dpi   −   −   −   −   2.0   3.5   1.5       16W   25 dpi   −   −   −   −   2.0   3.0   −           40 dpi   −   −   −   −   2.0   3.0   −           64 dpi   −   −   −   −   2.5   2.5   1.5       16Y   36 dpi   −   −   −   −   1.0   3.0   1.0           64 dpi   −   −   −   −   2.5   3.0   −                                    
         [0159]    [0159]                                                           TABLE 9                           Characteristics of the ORFs of Lelystad Virus.                        Calculated                   No. of   size of the   number of           Nucleotides   amino   unmodified   glycosylation       ORF   (first-last)   acids   peptide (kDa)   sites                    ORF1A    212-7399   2396   260.0   3 (SEQ ID NO: 2)       ORF1B    7384-11772   1463   161.8   3 (SEQ ID NO: 3)       ORF2   11786-12532   249   28.4   2 (SEQ ID NO: 4)       ORF3   12394-13188   265   30.6   7 (SEQ ID NO: 5)           12556-13188   211   24.5   4       ORF4   12936-13484   183   20.0   4 (SEQ ID NO: 6)           12981-13484   168   18.4   4           13068-13484   139   15.4   3       ORF5   13484-14086   201   22.4   2 (SEQ ID NO: 7)       ORF6   14077-14595   173   18.9   2 (SEQ ID NO: 8)       ORF7   14588-14971   128   13.8   1 (SEQ ID NO: 9)                    
       REFERENCES  
       [0160]    Boer, G. F. de, Back, W., and Osterhaus, A. D. M. E. (1990), An ELISA for detection of antibodies against influenza A nucleoprotein in human and various animal species, Arch. Virol. 115, 47-61.  
         [0161]    Boursnell, M. E. G., Brown, T. D. K., and Binns, M. M. (1984), Sequence of the membrane protein gene from avian coronavirus IBV, Virus Res. 1, 303-314.  
         [0162]    Boursnell, M. E. G., Brown, T. D. K., Foulds, I. J., Green, P. F., Tomley, F. M., and Binns, M. M. (1987), Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus, J. Gen. Virol. 68, 57-77.  
         [0163]    Brakke, M. K. (1967), In: Methods in Virology, Volume II, pp. 93-117 (Edited by K. Maramorosch and H. Koprowski) New York, Academic Press.  
         [0164]    Bredenbeek, P. J., Pachuk, C. J., Noten, J. F. H., Charite, J., Luytjes, W., Weiss, S. R., and Spaan, W. J. M. (1990), The primary structure and expression of the second open reading frame of the polymerase gene of coronavirus MHV-A59. Nucleic Acids Res. 18, 1825-1832.  
         [0165]    Brenner, S., and Home, R. W. (1959), A negative staining method for high resolution electron microscopy of viruses, Biochimica et Biophysica Acta 34, 103-110.  
         [0166]    Brinton-Damell, M., and Plagemann, P. G. (1975), Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA, J. Virol. 16, 420-433.  
         [0167]    Favaloro, J., Treisman, R. &amp; Kamen, R. (1980), In: Methods in Enzymology, vol. 65, 718-749 (eds. Grossman, L. &amp; Moldave, K.) Academic Press, New York.  
         [0168]    Godeny, E. K., Speicher, D. W., and Brinton, M. A. (1990), Map location of lactate dehydrogenase-elevating virus (LDV) capsid protein (VpI) gene, Virology, 177, 768-771.  
         [0169]    Grist, N. R., Ross, C. A., and Bell, E. J. (1974), In: Diagnostic Methods in Clinical Virology, p. 120, Oxford, Blackwell Scientific Publications.  
         [0170]    Güibler, U., and Hoffman, B. J. (1983), A simple and very efficient method for generating cDNA libraries, Gene 25, 263-269.  
         [0171]    Hanahan, D. (1985), In: DNA Cloning I; A Practical Approach, Chapter 6, 109-135.  
         [0172]    Hill, H. (1990), Overview and History of Mystery Swine Disease (Swine Infertility Respiratory Syndrome), In: Proceedings of the Mystery Swine Disease Committee Meeting, Oct. 6, 1990, Denver, Colo., Livestock Conservation Institute, Madison, Wis., U.S.A.  
         [0173]    Hirsch, J. G. &amp; Fedorko, M. E. (1968), Ultrastructure of human leucocytes after simultanous fixation with glutaraldehyde and osmiumtetroxide and postfixation in uranylacetate, Journal of Cellular Biology 38, 615.  
         [0174]    Horzinek, M. C., Maess, J., and Laufs, R. (1971), Studies on the substructure of togaviruses II. Analysis of equine arteritis, rubella, bovine viral diarrhea and hog cholera viruses, Arch. Gesamte Virusforsch. 33, 306-318.  
         [0175]    Hyllseth, B. (1973), Structural proteins of equine arteritis virus, Arch. Gesamte Virusforsch. 40, 177-188.  
         [0176]    Kasza, L., Shadduck, J. A., and Christoffinis, G. J. (1972), Establishment, viral susceptibility and biological characteristics of a swine kidney cell line SK-6, Res. Vet. Sci. 13, 46-51.  
         [0177]    Loula, T. (1990), Clinical Presentation of Mystery Pig Disease in the breeding herd and suckling piglets, In: Proceedings of the Mystery Swine Disease Committee Meeting, Oct. 6, 1990, Denver, Colo., Livestock Conservation Institute, Madison, Wis., U.S.A.  
         [0178]    Masurel, N. (1976), Swine influenza virus and the recycling of influenza A viruses in man, Lancet ii, 244-247.  
         [0179]    Mazancourt, A. de, Waxham. M. N., Nicholas, J. C., &amp; Wolinsky, J. S. (1986), Antibody response to the rubella virus structural proteins in infants with the congenital rubella syndrome. J. Med. Virol. 19, 111-122.  
         [0180]    Mengeling, W. L., and Lager, K. M. (1990), Mystery Pig Disease: Evidence and considerations for its etiology, In: Proceedings of the Mystery Swine Disease Committee Meeting, Oct. 6, 1990, Denver, Colo., Livestock Conservation Institute, Madison, Wis., U.S.A.  
         [0181]    Moormann, R. J. M., and Hulst, M. M. (1988), Hog cholera virus: identification and characterization of the viral RNA and virus-specific RNA synthesized in infected swine kidney cells, Virus Res. 11, 281-291.  
         [0182]    Moormann, R. J. M., Warmerdam, P. A. M., van derMeer, B., Schaaper, W. M. M., Wensvoort, G., and Hulst, M. M. (1990), Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein E 1, Virology, 177, 184-198.  
         [0183]    Oirschot, J. T. van, Houwers, D. J., Rziha, H. J., and Moonen, P. J. L. M. (1988), Development of an ELISA for detection of antibodies to glycoprotein I of Aujeszky&#39;s disease virus: a method for the serological differentiation between infected and vaccinated pigs, J. Virol. Meth. 22, 191-206.  
         [0184]    Pearson, W. R., and Lipman, D. J. (1988), Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448.  
         [0185]    Reed, L. J., and Muench, H. (1938), A simple method of estimating fifty percent endpoints, Am. J. Hyg. 27, 493-497.  
         [0186]    Rottier, P. J. M., Welling, G. W., Welling-Wester, S., Niesters, H. G. M., Lenstra, J. M., and van der Zeijst, B. A. M. (1986), Predicted membrane topology of the coronavirus protein E 1. Biochemistry 25, 1335-1339.  
         [0187]    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.  
         [0188]    Sethna, P. B., Hung, S. L., and Brian, D. A. (1989), Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons, Proc. Natl. Acad. Sci. USA, 86, 5626-5630.  
         [0189]    Setzer, D. R., McGrogan, M., Nunberg, J. H. &amp; Schimke, R. T. (1980), Size heterogeneity in the 3′-end of the dehydrofolate reductase messenger RNA&#39;s in mouse cells, Cell 22, 361-370.  
         [0190]    Snijder, E. J., den Boon, J. A., Bredenbeek, P. J., Horzinek, M. C., Rijnbrand, R., and Spaan, W. J. M. (1990a), The carboxyl-terminal part of the putative Beme virus polymerase is expressed by ribosomal frameshifting and contains sequence motifs which indicate that toro- and coronaviruses are evolutionary related, Nucleic Acids Res. 18, 4535-4542.  
         [0191]    Snijder, E. J., Horzinek, M. C., and Spaan, W. J. M. (1990b), A 3′-coterminal nested set of independently transcribed messenger RNAs is generated during Beme virus replication. J. Virol. 64, 355-363.  
         [0192]    Spaan, W. J. M., Cavanagh, D., and Horzinek, M. C. (1988), Coronaviruses: structure and genome expression. J. Gen. Virol. 69, 2939-2952.  
         [0193]    Strauss, W. M. (1987), Preparation of genomic DNA from mammalian tissue, In: Current protocols in molecular biology (eds. Ausubel F. M., et al.) 2.2.1 John Wiley &amp; Sons, New York.  
         [0194]    Terpstra, C. (1978), Detection of Border disease antigen in tissues of affected sheep and in cell cultures by immunofluorescence, Res. Vet. Sci. 25, 350-355.  
         [0195]    Venable, J. H. &amp; Coggeshall, R. (1965), A simplified lead citrate stain for use in electronmicroscopy, Journal of Cellular Biology 25, 407.  
         [0196]    Vries, A. A. P. de, Chirnside, E. D., Bredenbeek, P. J., Gravestein, L. A., Horzinek, M. C., and Spaan, W. J. M. (1990), All subgenomic mRNAs of equine arteritis virus contain a common leader sequence, Nucleic Acids Res. 18, 3241-3247.  
         [0197]    Wensvoort, G., and Terpstra, C. (1988), Bovine viral diarrhea infections in piglets from sows vaccinated against swine fever with contaminated vaccine, Res. Vet. Sci. 45, 143-148.  
         [0198]    Wensvoort, G., Terpstra, C., and Bloemraad, M. (1988), An enzyme immunoassay, employing monoclonal antibodies and detecting specifically antibodies against classical swine fever virus, Vet. Microbiol. 17, 129-140.  
         [0199]    Wensvoort, G., Terpstra, C., Boonsta, J., Bloemraad, M., and Zaane, D. van (1986), Production ofmonoclonal antibodies against swine fever virus and their use in laboratory diagnosis, Vet. Microbiol. 12, 101-108.  
         [0200]    Wensvoort, G., Terpstra. C., and Kluyver, E. P. de (1989), Characterization of porcine and some ruminant pestiviruses by cross-neutralization, Vet. Microbiol. 20, 291-306.  
         [0201]    Westenbrink, F., Middel. W. G. J., Straver, P., and Leeuw, P. W. de (1986), A blocking enzyme-linked immunosorbent assay (ELISA) for bovine virus diarrhea virus serology, J. Vet. Med. B33, 354-361.  
         [0202]    Westenbrink, F., Veldhuis, M. A., and Brinkhof, J. M. A. (1989), An enzyme-linked immunosorbent assay for detection of antibodies to porcine parvo virus, J. Virol. Meth. 23, 169-178.  
         [0203]    Zeijst. B. A. M. van der, Horzinek, M. C., and Moennig, V. (1975), The genome of equine arteritis virus, Virology, 68, 418-425.   
     
       
       
         1 
         
           
             
9 
 
           
           
             
               15108 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA (genomic)  
             
             
               CDS  
                212..7399  
                
 
             
             
               CDS  
                7384..11772  
                
 
             
             
               CDS  
                11786..12532  
                
 
             
             
               CDS  
                12394..13188  
                
 
             
             
               CDS  
                12936..13484  
                
 
             
             
               CDS  
                13484..14086  
                
 
             
             
               CDS  
                14077..14595  
                
 
             
             
               CDS  
                14588..14971  
                
 
             
              1 

GGGTATTCCC CCTACATACA CGACACTTCT AGTGTTTGTG TACCTTGGAG GCGTGGGTAC     60 

AGCCCCGCCC CACCCCTTGG CCCCTGTTCT AGCCCAACAG GTATCCTTCT CTCTCGGGGC    120 

GAGTGCGCCG CCTGCTGCTC CCTTGCAGCG GGAAGGACCT CCCGAGTATT TCCGGAGAGC    180 

ACCTGCTTTA CGGGATCTCC ACCCTTTAAC C ATGTCTGGGA CGTTCTCCCG             231 

GTGCATGTGC ACCCCGGCTG CCCGGGTATT TTGGAACGCC GGCCAAGTCT TTTGCACACG    291 

GTGTCTCAGT GCGCGGTCTC TTCTCTCTCC AGAGCTTCAG GACACTGACC TCGGTGCAGT    351 

TGGCTTGTTT TACAAGCCTA GGGACAAGCT TCACTGGAAA GTCCCTATCG GCATCCCTCA    411 

GGTGGAATGT ACTCCATCCG GGTGCTGTTG GCTCTCAGCT GTTTTCCCTT TGGCGCGTAT    471 

GACCTCCGGC AATCACAACT TCCTCCAACG ACTTGTGAAG GTTGCTGATG TTTTGTACCG    531 

TGACGGTTGC TTGGCACCTC GACACCTTCG TGAACTCCAA GTTTACGAGC GCGGCTGCAA    591 

CTGGTACCCG ATCACGGGGC CCGTGCCCGG GATGGGTTTG TTTGCGAACT CCATGCACGT    651 

ATCCGACCAG CCGTTCCCTG GTGCCACCCA TGTGTTGACT AACTCGCCTT TGCCTCAACA    711 

GGCTTGTCGG CAGCCGTTCT GTCCATTTGA GGAGGCTCAT TCTAGCGTGT ACAGGTGGAA    771 

GAAATTTGTG GTTTTCACGG ACTCCTCCCT CAACGGTCGA TCTCGCATGA TGTGGACGCC    831 

GGAATCCGAT GATTCAGCCG CCCTGGAGGT ACTACCGCCT GAGTTAGAAC GTCAGGTCGA    891 

AATCCTCATT CGGAGTTTTC CTGCTCATCA CCCTGTCGAC CTGGCCGACT GGGAGCTCAC    951 

TGAGTCCCCT GAGAACGGTT TTTCCTTCAA CACGTCTCAT TCTTGCGGTC ACCTTGTCCA   1011 

GAACCCCGAC GTGTTTGATG GCAAGTGCTG GCTCTCCTGC TTTTTGGGCC AGTCGGTCGA   1071 

AGTGCGCTGC CATGAGGAAC ATCTAGCTGA CGCCTTCGGT TACCAAACCA AGTGGGGCGT   1131 

GCATGGTAAG TACCTCCAGC GCAGGCTTCA AGTTCGCGGC ATTCGTGCTG TAGTCGATCC   1191 

TGATGGTCCC ATTCACGTTG AAGCGCTGTC TTGCCCCCAG TCTTGGATCA GGCACCTGAC   1251 

TCTGGATGAT GATGTCACCC CAGGATTCGT TCGCCTGACA TCCCTTCGCA TTGTGCCGAA   1311 

CACAGAGCCT ACCACTTCCC GGATCTTTCG GTTTGGAGCG CATAAGTGGT ATGGCGCTGC   1371 

CGGCAAACGG GCTCGTGCTA AGCGTGCCGC TAAAAGTGAG AAGGATTCGG CTCCCACCCC   1431 

CAAGGTTGCC CTGCCGGTCC CCACCTGTGG AATTACCACC TACTCTCCAC CGACAGACGG   1491 

GTCTTGTGGT TGGCATGTCC TTGCCGCCAT AATGAACCGG ATGATAAATG GTGACTTCAC   1551 

GTCCCCTCTG ACTCAGTACA ACAGACCAGA GGATGATTGG GCTTCTGATT ATGATCTTGT   1611 

TCAGGCGATT CAATGTCTAC GACTGCCTGC TACCGTGGTT CGGAATCGCG CCTGTCCTAA   1671 

CGCCAAGTAC CTTATAAAAC TTAACGGAGT TCACTGGGAG GTAGAGGTGA GGTCTGGAAT   1731 

GGCTCCTCGC TCCCTTTCTC GTGAATGTGT GGTTGGCGTT TGCTCTGAAG GCTGTGTCGC   1791 

ACCGCCTTAT CCAGCAGACG GGCTACCTAA ACGTGCACTC GAGGCCTTGG CGTCTGCTTA   1851 

CAGACTACCC TCCGATTGTG TTAGCTCTGG TATTGCTGAC TTTCTTGCTA ATCCACCTCC   1911 

TCAGGAATTC TGGACCCTCG ACAAAATGTT GACCTCCCCG TCACCAGAGC GGTCCGGCTT   1971 

CTCTAGTTTG TATAAATTAC TATTAGAGGT TGTTCCGCAA AAATGCGGTG CCACGGAAGG   2031 

GGCTTTCATC TATGCTGTTG AGAGGATGTT GAAGGATTGT CCGAGCTCCA AACAGGCCAT   2091 

GGCCCTTCTG GCAAAAATTA AAGTTCCATC CTCAAAGGCC CCGTCTGTGT CCCTGGACGA   2151 

GTGTTTCCCT ACGGATGTTT TAGCCGACTT CGAGCCAGCA TCTCAGGAAA GGCCCCAAAG   2211 

TTCCGGCGCT GCTGTTGTCC TGTGTTCACC GGATGCAAAA GAGTTCGAGG AAGCAGCCCC   2271 

RGAAGAAGTT CAAGAGAGTG GCCACAAGGC CGTCCACTCT GCACTCCTTG CCGAGGGTCC   2331 

TAACAATGAG CAGGTACAGG TGGTTGCCGG TGAGCAACTG AAGCTCGGCG GTTGTGGTTT   2391 

GGCAGTCGGG AATGCTCATG AAGGTGCTCT GGTCTCAGCT GGTCTAATTA ACCTGGTAGG   2451 

CGGGAATTTG TCCCCCTCAG ACCCCATGAA AGAAAACATG CTCAATAGCC GGGAAGACGA   2511 

ACCACTGGAT TTGTCCCAAC CAGCACCAGC TTCCACAACG ACCCTTGTGA GAGAGCAAAC   2571 

ACCCGACAAC CCAGGTTCTG ATGCCGGTGC CCTCCCCGTC ACCGTTCGAG AATTTGTCCC   2631 

GACGGGGCCT ATACTCTGTC ATGTTGAGCA CTGCGGCACG GAGTCGGGCG ACAGCAGTTC   2691 

GCCTTTGGAT CTATCTGATG CGCAAACCCT GGACCAGCCT TTAAATCTAT CCCTGGCCGC   2751 

TTGGCCAGTG AGGGCCACCG CGTCTGACCC TGGCTGGGTC CACGGTAGGC GCGAGCCTGT   2811 

CTTTGTAAAG CCTCGAAATG CTTTCTCTGA TGGCGATTCA GCCCTTCAGT TCGGGGAGCT   2871 

TTCTGAATCC AGCTCTGTCA TCGAGTTTGA CCGGACAAAA GATGCTCCGG TGGTTGACGC   2931 

CCCTGTCGAC TTGACGACTT CGAACGAGGC CCTCTCTGTA GTCGATCCTT TCGAATTTGC   2991 

CGAACTCAAG CGCCCGCGTT TCTCCGCACA AGCCTTAATT GACCGAGGCG GTCCACTTGC   3051 

CGATGTCCAT GCAAAAATAA AGAACCGGGT ATATGAACAG TGCCTCCAAG CTTGTGAGCC   3111 

CGGTAGTCGT GCAACCCCAG CCACCAGGGA GTGGCTCGAC AAAATGTGGG ATAGGGTGGA   3171 

CATGAAAACT TGGCGCTGCA CCTCGCAGTT CCAAGCTGGT CGCATTCTTG CGTCCCTCAA   3231 

ATTCCTCCCT GACATGATTC AAGACACACC GCCTCCTGTT CCCAGGAAGA ACCGAGCTAG   3291 

TGACAATGCC GGCCTGAAGC AACTGGTGGC ACAGTGGGAT AGGAAATTGA GTGTGACCCC   3351 

CCCCCCAAAA CCGGTTGGGC CAGTGCTTGA CCAGATCGTC CCTCCGCCTA CGGATATCCA   3411 

GCAAGAAGAT GTCACCCCCT CCGATGGGCC ACCCCATGCG CCGGATTTTC CTAGTCGAGT   3471 

GAGCACGGGC GGGAGTTGGA AAGGCCTTAT GCTTTCCGGC ACCCGTCTCG CGGGGTCTAT   3531 

CAGCCAGCGC CTTATGACAT GGGTTTTTGA AGTTTTCTCC CACCTCCCAG CTTTTATGCT   3591 

CACACTTTTC TCGCCGCGGG GCTCTATGGC TCCAGGTGAT TGGTTGTTTG CAGGTGTCGT   3651 

TTTACTTGCT CTCTTGCTCT GTCGTTCTTA CCCGATACTC GGATGCCTTC CCTTATTGGG   3711 

TGTCTTTTCT GGTTCTTTGC GGCGTGTTCG TCTGGGTGTT TTTGGTTCTT GGATGGCTTT   3771 

TGCTGTATTT TTATTCTCGA CTCCATCCAA CCCAGTCGGT TCTTCTTGTG ACCACGATTC   3831 

GCCGGAGTGT CATGCTGAGC TTTTGGCTCT TGAGCAGCGC CAACTTTGGG AACCTGTGCG   3891 

CGGCCTTGTG GTCGGCCCCT CAGGCCTCTT ATGTGTCATT CTTGGCAAGT TACTCGGTGG   3951 

GTCACGTTAT CTCTGGCATG TTCTCCTACG TTTATGCATG CTTGCAGATT TGGCCCTTTC   4011 

TCTTGTTTAT GTGGTGTCCC AGGGGCGTTG TCACAAGTGT TGGGGAAAGT GTATAAGGAC   4071 

AGCTCCTGCG GAGGTGGCTC TTAATGTATT TCCTTTCTCG CGCGCCACCC GTGTCTCTCT   4131 

TGTATCCTTG TGTGATCGAT TCCAAACGCC AAAAGGGGTT GATCCTGTGC ACTTGGCAAC   4191 

GGGTTGGCGC GGGTGCTGGC GTGGTGAGAG CCCCATCCAT CAACCACACC AAAAGCCCAT   4251 

AGCTTATGCC AATTTGGATG AAAAGAAAAT GTCTGCCCAA ACGGTGGTTG CTGTCCCATA   4311 

CGATCCCAGT CAGGCTATCA AATGCCTGAA AGTTCTGCAG GCGGGAGGGG CCATCGTGGA   4371 

CCAGCCTACA CCTGAGGTCG TTCGTGTGTC CGAGATCCCC TTCTCAGCCC CATTTTTCCC   4431 

AAAAGTTCCA GTCAACCCAG ATTGCAGGGT TGTGGTAGAT TCGGACACTT TTGTGGCTGC   4491 

GGTTCGCTGC GGTTACTCGA CAGCACAACT GGTYCTGGGC CGGGGCAACT TTGCCAAGTT   4551 

AAATCAGACC CCCCCCAGGA ACTCTATCTC CACCAAAACG ACTGGTGGGG CCTCTTACAC   4611 

CCTTGCTGTG GCTCAAGTGT CTGCGTGGAC TCTTGTTCAT TTCATCCTCG GTCTTTGGTT   4671 

CACATCACCT CAAGTGTGTG GCCGAGGAAC CGCTGACCCA TGGTGTTCAA ATCCTTTTTC   4731 

ATATCCTACC TATGGCCCCG GAGTTGTGTG CTCCTCTCGA CTTTGTGTGT CTGCCGACGG   4791 

GGTCACCCTG CCATTGTTCT CAGCCGTGGC ACAACTCTCC GGTAGAGAGG TGGGGATTTT   4851 

TATTTTGGTG CTCGTCTCCT TGACTGCTTT GGCCCACCGC ATGGCTCTTA AGGCAGACAT   4911 

GTTAGTGGTC TTTTCGGCTT TTTGTGCTTA CGCCTGGCCC ATGAGCTCCT GGTTAATCTG   4971 

CTTCTTTCCT ATACTCTTGA AGTGGGTTAC CCTTCACCCT CTTACTATGC TTTGGGTGCA   5031 

CTCATTCTTG GTGTTTTGTC TGCCAGCAGC CGGCATCCTC TCACTAGGGA TAACTGGCCT   5091 

TCTTTGGGCA ATTGGCCGCT TTACCCAGGT TGCCGGAATT ATTACACCTT ATGACATCCA   5151 

CCAGTACACC TCTGGGCCAC GTGGTGCAGC TGCTGTGGCC ACAGCCCCAG AAGGCACTTA   5211 

TATGGCCGCC GTCCGGAGAG CTGCTTTAAC TGGGCGAACT TTAATCTTCA CCCCGTCTGC   5271 

AGTTGGATCC CTTCTCGAAG GTGCTTTCAG GACTCATAAA CCCTGCCTTA ACACCGTGAA   5331 

TGTTGTAGGC TCTTCCCTTG GTTCCGGAGG GGTTTTCACC ATTGATGGCA GAAGAACTGT   5391 

CGTCACTGCT GCCCATGTGT TGAACGGCGA CACAGCTAGA GTCACCGGCG ACTCCTACAA   5451 

CCGCATGCAC ACTTTCAAGA CCAATGGTGA TTATGCCTGG TCCCATGCTG ATGACTGGCA   5511 

GGGCGTTGCC CCTGTGGTCA AGGTTGCGAA GGGGTACCGC GGTCGTGCCT ACTGGCAAAC   5571 

ATCAACTGGT GTCGAACCCG GTATCATTGG GGAAGGGTTC GCCTTCTGTT TTACTAACTG   5631 

CGGCGATTCG GGGTCACCCG TCATCTCAGA ATCTGGTGAT CTTATTGGAA TCCACACCGG   5691 

TTCAAACAAA CTTGGTTCTG GTCTTGTGAC AACCCCTGAA GGGGAGACCT GCACCATCAA   5751 

AGAAACCAAG CTCTCTGACC TTTCCAGACA TTTTGCAGGC CCAAGCGTTC CTCTTGGGGA   5811 

CATTAAATTG AGTCCGGCCA TCATCCCTGA TGTAACATCC ATTCCGAGTG ACTTGGCATC   5871 

GCTCCTAGCC TCCGTCCCTG TAGTGGAAGG CGGCCTCTCG ACCGTTCAAC TTTTGTGTGT   5931 

CTTTTTCCTT CTCTGGCGCA TGATGGGCCA TGCCTGGACA CCCATTGTTG CCGTGGGCTT   5991 

CTTTTTGCTG AATGAAATTC TTCCAGCAGT TTTGGTCCGA GCCGTGTTTT CTTTTGCACT   6051 

CTTTGTGCTT GCATGGGCCA CCCCCTGGTC TGCACAGGTG TTGATGATTA GACTCCTCAC   6111 

GGCATCTCTC AACCGCAACA AGCTTTCTCT GGCGTTCTAC GCACTCGGGG GTGTCGTCGG   6171 

TTTGGCAGCT GAAATCGGGA CTTTTGCTGG CAGATTGTCT GAATTGTCTC AAGCTCTTTC   6231 

GACATACTGC TTCTTACCTA GGGTCCTTGC TATGACCAGT TGTGTTCCCA CCATCATCAT   6291 

TGGTGGACTC CATACCCTCG GTGTGATTCT GTGGTTRTTC AAATACCGGT GCCTCCACAA   6351 

CATGCTGGTT GGTGATGGGA GTTTTTCAAG CGCCTTCTTC CTACGGTATT TTGCAGAGGG   6411 

TAATCTCAGA AAAGGTGTTT CACAGTCCTG TGGCATGAAT AACGAGTCCC TAACGGCTGC   6471 

TTTAGCTTGC AAGTTGTCAC AGGCTGACCT TGATTTTTTG TCCAGCTTAA CGAACTTCAA   6531 

GTGCTTTGTA TCTGCTTCAA ACATGAAAAA TGCTGCCGGC CAGTACATTG AAGCAGCGTA   6591 

TGCCAAGGCC CTGCGCCAAG AGTTGGCCTC TCTAGTTCAG ATTGACAAAA TGAAAGGAGT   6651 

TTTGTCCAAG CTCGAGGCCT TTGCTGAAAC AGCCACCCCG TCCCTTGACA TAGGTGACGT   6711 

GATTGTTCTG CTTGGGCAAC ATCCTCACGG ATCCATCCTC GATATTAATG TGGGGACTGA   6771 

AAGGAAAACT GTGTCCGTGC AAGAGACCCG GAGCCTAGGC GGCTCCAAAT TCAGTGTTTG   6831 

TACTGTCGTG TCCAACACAC CCGTGGACGC CTTRACCGGC ATCCCACTCC AGACACCAAC   6891 

CCCTCTTTTT GAGAATGGTC CGCGTCATCG CAGCGAGGAA GACGATCTTA AAGTCGAGAG   6951 

GATGAAGAAA CACTGTGTAT CCCTCGGCTT CCACAACATC AATGGCAAAG TTTACTGCAA   7011 

AATTTGGGAC AAGTCTACCG GTGACACCTT TTACACGGAT GATTCCCGGT ACACCCAAGA   7071 

CCATGCTTTT CAGGACAGGT CAGCCGACTA CAGAGACAGG GACTATGAGG GTGTGCAAAC   7131 

CACCCCCCAA CAGGGATTTG ATCCAAAGTC TGAAACCCCT GTTGGCACTG TTGTGATCGG   7191 

CGGTATTACG TATAACAGGT ATCTGATCAA AGGTAAGGAG GTTCTGGTCC CCAAGCCTGA   7251 

CAACTGCCTT GAAGCTGCCA AGCTGTCCCT TGAGCAAGCT CTCGCTGGGA TGGGCCAAAC   7311 

TTGCGACCTT ACAGCTGCCG AGGTGGAAAA GCTAAAGCGC ATCATTAGTC AACTCCAAGG   7371 

TTTGACCACT GAACAGGCTT TAAACTGT TAGCCGCCAG CGGCTTGACC CGCTGTGGCC     7429 

GCGGCGGCCT AGTTGTGACT GAAACGGCGG TAAAAATTAT AAAATACCAC AGCAGAACTT   7489 

TCACCTTAGG CCCTTTAGAC CTAAAAGTCA CTTCCGAGGT GGAGGTAAAG AAATCAACTG   7549 

AGCAGGGCCA CGCTGTTGTG GCAAACTTAT GTTCCGGTGT CATCTTGATG AGACCTCACC   7609 

CACCGTCCCT TGTCGACGTT CTTCTGAAAC CCGGACTTGA CACAATACCC GGCATTCAAC   7669 

CAGGGCATGG GGCCGGGAAT ATGGGCGTGG ACGGTTCTAT TTGGGATTTT GAAACCGCAC   7729 

CCACAAAGGC AGAACTCGAG TTATCCAAGC AAATAATCCA AGCATGTGAA GTTAGGCGCG   7789 

GGGACGCCCC GAACCTCCAA CTCCCTTACA AGCTCTATCC TGTTAGGGGG GATCCTGAGC   7849 

GGCATAAAGG CCGCCTTATC AATACCAGGT TTGGAGATTT ACCTTACAAA ACTCCTCAAG   7909 

ACACCAAGTC CGCAATCCAC GCGGCTTGTT GCCTGCACCC CAACGGGGCC CCCGTGTCTG   7969 

ATGGTAAATC CACACTAGGT ACCACTCTTC AACATGGTTT CGAGCTTTAT GTCCCTACTG   8029 

TGCCCTATAG TGTCATGGAG TACCTTGATT CACGCCCTGA CACCCCTTTT ATGTGTACTA   8089 

AACATGGCAC TTCCAAGGCT GCTGCAGAGG ACCTCCAAAA ATACGACCTA TCCACCCAAG   8149 

GATTTGTCCT GCCTGGGGTC CTACGCCTAG TACGCAGATT CATCTTTGGC CATATTGGTA   8209 

AGGCGCCGCC ATTGTTCCTC CCATCAACCT ATCCCGCCAA GAACTCTATG GCAGGGATCA   8269 

ATGGCCAGAG GTTCCCAACA AAGGACGTTC AGAGCATACC TGAAATTGAT GAAATGTGTG   8329 

CCCGCGCTGT CAAGGAGAAT TGGCAAACTG TGACACCTTG CACCCTCAAG AAACAGTACT   8389 

GTTCCAAGCC CAAAACCAGG ACCATCCTGG GCACCAACAA CTTTATTGCC TTGGCTCACA   8449 

GATCGGCGCT CAGTGGTGTC ACCCAGGCAT TCATGAAGAA GGCTTGGAAG TCCCCAATTG   8509 

CCTTGGGGAA AAACAAATTC AAGGAGCTGC ATTGCACTGT CGCCGGCAGG TGTCTTGAGG   8569 

CCGACTTGGC CTCCTGTGAC CGCAGCACCC CCGCCATTGT AAGATGGTTT GTTGCCAACC   8629 

TCCTGTATGA ACTTGCAGGA TGTGAAGAGT ACTTGCCTAG CTATGTGCTT AATTGCTGCC   8689 

ATGACCTCGT GGCAACACAG GATGGTGCCT TCACAAAACG CGGTGGCCTG TCGTCCGGGG   8749 

ACCCCGTCAC CAGTGTGTCC AACACCGTAT ATTCACTGGT AATTTATGCC CAGCACATGG   8809 

TATTGTCGGC CTTGAAAATG GGTCATGAAA TTGGTCTTAA GTTCCTCGAG GAACAGCTCA   8869 

AGTTCGAGGA CCTCCTTGAA ATTCAGCCTA TGTTGGTATA CTCTGATGAT CTTGTCTTGT   8929 

ACGCTGAAAG ACCCACMTTT CCCAATTACC ACTGGTGGGT CGAGCACCTT GACCTGATGC   8989 

TGGGTTTCAG AACGGACCCA AAGAAAACCG TCATAACTGA TAAACCCAGC TTCCTCGGCT   9049 

GCAGAATTGA GGCAGGGCGA CAGCTAGTCC CCAATCGCGA CCGCATCCTG GCTGCTCTTG   9109 

CATATCACAT GAAGGCGCAG AACGCCTCAG AGTATTATGC GTCTGCTGCC GCAATCCTGA   9169 

TGGATTCATG TGCTTGCATT GACCATGACC CTGAGTGGTA TGAGGACCTC ATCTGCGGTA   9229 

TTGCCCGGTG CGCCCGCCAG GATGGTTATA GCTTCCCAGG TCCGGCATTT TTCATGTCCA   9289 

TGTGGGAGAA GCTGAGAAGT CATAATGAAG GGAAGAAATT CCGCCACTGC GGCATCTGCG   9349 

ACGCCAAAGC CGACTATGCG TCCGCCTGTG GGCTTGATTT GTGTTTGTTC CATTCGCACT   9409 

TTCATCAACA CTGCCCYGTC ACTCTGAGCT GCGGTCACCA TGCCGGTTCA AAGGAATGTT   9469 

CGCAGTGTCA GTCACCTGTT GGGGCTGGCA GATCCCCTCT TGATGCCGTG CTAAAACAAA   9529 

TTCCATACAA ACCTCCTCGT ACTGTCATCA TGAAGGTGGG TAATAAAACA ACGGCCCTCG   9589 

ATCCGGGGAG GTACCAGTCC CGTCGAGGTC TCGTTGCAGT CAAGAGGGGT ATTGCAGGCA   9649 

ATGAAGTTGA TCTTTCTGAT GGRGACTACC AAGTGGTGCC TCTTTTGCCG ACTTGCAAAG   9709 

ACATAAACAT GGTGAAGGTG GCTTGCAATG TACTACTCAG CAAGTTCATA GTAGGGCCAC   9769 

CAGGTTCCGG AAAGACCACC TGGCTACTGA GTCAAGTCCA GGACGATGAT GTCATTTACA   9829 

YACCCACCCA TCAGACTATG TTTGATATAG TCAGTGCTCT CAAAGTTTGC AGGTATTCCA   9889 

TTCCAGGAGC CTCAGGACTC CCTTTCCCAC CACCTGCCAG GTCCGGGCCG TGGGTTAGGC   9949 

TTATTGCCAG CGGGCACGTC CCTGGCCGAG TATCATACCT CGATGAGGCT GGATATTGTA  10009 

ATCATCTGGA CATTCTTAGA CTGCTTTCCA AAACACCCCT TGTGTGTTTG GGTGACCTTC  10069 

AGCAACTTCA CCCTGTCGGC TTTGATTCCT ACTGTTATGT GTTCGATCAG ATGCCTCAGA  10129 

AGCAGCTGAC CACTATTTAC AGATTTGGCC CTAACATCTG CGCACGCATC CAGCCTTGTT  10189 

ACAGGGAGAA ACTTGAATCT AAGGCTAGGA ACACTAGGGT GGTTTTTACC ACCCGGCCTG  10249 

TGGCCTTTGG TCAGGTGCTG ACACCATACC ATAAAGATCG CATCGGCTCT GCGATAACCA  10309 

TAGATTCATC CCAGGGGGCC ACCTTTGATA TTGTGACATT GCATCTACCA TCGCCAAAGT  10369 

CCCTAAATAA ATCCCGAGCA CTTGTAGCCA TCACTCGGGC AAGACACGGG TTGTTCATTT  10429 

ATGACCCTCA TAACCAGCTC CAGGAGTTTT TCAACTTAAC CCCTGAGCGC ACTGATTGTA  10489 

ACCTTGTGTT CAGCCGTGGG GATGAGCTGG TAGTTCTGAA TGCGGATAAT GCAGTCACAA  10549 

CTGTAGCGAA GGCCCTTGAG ACAGGTCCAT CTCGATTTCG AGTATCAGAC CCGAGGTGCA  10609 

AGTCTCTCTT AGCCGCTTGT TCGGCCAGTC TGGAAGGGAG CTGTATGCCA CTACCGCAAG  10669 

TGGCACATAA CCTGGGGTTT TACTTTTCCC CGGACAGTCC AACATTTGCA CCTCTGCCAA  10729 

AAGAGTTGGC GCCACATTGG CCAGTGGTTA CCCACCAGAA TAATCGGGCG TGGCCTGATC  10789 

GACTTGTCGC TAGTATGCGC CCAATTGATG CCCGCTACAG CAAGCCAATG GTCGGTGCAG  10849 

GGTATGTGGT CGGGCCGTCC ACCTTTCTTG GTACTCCTGG TGTGGTGTCA TACTATCTCA  10909 

CACTATACAT CAGGGGTGAG CCCCAGGCCT TGCCAGAAAC ACTCGTTTCA ACAGGGCGTA  10969 

TAGCCACAGA TTGTCGGGAG TATCTCGACG CGGCTGAGGA AGAGGCAGCA AAAGAACTCC  11029 

CCCACGCATT CATTGGCGAT GTCAAAGGTA CCACGGTTGG GGGGTGTCAT CACATTACAT  11089 

CAAAATACCT ACCTAGGTCC CTGCCTAAGG ACTCTGTTGC CGTAGTTGGA GTAAGTTCGC  11149 

CCGGCAGGGC TGCTAAAGCC GTGTGCACTC TCACCGATGT GTACCTCCCC GAACTCCGGC  11209 

CATATCTGCA ACCTGAGACG GCATCAAAAT GCTGGAAACT CAAATTAGAC TTCAGGGACG  11269 

TCCGACTAAT GGTCTGGAAA GGAGCCACCG CCTATTTCCA GTTGGAAGGG CTTACATGGT  11329 

CGGCGCTGCC CGACTATGCC AGGTTYATTC AGCTGCCCAA GGATGCCGTT GTATACATTG  11389 

ATCCGTGTAT AGGACCGGCA ACAGCCAACC GTAAGGTCGT GCGAACCACA GACTGGCGGG  11449 

CCGACCTGGC AGTGACACCG TATGATTACG GTGCCCAGAA CATTTTGACA ACAGCCTGGT  11509 

TCGAGGACCT CGGGCCGCAG TGGAAGATTT TGGGGTTGCA GCCCTTTAGG CGAGCATTTG  11569 

GCTTTGAAAA CACTGAGGAT TGGGCAATCC TTGCACGCCG TATGAATGAC GGCAAGGACT  11629 

ACACTGACTA TAACTGGAAC TGTGTTCGAG AACGCCCACA CGCCATCTAC GGGCGTGCTC  11689 

GTGACCATAC GTATCATTTT GCCCCTGGCA CAGAATTGCA GGTAGAGCTA GGTAAACCCC  11749 

GGCTGCCGCC TGGGCAAGTG CCG TGAATTCGGG GTGATGCAAT GGGGTCACTG         11802 

TGGAGTAAAA TCAGCCAGCT GTTCGTGGAC GCCTTCACTG AGTTCCTTGT TAGTGTGGTT  11862 

GATATTGYCA TTTTCCTTGC CATACTGTTT GGGTTCACCG TCGCAGGATG GTTACTGGTC  11922 

TTTCTTCTCA GAGTGGTTTG CTCCGCGCTT CTCCGTTCGC GCTCTGCCAT TCACTCTCCC  11982 

GAACTATCGA AGGTCCTATG AAGGCTTGTT GCCCAACTGC AGACCGGATG TCCCACAATT  12042 

TGCAGTCAAG CACCCATTGG GYATGTTTTG GCACATGCGA GTTTCCCACT TGATTGATGA  12102 

GRTGGTCTCT CGTCGCATTT ACCAGACCAT GGAACATTCA GGTCAAGCGG CCTGGAAGCA  12162 

GGTGGTTGGT GAGGCCACTC TCACGAAGCT GTCAGGGCTC GATATAGTTA CTCATTTCCA  12222 

ACACCTGGCC GCAGTGGAGG CGGATTCTTG CCGCTTTCTC AGCTCACGAC TCGTGATGCT  12282 

AAAAAATCTT GCCGTTGGCA ATGTGAGCCT ACAGTACAAC ACCACGTTGG ACCGCGTTGA  12342 

GCTCATCTTC CCCACGCCAG GTACGAGGCC CAAGTTGACC GATTTCAGAC AATGGCTCAT  12402 

CAGTGTGCAC GCTTCCATTT TTTCCTCTGT GGCTTCATCT GTTACCTTGT TCATAGTGCT  12462 

TTGGCTTCGA ATTCCAGCTC TACGCTATGT TTTTGGTTTC CATTGGCCCA CGGCAACACA  12522 

TCATTCGAGC TGACCATCAA CTACACCATA TGCATGCCCT GTTCTACCAG TCAAGCGGCT  12582 

CGCCAAAGGC TCGAGCCCGG TCGTAACATG TGGTGCAAAA TAGGGCATGA CAGGTGTGAG  12642 

GAGCGTGACC ATGATGAGTT GTTAATGTCC ATCCCGTCCG GGTACGACAA CCTCAAACTT  12702 

GAGGGTTATT ATGCTTGGCT GGCTTTTTTG TCCTTTTCCT ACGCGGCCCA ATTCCATCCG  12762 

GAGTTGTTCG GGATAGGGAA TGTGTCGCGC GTCTTCGTGG ACAAGCGACA CCAGTTCATT  12822 

TGTGCCGAGC ATGATGGACA CAATTCAACC GTATCTACCG GACACAACAT CTCCGCATTA  12882 

TATGCGGCAT ATTACCACCA CCAAATAGAC GGGGGCAATT GGTTCCATTT GGAATGGCTG  12942 

CGGCCACTCT TTTCTTCCTG GCTGGTGCTC AACATATCAT GGTTTCTGAG GCGTTCGCCT  13002 

GTAAGCCCTG TTTCTCGACG CATCTATCAG ATATTGAGAC CAACACGACC GCGGCTGCCG  13062 

GTTTCATGGT CCTTCAGGAC ATCAATTGTT TCCGACCTCA CGGGGTCTCA GCAGCGCAAG  13122 

AGAAAATTTC CTTCGGAAAG TCGTCCCAAT GTCGTGAAGC CGTCGGTACT CCCCAGTACA  13182 

TCACGA TAACGGCTAA CGTGACCGAC GAATCATACT TGTACAACGC GGACCTGCTG      13238 

ATGCTTTCTG CGTGCCTTTT CTACGCCTCA GAAATGAGCG AGAAAGGCTT CAAAGTCATC  13298 

TTTGGGAATG TCTCTGGCGT TGTTTCTGCT TGTGTCAATT TCACAGATTA TGTGGCCCAT  13358 

GTGACCCAAC ATACCCAGCA GCATCATCTG GTAATTGATC ACATTCGGTT GCTGCATTTC  13418 

CTGACACCAT CTGCAATGAG GTGGGCTACA ACCATTGCTT GTTTGTTCGC CATTCTCTTG  13478 

GCAATA TGAGATGTTC TCACAAATTG GGGCGTTTCT TGACTCCGCA CTCTTGCTTC      13534 

TGGTGGCTTT TTTTGCTGTG TACCGGCTTG TCCTGGTCCT TTGCCGATGG CAACGGCGAC  13594 

AGCTCGACAT ACCAATACAT ATATAACTTG ACGATATGCG AGCTGAATGG GACCGACTGG  13654 

TTGTCCAGCC ATTTTGGTTG GGCAGTCGAG ACCTTTGTGC TTTACCCGGT TGCCACTCAT  13714 

ATCCTCTCAC TGGGTTTTCT CACAACAAGC CATTTTTTTG ACGCGCTCGG TCTCGGCGCT  13774 

GTATCCACTG CAGGATTTGT TGGCGGGCGG TACGTACTCT GCAGCGTCTA CGGCGCTTGT  13834 

GCTTTCGCAG CGTTCGTATG TTTTGTCATC CGTGCTGCTA AAAATTGCAT GGCCTGCCGC  13894 

TATGCCCGTA CCCGGTTTAC CAACTTCATT GTGGACGACC GGGGGAGAGT TCATCGATGG  13954 

AAGTCTCCAA TAGTGGTAGA AAAATTGGGC AAAGCCGAAG TCGATGGCAA CCTCGTCACC  14014 

ATCAAACATG TCGTCCTCGA AGGGGTTAAA GCTCAACCCT TGACGAGGAC TTCGGCTGAG  14074 

CAATGGGAGG CC TAGACGATTT TTGCAACGAT CCTATCGCCG CACAAAAGCT          14126 

CGTGCTAGCC TTTAGCATCA CATACACACC TATAATGATA TACGCCCTTA AGGTGTCACG  14186 

CGGCCGACTC CTGGGGCTGT TGCACATCCT AATATTTCTG AACTGTTCCT TTACATTCGG  14246 

ATACATGACA TATGTGCATT TTCAATCCAC CAACCGTGTC GCACTTACCC TGGGGGCTGT  14306 

TGTCGCCCTT CTGTGGGGTG TTTACAGCTT CACAGAGTCA TGGAAGTTTA TCACTTCCAG  14366 

ATGCAGATTG TGTTGCCTTG GCCGGCGATA CATTCTGGCC CCTGCCCATC ACGTAGAAAG  14426 

TGCTGCAGGT CTCCATTCAA TCTCAGCGTC TGGTAACCGA GCATACGCTG TGAGAAAGCC  14486 

CGGACTAACA TCAGTGAACG GCACTCTAGT ACCAGGACTT CGGAGCCTCG TGCTGGGCGG  14546 

CAAACGAGCT GTTAAACGAG GAGTGGTTAA CCTCGTCAAG TATGGCCGG TAAAAACCAG   14605 

AGCCAGAAGA AAAAGAAAAG TACAGCTCCG ATGGGGAATG GCCAGCCAGT CAATCAACTG  14665 

TGCCAGTTGC TGGGTGCAAT GATAAAGTCC CAGCGCCAGC AACCTAGGGG AGGACAGGCY  14725 

AAAAAGAAAA AGCCTGAGAA GCCACATTTT CCCCTGGCTG CTGAAGATGA CATCCGGCAC  14785 

CACCTCACCC AGACTGAACG CTCCCTCTGC TTGCAATCGA TCCAGACGGC TTTCAATCAA  14845 

GGCGCAGGAA CTGCGTCRCT TTCATCCAGC GGGAAGGTCA GTTTTCAGGT TGAGTTTATG  14905 

CTGCCGGTTG CTCATACAGT GCGCCTGATT CGCGTGACTT CTACATCCGC CAGTCAGGGT  14965 

GCAAGT TAATTTGACA GTCAGGTGAA TGGCCGCGAT GGCGTGTGGC CTCTGAGTCA      15021 

CCTATTCAAT TAGGGCGATC ACATGGGGGT CATACTTAAT TCAGGCAGGA ACCATGTGAC  15081 

CGAAATTAAA AAAAAAAAAA AAAAAAA                                      15108 

 
           
           
             
               2396 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              2 

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 Xaa 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 Asn 
    1010                1015                1020 

Arg Ala Ser Asp Asn Ala Gly Leu Lys Gln Leu Val Ala Gln Trp Asp 
1025                1030                1035                1040 

Arg Lys Leu Ser Val Thr Pro Pro Pro Lys Pro Val Gly Pro Val Leu 
                1045                1050                1055 

Asp Gln Ile Val Pro Pro Pro Thr Asp Ile Gln Gln Glu Asp Val Thr 
            1060                1065                1070 

Pro Ser Asp Gly Pro Pro His Ala Pro Asp Phe Pro Ser Arg Val Ser 
        1075                1080                1085 

Thr Gly Gly Ser Trp Lys Gly Leu Met Leu Ser Gly Thr Arg Leu Ala 
    1090                1095                1100 

Gly Ser Ile Ser Gln Arg Leu Met Thr Trp Val Phe Glu Val Phe Ser 
1105                1110                1115                1120 

His Leu Pro Ala Phe Met Leu Thr Leu Phe Ser Pro Arg Gly Ser Met 
                1125                1130                1135 

Ala Pro Gly Asp Trp Leu Phe Ala Gly Val Val Leu Leu Ala Leu Leu 
            1140                1145                1150 

Leu Cys Arg Ser Tyr Pro Ile Leu Gly Cys Leu Pro Leu Leu Gly Val 
        1155                1160                1165 

Phe Ser Gly Ser Leu Arg Arg Val Arg Leu Gly Val Phe Gly Ser Trp 
    1170                1175                1180 

Met Ala Phe Ala Val Phe Leu Phe Ser Thr Pro Ser Asn Pro Val Gly 
1185                1190                1195                1200 

Ser Ser Cys Asp His Asp Ser Pro Glu Cys His Ala Glu Leu Leu Ala 
                1205                1210                1215 

Leu Glu Gln Arg Gln Leu Trp Glu Pro Val Arg Gly Leu Val Val Gly 
            1220                1225                1230 

Pro 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 Leu 
    1250                1255                1260 

Ala Leu Ser Leu Val Tyr Val Val Ser Gln Gly Arg Cys His Lys Cys 
1265                1270                1275                1280 

Trp Gly Lys Cys Ile Arg Thr Ala Pro Ala Glu Val Ala Leu Asn Val 
                1285                1290                1295 

Phe Pro Phe Ser Arg Ala Thr Arg Val Ser Leu Val Ser Leu Cys Asp 
            1300                1305                1310 

Arg Phe Gln Thr Pro Lys Gly Val Asp Pro Val His Leu Ala Thr Gly 
        1315                1320                1325 

Trp Arg Gly Cys Trp Arg Gly Glu Ser Pro Ile His Gln Pro His Gln 
    1330                1335                1340 

Lys Pro Ile Ala Tyr Ala Asn Leu Asp Glu Lys Lys Met Ser Ala Gln 
1345                1350                1355                1360 

Thr Val Val Ala Val Pro Tyr Asp Pro Ser Gln Ala Ile Lys Cys Leu 
                1365                1370                1375 

Lys Val Leu Gln Ala Gly Gly Ala Ile Val Asp Gln Pro Thr Pro Glu 
            1380                1385                1390 

Val Val Arg Val Ser Glu Ile Pro Phe Ser Ala Pro Phe Phe Pro Lys 
        1395                1400                1405 

Val Pro Val Asn Pro Asp Cys Arg Val Val Val Asp Ser Asp Thr Phe 
    1410                1415                1420 

Val Ala Ala Val Arg Cys Gly Tyr Ser Thr Ala Gln Leu Xaa Leu Gly 
1425                1430                1435                1440 

Arg Gly Asn Phe Ala Lys Leu Asn Gln Thr Pro Pro Arg Asn Ser Ile 
                1445                1450                1455 

Ser Thr Lys Thr Thr Gly Gly Ala Ser Tyr Thr Leu Ala Val Ala Gln 
            1460                1465                1470 

Val 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 Asn 
    1490                1495                1500 

Pro Phe Ser Tyr Pro Thr Tyr Gly Pro Gly Val Val Cys Ser Ser Arg 
1505                1510                1515                1520 

Leu Cys Val Ser Ala Asp Gly Val Thr Leu Pro Leu Phe Ser Ala Val 
                1525                1530                1535 

Ala Gln Leu Ser Gly Arg Glu Val Gly Ile Phe Ile Leu Val Leu Val 
            1540                1545                1550 

Ser Leu Thr Ala Leu Ala His Arg Met Ala Leu Lys Ala Asp Met Leu 
        1555                1560                1565 

Val Val Phe Ser Ala Phe Cys Ala Tyr Ala Trp Pro Met Ser Ser Trp 
    1570                1575                1580 

Leu Ile Cys Phe Phe Pro Ile Leu Leu Lys Trp Val Thr Leu His Pro 
1585                1590                1595                1600 

Leu Thr Met Leu Trp Val His Ser Phe Leu Val Phe Cys Leu Pro Ala 
                1605                1610                1615 

Ala Gly Ile Leu Ser Leu Gly Ile Thr Gly Leu Leu Trp Ala Ile Gly 
            1620                1625                1630 

Arg Phe Thr Gln Val Ala Gly Ile Ile Thr Pro Tyr Asp Ile His Gln 
        1635                1640                1645 

Tyr Thr Ser Gly Pro Arg Gly Ala Ala Ala Val Ala Thr Ala Pro Glu 
    1650                1655                1660 

Gly Thr Tyr Met Ala Ala Val Arg Arg Ala Ala Leu Thr Gly Arg Thr 
1665                1670                1675                1680 

Leu Ile Phe Thr Pro Ser Ala Val Gly Ser Leu Leu Glu Gly Ala Phe 
                1685                1690                1695 

Arg Thr His Lys Pro Cys Leu Asn Thr Val Asn Val Val Gly Ser Ser 
            1700                1705                1710 

Leu 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 Asp 
    1730                1735                1740 

Ser Tyr Asn Arg Met His Thr Phe Lys Thr Asn Gly Asp Tyr Ala Trp 
1745                1750                1755                1760 

Ser His Ala Asp Asp Trp Gln Gly Val Ala Pro Val Val Lys Val Ala 
                1765                1770                1775 

Lys Gly Tyr Arg Gly Arg Ala Tyr Trp Gln Thr Ser Thr Gly Val Glu 
            1780                1785                1790 

Pro Gly Ile Ile Gly Glu Gly Phe Ala Phe Cys Phe Thr Asn Cys Gly 
        1795                1800                1805 

Asp Ser Gly Ser Pro Val Ile Ser Glu Ser Gly Asp Leu Ile Gly Ile 
    1810                1815                1820 

His Thr Gly Ser Asn Lys Leu Gly Ser Gly Leu Val Thr Thr Pro Glu 
1825                1830                1835                1840 

Gly Glu Thr Cys Thr Ile Lys Glu Thr Lys Leu Ser Asp Leu Ser Arg 
                1845                1850                1855 

His Phe Ala Gly Pro Ser Val Pro Leu Gly Asp Ile Lys Leu Ser Pro 
            1860                1865                1870 

Ala Ile Ile Pro Asp Val Thr Ser Ile Pro Ser Asp Leu Ala Ser Leu 
        1875                1880                1885 

Leu Ala Ser Val Pro Val Val Glu Gly Gly Leu Ser Thr Val Gln Leu 
    1890                1895                1900 

Leu Cys Val Phe Phe Leu Leu Trp Arg Met Met Gly His Ala Trp Thr 
1905                1910                1915                1920 

Pro Ile Val Ala Val Gly Phe Phe Leu Leu Asn Glu Ile Leu Pro Ala 
                1925                1930                1935 

Val Leu Val Arg Ala Val Phe Ser Phe Ala Leu Phe Val Leu Ala Trp 
            1940                1945                1950 

Ala 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 Gly 
    1970                1975                1980 

Val Val Gly Leu Ala Ala Glu Ile Gly Thr Phe Ala Gly Arg Leu Ser 
1985                1990                1995                2000 

Glu Leu Ser Gln Ala Leu Ser Thr Tyr Cys Phe Leu Pro Arg Val Leu 
                2005                2010                2015 

Ala Met Thr Ser Cys Val Pro Thr Ile Ile Ile Gly Gly Leu His Thr 
            2020                2025                2030 

Leu Gly Val Ile Leu Trp Xaa Phe Lys Tyr Arg Cys Leu His Asn Met 
        2035                2040                2045 

Leu Val Gly Asp Gly Ser Phe Ser Ser Ala Phe Phe Leu Arg Tyr Phe 
    2050                2055                2060 

Ala Glu Gly Asn Leu Arg Lys Gly Val Ser Gln Ser Cys Gly Met Asn 
2065                2070                2075                2080 

Asn Glu Ser Leu Thr Ala Ala Leu Ala Cys Lys Leu Ser Gln Ala Asp 
                2085                2090                2095 

Leu Asp Phe Leu Ser Ser Leu Thr Asn Phe Lys Cys Phe Val Ser Ala 
            2100                2105                2110 

Ser Asn Met Lys Asn Ala Ala Gly Gln Tyr Ile Glu Ala Ala Tyr Ala 
        2115                2120                2125 

Lys Ala Leu Arg Gln Glu Leu Ala Ser Leu Val Gln Ile Asp Lys Met 
    2130                2135                2140 

Lys Gly Val Leu Ser Lys Leu Glu Ala Phe Ala Glu Thr Ala Thr Pro 
2145                2150                2155                2160 

Ser Leu Asp Ile Gly Asp Val Ile Val Leu Leu Gly Gln His Pro His 
                2165                2170                2175 

Gly Ser Ile Leu Asp Ile Asn Val Gly Thr Glu Arg Lys Thr Val Ser 
            2180                2185                2190 

Val 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 Xaa Thr Gly Ile Pro Leu Gln 
    2210                2215                2220 

Thr Pro Thr Pro Leu Phe Glu Asn Gly Pro Arg His Arg Ser Glu Glu 
2225                2230                2235                2240 

Asp Asp Leu Lys Val Glu Arg Met Lys Lys His Cys Val Ser Leu Gly 
                2245                2250                2255 

Phe His Asn Ile Asn Gly Lys Val Tyr Cys Lys Ile Trp Asp Lys Ser 
            2260                2265                2270 

Thr Gly Asp Thr Phe Tyr Thr Asp Asp Ser Arg Tyr Thr Gln Asp His 
        2275                2280                2285 

Ala Phe Gln Asp Arg Ser Ala Asp Tyr Arg Asp Arg Asp Tyr Glu Gly 
    2290                2295                2300 

Val Gln Thr Thr Pro Gln Gln Gly Phe Asp Pro Lys Ser Glu Thr Pro 
2305                2310                2315                2320 

Val Gly Thr Val Val Ile Gly Gly Ile Thr Tyr Asn Arg Tyr Leu Ile 
                2325                2330                2335 

Lys Gly Lys Glu Val Leu Val Pro Lys Pro Asp Asn Cys Leu Glu Ala 
            2340                2345                2350 

Ala Lys Leu Ser Leu Glu Gln Ala Leu Ala Gly Met Gly Gln Thr Cys 
        2355                2360                2365 

Asp Leu Thr Ala Ala Glu Val Glu Lys Leu Lys Arg Ile Ile Ser Gln 
    2370                2375                2380 

Leu Gln Gly Leu Thr Thr Glu Gln Ala Leu Asn Cys 
2385                2390                2395 

 
           
           
             
               1463 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              3 

Thr Gly Phe Lys Leu Leu Ala Ala Ser Gly Leu Thr Arg Cys Gly Arg 
  1               5                  10                  15 

Gly Gly Leu Val Val Thr Glu Thr Ala Val Lys Ile Ile Lys Tyr His 
             20                  25                  30 

Ser Arg Thr Phe Thr Leu Gly Pro Leu Asp Leu Lys Val Thr Ser Glu 
         35                  40                  45 

Val Glu Val Lys Lys Ser Thr Glu Gln Gly His Ala Val Val Ala Asn 
     50                  55                  60 

Leu Cys Ser Gly Val Ile Leu Met Arg Pro His Pro Pro Ser Leu Val 
 65                  70                  75                  80 

Asp Val Leu Leu Lys Pro Gly Leu Asp Thr Ile Pro Gly Ile Gln Pro 
                 85                  90                  95 

Gly His Gly Ala Gly Asn Met Gly Val Asp Gly Ser Ile Trp Asp Phe 
            100                 105                 110 

Glu Thr Ala Pro Thr Lys Ala Glu Leu Glu Leu Ser Lys Gln Ile Ile 
        115                 120                 125 

Gln Ala Cys Glu Val Arg Arg Gly Asp Ala Pro Asn Leu Gln Leu Pro 
    130                 135                 140 

Tyr Lys Leu Tyr Pro Val Arg Gly Asp Pro Glu Arg His Lys Gly Arg 
145                 150                 155                 160 

Leu Ile Asn Thr Arg Phe Gly Asp Leu Pro Tyr Lys Thr Pro Gln Asp 
                165                 170                 175 

Thr Lys Ser Ala Ile His Ala Ala Cys Cys Leu His Pro Asn Gly Ala 
            180                 185                 190 

Pro Val Ser Asp Gly Lys Ser Thr Leu Gly Thr Thr Leu Gln His Gly 
        195                 200                 205 

Phe Glu Leu Tyr Val Pro Thr Val Pro Tyr Ser Val Met Glu Tyr Leu 
    210                 215                 220 

Asp Ser Arg Pro Asp Thr Pro Phe Met Cys Thr Lys His Gly Thr Ser 
225                 230                 235                 240 

Lys Ala Ala Ala Glu Asp Leu Gln Lys Tyr Asp Leu Ser Thr Gln Gly 
                245                 250                 255 

Phe Val Leu Pro Gly Val Leu Arg Leu Val Arg Arg Phe Ile Phe Gly 
            260                 265                 270 

His Ile Gly Lys Ala Pro Pro Leu Phe Leu Pro Ser Thr Tyr Pro Ala 
        275                 280                 285 

Lys Asn Ser Met Ala Gly Ile Asn Gly Gln Arg Phe Pro Thr Lys Asp 
    290                 295                 300 

Val Gln Ser Ile Pro Glu Ile Asp Glu Met Cys Ala Arg Ala Val Lys 
305                 310                 315                 320 

Glu Asn Trp Gln Thr Val Thr Pro Cys Thr Leu Lys Lys Gln Tyr Cys 
                325                 330                 335 

Ser Lys Pro Lys Thr Arg Thr Ile Leu Gly Thr Asn Asn Phe Ile Ala 
            340                 345                 350 

Leu Ala His Arg Ser Ala Leu Ser Gly Val Thr Gln Ala Phe Met Lys 
        355                 360                 365 

Lys Ala Trp Lys Ser Pro Ile Ala Leu Gly Lys Asn Lys Phe Lys Glu 
    370                 375                 380 

Leu His Cys Thr Val Ala Gly Arg Cys Leu Glu Ala Asp Leu Ala Ser 
385                 390                 395                 400 

Cys Asp Arg Ser Thr Pro Ala Ile Val Arg Trp Phe Val Ala Asn Leu 
                405                 410                 415 

Leu Tyr Glu Leu Ala Gly Cys Glu Glu Tyr Leu Pro Ser Tyr Val Leu 
            420                 425                 430 

Asn Cys Cys His Asp Leu Val Ala Thr Gln Asp Gly Ala Phe Thr Lys 
        435                 440                 445 

Arg Gly Gly Leu Ser Ser Gly Asp Pro Val Thr Ser Val Ser Asn Thr 
    450                 455                 460 

Val Tyr Ser Leu Val Ile Tyr Ala Gln His Met Val Leu Ser Ala Leu 
465                 470                 475                 480 

Lys Met Gly His Glu Ile Gly Leu Lys Phe Leu Glu Glu Gln Leu Lys 
                485                 490                 495 

Phe Glu Asp Leu Leu Glu Ile Gln Pro Met Leu Val Tyr Ser Asp Asp 
            500                 505                 510 

Leu Val Leu Tyr Ala Glu Arg Pro Xaa Phe Pro Asn Tyr His Trp Trp 
        515                 520                 525 

Val Glu His Leu Asp Leu Met Leu Gly Phe Arg Thr Asp Pro Lys Lys 
    530                 535                 540 

Thr Val Ile Thr Asp Lys Pro Ser Phe Leu Gly Cys Arg Ile Glu Ala 
545                 550                 555                 560 

Gly Arg Gln Leu Val Pro Asn Arg Asp Arg Ile Leu Ala Ala Leu Ala 
                565                 570                 575 

Tyr His Met Lys Ala Gln Asn Ala Ser Glu Tyr Tyr Ala Ser Ala Ala 
            580                 585                 590 

Ala Ile Leu Met Asp Ser Cys Ala Cys Ile Asp His Asp Pro Glu Trp 
        595                 600                 605 

Tyr Glu Asp Leu Ile Cys Gly Ile Ala Arg Cys Ala Arg Gln Asp Gly 
    610                 615                 620 

Tyr Ser Phe Pro Gly Pro Ala Phe Phe Met Ser Met Trp Glu Lys Leu 
625                 630                 635                 640 

Arg Ser His Asn Glu Gly Lys Lys Phe Arg His Cys Gly Ile Cys Asp 
                645                 650                 655 

Ala Lys Ala Asp Tyr Ala Ser Ala Cys Gly Leu Asp Leu Cys Leu Phe 
            660                 665                 670 

His Ser His Phe His Gln His Cys Xaa Val Thr Leu Ser Cys Gly His 
        675                 680                 685 

His Ala Gly Ser Lys Glu Cys Ser Gln Cys Gln Ser Pro Val Gly Ala 
    690                 695                 700 

Gly Arg Ser Pro Leu Asp Ala Val Leu Lys Gln Ile Pro Tyr Lys Pro 
705                 710                 715                 720 

Pro Arg Thr Val Ile Met Lys Val Gly Asn Lys Thr Thr Ala Leu Asp 
                725                 730                 735 

Pro Gly Arg Tyr Gln Ser Arg Arg Gly Leu Val Ala Val Lys Arg Gly 
            740                 745                 750 

Ile Ala Gly Asn Glu Val Asp Leu Ser Asp Xaa Asp Tyr Gln Val Val 
        755                 760                 765 

Pro Leu Leu Pro Thr Cys Lys Asp Ile Asn Met Val Lys Val Ala Cys 
    770                 775                 780 

Asn Val Leu Leu Ser Lys Phe Ile Val Gly Pro Pro Gly Ser Gly Lys 
785                 790                 795                 800 

Thr Thr Trp Leu Leu Ser Gln Val Gln Asp Asp Asp Val Ile Tyr Xaa 
                805                 810                 815 

Pro Thr His Gln Thr Met Phe Asp Ile Val Ser Ala Leu Lys Val Cys 
            820                 825                 830 

Arg Tyr Ser Ile Pro Gly Ala Ser Gly Leu Pro Phe Pro Pro Pro Ala 
        835                 840                 845 

Arg Ser Gly Pro Trp Val Arg Leu Ile Ala Ser Gly His Val Pro Gly 
    850                 855                 860 

Arg Val Ser Tyr Leu Asp Glu Ala Gly Tyr Cys Asn His Leu Asp Ile 
865                 870                 875                 880 

Leu Arg Leu Leu Ser Lys Thr Pro Leu Val Cys Leu Gly Asp Leu Gln 
                885                 890                 895 

Gln Leu His Pro Val Gly Phe Asp Ser Tyr Cys Tyr Val Phe Asp Gln 
            900                 905                 910 

Met Pro Gln Lys Gln Leu Thr Thr Ile Tyr Arg Phe Gly Pro Asn Ile 
        915                 920                 925 

Cys Ala Arg Ile Gln Pro Cys Tyr Arg Glu Lys Leu Glu Ser Lys Ala 
    930                 935                 940 

Arg Asn Thr Arg Val Val Phe Thr Thr Arg Pro Val Ala Phe Gly Gln 
945                 950                 955                 960 

Val Leu Thr Pro Tyr His Lys Asp Arg Ile Gly Ser Ala Ile Thr Ile 
                965                 970                 975 

Asp Ser Ser Gln Gly Ala Thr Phe Asp Ile Val Thr Leu His Leu Pro 
            980                 985                 990 

Ser Pro Lys Ser Leu Asn Lys Ser Arg Ala Leu Val Ala Ile Thr Arg 
        995                 1000                1005 

Ala Arg His Gly Leu Phe Ile Tyr Asp Pro His Asn Gln Leu Gln Glu 
    1010                1015                1020 

Phe Phe Asn Leu Thr Pro Glu Arg Thr Asp Cys Asn Leu Val Phe Ser 
1025                1030                1035                1040 

Arg Gly Asp Glu Leu Val Val Leu Asn Ala Asp Asn Ala Val Thr Thr 
                1045                1050                1055 

Val Ala Lys Ala Leu Glu Thr Gly Pro Ser Arg Phe Arg Val Ser Asp 
            1060                1065                1070 

Pro Arg Cys Lys Ser Leu Leu Ala Ala Cys Ser Ala Ser Leu Glu Gly 
        1075                1080                1085 

Ser Cys Met Pro Leu Pro Gln Val Ala His Asn Leu Gly Phe Tyr Phe 
    1090                1095                1100 

Ser Pro Asp Ser Pro Thr Phe Ala Pro Leu Pro Lys Glu Leu Ala Pro 
1105                1110                1115                1120 

His Trp Pro Val Val Thr His Gln Asn Asn Arg Ala Trp Pro Asp Arg 
                1125                1130                1135 

Leu Val Ala Ser Met Arg Pro Ile Asp Ala Arg Tyr Ser Lys Pro Met 
            1140                1145                1150 

Val Gly Ala Gly Tyr Val Val Gly Pro Ser Thr Phe Leu Gly Thr Pro 
        1155                1160                1165 

Gly Val Val Ser Tyr Tyr Leu Thr Leu Tyr Ile Arg Gly Glu Pro Gln 
    1170                1175                1180 

Ala Leu Pro Glu Thr Leu Val Ser Thr Gly Arg Ile Ala Thr Asp Cys 
1185                1190                1195                1200 

Arg Glu Tyr Leu Asp Ala Ala Glu Glu Glu Ala Ala Lys Glu Leu Pro 
                1205                1210                1215 

His Ala Phe Ile Gly Asp Val Lys Gly Thr Thr Val Gly Gly Cys His 
            1220                1225                1230 

His Ile Thr Ser Lys Tyr Leu Pro Arg Ser Leu Pro Lys Asp Ser Val 
        1235                1240                1245 

Ala Val Val Gly Val Ser Ser Pro Gly Arg Ala Ala Lys Ala Val Cys 
    1250                1255                1260 

Thr Leu Thr Asp Val Tyr Leu Pro Glu Leu Arg Pro Tyr Leu Gln Pro 
1265                1270                1275                1280 

Glu Thr Ala Ser Lys Cys Trp Lys Leu Lys Leu Asp Phe Arg Asp Val 
                1285                1290                1295 

Arg Leu Met Val Trp Lys Gly Ala Thr Ala Tyr Phe Gln Leu Glu Gly 
            1300                1305                1310 

Leu Thr Trp Ser Ala Leu Pro Asp Tyr Ala Arg Xaa Ile Gln Leu Pro 
        1315                1320                1325 

Lys Asp Ala Val Val Tyr Ile Asp Pro Cys Ile Gly Pro Ala Thr Ala 
    1330                1335                1340 

Asn Arg Lys Val Val Arg Thr Thr Asp Trp Arg Ala Asp Leu Ala Val 
1345                1350                1355                1360 

Thr Pro Tyr Asp Tyr Gly Ala Gln Asn Ile Leu Thr Thr Ala Trp Phe 
                1365                1370                1375 

Glu Asp Leu Gly Pro Gln Trp Lys Ile Leu Gly Leu Gln Pro Phe Arg 
            1380                1385                1390 

Arg Ala Phe Gly Phe Glu Asn Thr Glu Asp Trp Ala Ile Leu Ala Arg 
        1395                1400                1405 

Arg Met Asn Asp Gly Lys Asp Tyr Thr Asp Tyr Asn Trp Asn Cys Val 
    1410                1415                1420 

Arg Glu Arg Pro His Ala Ile Tyr Gly Arg Ala Arg Asp His Thr Tyr 
1425                1430                1435                1440 

His Phe Ala Pro Gly Thr Glu Leu Gln Val Glu Leu Gly Lys Pro Arg 
                1445                1450                1455 

Leu Pro Pro Gly Gln Val Pro 
            1460 

 
           
           
             
               249 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              4 

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 Xaa 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 Xaa Met Phe Trp 
                 85                  90                  95 

His Met Arg Val Ser His Leu Ile Asp Glu Xaa 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 

 
           
           
             
               265 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              5 

Met Ala His Gln Cys Ala Arg Phe His Phe Phe Leu Cys Gly Phe Ile 
  1               5                  10                  15 

Cys Tyr Leu Val His Ser Ala Leu Ala Ser Asn Ser Ser Ser Thr Leu 
             20                  25                  30 

Cys Phe Trp Phe Pro Leu Ala His Gly Asn Thr Ser Phe Glu Leu Thr 
         35                  40                  45 

Ile Asn Tyr Thr Ile Cys Met Pro Cys Ser Thr Ser Gln Ala Ala Arg 
     50                  55                  60 

Gln Arg Leu Glu Pro Gly Arg Asn Met Trp Cys Lys Ile Gly His Asp 
 65                  70                  75                  80 

Arg Cys Glu Glu Arg Asp His Asp Glu Leu Leu Met Ser Ile Pro Ser 
                 85                  90                  95 

Gly Tyr Asp Asn Leu Lys Leu Glu Gly Tyr Tyr Ala Trp Leu Ala Phe 
            100                 105                 110 

Leu Ser Phe Ser Tyr Ala Ala Gln Phe His Pro Glu Leu Phe Gly Ile 
        115                 120                 125 

Gly Asn Val Ser Arg Val Phe Val Asp Lys Arg His Gln Phe Ile Cys 
    130                 135                 140 

Ala Glu His Asp Gly His Asn Ser Thr Val Ser Thr Gly His Asn Ile 
145                 150                 155                 160 

Ser Ala Leu Tyr Ala Ala Tyr Tyr His His Gln Ile Asp Gly Gly Asn 
                165                 170                 175 

Trp Phe His Leu Glu Trp Leu Arg Pro Leu Phe Ser Ser Trp Leu Val 
            180                 185                 190 

Leu Asn Ile Ser Trp Phe Leu Arg Arg Ser Pro Val Ser Pro Val Ser 
        195                 200                 205 

Arg Arg Ile Tyr Gln Ile Leu Arg Pro Thr Arg Pro Arg Leu Pro Val 
    210                 215                 220 

Ser Trp Ser Phe Arg Thr Ser Ile Val Ser Asp Leu Thr Gly Ser Gln 
225                 230                 235                 240 

Gln Arg Lys Arg Lys Phe Pro Ser Glu Ser Arg Pro Asn Val Val Lys 
                245                 250                 255 

Pro Ser Val Leu Pro Ser Thr Ser Arg 
            260                 265 

 
           
           
             
               183 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              6 

Met Ala Ala Ala Thr Leu Phe Phe Leu Ala Gly Ala Gln His Ile Met 
  1               5                  10                  15 

Val Ser Glu Ala Phe Ala Cys Lys Pro Cys Phe Ser Thr His Leu Ser 
             20                  25                  30 

Asp Ile Glu Thr Asn Thr Thr Ala Ala Ala Gly Phe Met Val Leu Gln 
         35                  40                  45 

Asp Ile Asn Cys Phe Arg Pro His Gly Val Ser Ala Ala Gln Glu Lys 
     50                  55                  60 

Ile Ser Phe Gly Lys Ser Ser Gln Cys Arg Glu Ala Val Gly Thr Pro 
 65                  70                  75                  80 

Gln Tyr Ile Thr Ile Thr Ala Asn Val Thr Asp Glu Ser Tyr Leu Tyr 
                 85                  90                  95 

Asn Ala Asp Leu Leu Met Leu Ser Ala Cys Leu Phe Tyr Ala Ser Glu 
            100                 105                 110 

Met Ser Glu Lys Gly Phe Lys Val Ile Phe Gly Asn Val Ser Gly Val 
        115                 120                 125 

Val Ser Ala Cys Val Asn Phe Thr Asp Tyr Val Ala His Val Thr Gln 
    130                 135                 140 

His Thr Gln Gln His His Leu Val Ile Asp His Ile Arg Leu Leu His 
145                 150                 155                 160 

Phe Leu Thr Pro Ser Ala Met Arg Trp Ala Thr Thr Ile Ala Cys Leu 
                165                 170                 175 

Phe Ala Ile Leu Leu Ala Ile 
            180 

 
           
           
             
               201 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              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 

 
           
           
             
               173 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              8 

Met Gly Gly Leu Asp Asp Phe Cys Asn Asp Pro Ile Ala Ala Gln Lys 
  1               5                  10                  15 

Leu Val Leu Ala Phe Ser Ile Thr Tyr Thr Pro Ile Met Ile Tyr Ala 
             20                  25                  30 

Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Ile Leu Ile 
         35                  40                  45 

Phe Leu Asn Cys Ser Phe Thr Phe Gly Tyr Met Thr Tyr Val His Phe 
     50                  55                  60 

Gln Ser Thr Asn Arg Val Ala Leu Thr Leu Gly Ala Val Val Ala Leu 
 65                  70                  75                  80 

Leu Trp Gly Val Tyr Ser Phe Thr Glu Ser Trp Lys Phe Ile Thr Ser 
                 85                  90                  95 

Arg Cys Arg Leu Cys Cys Leu Gly Arg Arg Tyr Ile Leu Ala Pro Ala 
            100                 105                 110 

His His Val Glu Ser Ala Ala Gly Leu His Ser Ile Ser Ala Ser Gly 
        115                 120                 125 

Asn Arg Ala Tyr Ala Val Arg Lys Pro Gly Leu Thr Ser Val Asn Gly 
    130                 135                 140 

Thr Leu Val Pro Gly Leu Arg Ser Leu Val Leu Gly Gly Lys Arg Ala 
145                 150                 155                 160 

Val Lys Arg Gly Val Val Asn Leu Val Lys Tyr Gly Arg 
                165                 170 

 
           
           
             
               128 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              9 

Met Ala Gly Lys Asn Gln Ser Gln Lys Lys Lys Lys Ser Thr Ala Pro 
  1               5                  10                  15 

Met Gly Asn Gly Gln Pro Val Asn Gln Leu Cys Gln Leu Leu Gly Ala 
             20                  25                  30 

Met Ile Lys Ser Gln Arg Gln Gln Pro Arg Gly Gly Gln Xaa Lys Lys 
         35                  40                  45 

Lys Lys Pro Glu Lys Pro His Phe Pro Leu Ala Ala Glu Asp Asp Ile 
     50                  55                  60 

Arg His His Leu Thr Gln Thr Glu Arg Ser Leu Cys Leu Gln Ser Ile 
 65                  70                  75                  80 

Gln Thr Ala Phe Asn Gln Gly Ala Gly Thr Ala Xaa Leu Ser Ser Ser 
                 85                  90                  95 

Gly Lys Val Ser Phe Gln Val Glu Phe Met Leu Pro Val Ala His Thr 
            100                 105                 110 

Val Arg Leu Ile Arg Val Thr Ser Thr Ser Ala Ser Gln Gly Ala Ser 
        115                 120                 125

Technology Category: y