text: 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 . 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 . 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 . 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 ). 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 . 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 ). 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 ). 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 . 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 ( fig2 ). 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 ( fig1 seq id no : 1 ). in this sequence 8 open reading frames ( orfs ) were identified : orf 1a , orf 1b , and orfs 2 to 7 . 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 ). 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 . the complete nucleotide sequence of fig1 ( 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 fig1 ( 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . [ 0049 ] fig1 ( 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 fig2 and section “ results ”), is underlined . [ 0050 ] fig2 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 . 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 : ( 1 ) tissue samples and an oral swab from affected piglets from the field ( table 1a ); ( 2 ) blood samples and oral swabs from affected sows in the field ( tables 1b and 4 ); ( 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 ( 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 ). 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 . tissue samples were cut on a cryostat microtome and sections were submitted for direct immunofluorescence testing ( ift ) with conjugates directed against various pig pathogens . 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 . 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 . 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 . 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 . 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 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 ). 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 . 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 . 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 . 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 . 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 ). 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 . 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 . 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 ). 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 . 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 . 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 ). 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 . 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 ). 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 . 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 . 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 . 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 . virus isolation from spf pigs kept in contact with msd affected sows 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 ). virus isolation from spf pigs inoculated with blood of msd affected sows 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 . summarizing , four groups of pigs were tested for the presence of agents that could be associated with mystery swine disease ( msd ). in group one , msd affected piglets , the lelystad agent ( la ) was isolated from 16 out of 20 piglets ; one time pev 2 was isolated . 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 . 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 . 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 . 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 . serology of spf pigs kept in contact with msd affected sows 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 . 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 . la did not haemagglutinate with chicken , guinea pig , pig , sheep , or human o red blood cells . la did not react in ipma with sera directed against prv , tge , ped , asfv , etc . 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 . 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 . growth curve of la ( see fig3 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 fig2 . 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 fig1 ). 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 ). 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 . the consensus nucleotide sequence of overlapping cdna clones is shown in fig1 ( 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 . 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 , fig2 ). 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 ( fig2 ), 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 fig1 ( 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 . 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 , fig1 ) 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 ). 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 ). 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 ). 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 . 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 . 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 fig1 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 ). 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 . 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 . 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 . a schematic representation of the organization of the lv genome is shown in fig2 . 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 fig1 ( 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 . 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 . 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 . [ 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 ] 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 ] 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 ] 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 ] 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 ] 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 ] 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 ] 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 ) boer , g . f . de , back , w ., and osterhaus , a . d . m . e . 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( 1975 ), the genome of equine arteritis virus , virology , 68 , 418 - 425 . phe trp asn ala gly gln val phe cys thr arg cys leu ser ala arg leu phe tyr lys pro arg asp lys leu his trp lys val pro ile gly ile pro gln val glu cys thr pro ser gly cys cys trp leu ser ala val phe pro leu ala arg met thr ser gly asn his asn phe leu gln pro arg his leu arg glu leu gln val tyr glu arg gly cys asn trp tyr pro ile thr gly pro val pro gly met gly leu phe ala asn ser met his val ser asp gln pro phe pro gly ala thr his val leu thr thr asp ser ser leu asn gly arg ser arg met met trp thr pro glu gln val glu ile leu ile arg ser phe pro ala his his pro val asp leu ala asp trp glu leu thr glu ser pro glu asn gly phe ser phe asn thr ser his ser cys gly his leu val gln asn pro asp val phe asp gly lys cys trp leu ser cys phe leu gly gln ser val glu val arg cys his glu glu his leu ala asp ala phe gly tyr gln thr lys ser cys pro gln ser trp ile arg his leu thr leu asp asp asp val glu pro thr thr ser arg ile phe arg phe gly ala his lys trp tyr val leu ala ala ile met asn arg met ile asn gly asp phe thr ser pro leu thr gln tyr asn arg pro glu asp asp trp ala ser asp tyr val his trp glu val glu val arg ser gly met ala pro arg ser leu ser ala tyr arg leu pro ser asp cys val ser ser gly ile ala asp phe leu ala asn pro pro pro gln glu phe trp thr leu asp lys met phe ile tyr ala val glu arg met leu lys asp cys pro ser ser lys val leu cys ser pro asp ala lys glu phe glu glu ala ala xaa glu glu gln thr pro asp asn pro gly ser asp ala gly ala leu pro val thr val arg glu phe val pro thr gly pro ile leu cys his val glu glu pro val phe val lys pro arg asn ala phe ser asp gly asp ser leu lys arg pro arg phe ser ala gln ala leu ile asp arg gly gly pro leu ala asp val his ala lys ile lys asn arg val tyr glu gln cys thr ser gln phe gln ala gly arg ile leu ala ser leu lys phe leu pro asp met ile gln asp thr pro pro pro val pro arg lys asn arg ala ser asp asn ala gly leu lys gln leu val ala gln trp asp gly ser ile ser gln arg leu met thr trp val phe glu val phe ser ala leu ser leu val tyr val val ser gln gly arg cys his lys cys trp gly lys cys ile arg thr ala pro ala glu val ala leu asn val arg phe gln thr pro lys gly val asp pro val his leu ala thr gly lys pro ile ala tyr ala asn leu asp glu lys lys met ser ala gln thr val val ala val pro tyr asp pro ser gln ala ile lys cys leu lys val leu gln ala gly gly ala ile val asp gln pro thr pro glu val ala ala val arg cys gly tyr ser thr ala gln leu xaa leu gly arg gly asn phe ala lys leu asn gln thr pro pro arg asn ser ile ser pro gln val cys gly arg gly thr ala asp pro trp cys ser asn leu thr met leu trp val his ser phe leu val phe cys leu pro ala arg phe thr gln val ala gly ile ile thr pro tyr asp ile his gln arg thr his lys pro cys leu asn thr val asn val val gly ser ser ser tyr asn arg met his thr phe lys thr asn gly asp tyr ala trp lys gly tyr arg gly arg ala tyr trp gln thr ser thr gly val glu his phe ala gly pro ser val pro leu gly asp ile lys leu ser pro leu cys val phe phe leu leu trp arg met met gly his ala trp thr ala thr pro trp ser ala gln val leu met ile arg leu leu thr ala glu leu ser gln ala leu ser thr tyr cys phe leu pro arg val leu ala met thr ser cys val pro thr ile ile ile gly gly leu his thr leu gly val ile leu trp xaa phe lys tyr arg cys leu his asn met ala glu gly asn leu arg lys gly val ser gln ser cys gly met asn lys ala leu arg gln glu leu ala ser leu val gln ile asp lys met gly ser ile leu asp ile asn val gly thr glu arg lys thr val ser val gln glu thr arg ser leu gly gly ser lys phe ser val cys thr val val ser asn thr pro val asp ala xaa thr gly ile pro leu gln asp asp leu lys val glu arg met lys lys his cys val ser leu gly phe his asn ile asn gly lys val tyr cys lys ile trp asp lys ser asp leu thr ala ala glu val glu lys leu lys arg ile ile ser gln ser arg thr phe thr leu gly pro leu asp leu lys val thr ser glu gly his gly ala gly asn met gly val asp gly ser ile trp asp phe gln ala cys glu val arg arg gly asp ala pro asn leu gln leu pro leu ile asn thr arg phe gly asp leu pro tyr lys thr pro gln asp thr lys ser ala ile his ala ala cys cys leu his pro asn gly ala asp ser arg pro asp thr pro phe met cys thr lys his gly thr ser his ile gly lys ala pro pro leu phe leu pro ser thr tyr pro ala lys asn ser met ala gly ile asn gly gln arg phe pro thr lys asp val gln ser ile pro glu ile asp glu met cys ala arg ala val lys glu asn trp gln thr val thr pro cys thr leu lys lys gln tyr cys ser lys pro lys thr arg thr ile leu gly thr asn asn phe ile ala leu ala his arg ser ala leu ser gly val thr gln ala phe met lys lys ala trp lys ser pro ile ala leu gly lys asn lys phe lys glu leu his cys thr val ala gly arg cys leu glu ala asp leu ala ser cys asp arg ser thr pro ala ile val arg trp phe val ala asn leu asn cys cys his asp leu val ala thr gln asp gly ala phe thr lys phe glu asp leu leu glu ile gln pro met leu val tyr ser asp asp leu val leu tyr ala glu arg pro xaa phe pro asn tyr his trp trp val glu his leu asp leu met leu gly phe arg thr asp pro lys lys thr val ile thr asp lys pro ser phe leu gly cys arg ile glu ala ala ile leu met asp ser cys ala cys ile asp his asp pro glu trp tyr ser phe pro gly pro ala phe phe met ser met trp glu lys leu arg ser his asn glu gly lys lys phe arg his cys gly ile cys asp gly arg ser pro leu asp ala val leu lys gln ile pro tyr lys pro pro arg thr val ile met lys val gly asn lys thr thr ala leu asp ile ala gly asn glu val asp leu ser asp xaa asp tyr gln val val pro leu leu pro thr cys lys asp ile asn met val lys val ala cys pro thr his gln thr met phe asp ile val ser ala leu lys val cys arg val ser tyr leu asp glu ala gly tyr cys asn his leu asp ile leu arg leu leu ser lys thr pro leu val cys leu gly asp leu gln gln leu his pro val gly phe asp ser tyr cys tyr val phe asp gln met pro gln lys gln leu thr thr ile tyr arg phe gly pro asn ile cys ala arg ile gln pro cys tyr arg glu lys leu glu ser lys ala val leu thr pro tyr his lys asp arg ile gly ser ala ile thr ile asp ser ser gln gly ala thr phe asp ile val thr leu his leu pro ala arg his gly leu phe ile tyr asp pro his asn gln leu gln glu phe phe asn leu thr pro glu arg thr asp cys asn leu val phe ser val ala lys ala leu glu thr gly pro ser arg phe arg val ser asp ser cys met pro leu pro gln val ala his asn leu gly phe tyr phe leu val ala ser met arg pro ile asp ala arg tyr ser lys pro met gly val val ser tyr tyr leu thr leu tyr ile arg gly glu pro gln ala leu pro glu thr leu val ser thr gly arg ile ala thr asp cys his ile thr ser lys tyr leu pro arg ser leu pro lys asp ser val glu thr ala ser lys cys trp lys leu lys leu asp phe arg asp val arg leu met val trp lys gly ala thr ala tyr phe gln leu glu gly leu thr trp ser ala leu pro asp tyr ala arg xaa ile gln leu pro thr pro tyr asp tyr gly ala gln asn ile leu thr thr ala trp phe glu asp leu gly pro gln trp lys ile leu gly leu gln pro phe arg arg ala phe gly phe glu asn thr glu asp trp ala ile leu ala arg arg met asn asp gly lys asp tyr thr asp tyr asn trp asn cys val his phe ala pro gly thr glu leu gln val glu leu gly lys pro arg met gln trp gly his cys gly val lys ser ala ser cys ser trp thr pro asp val pro gln phe ala val lys his pro leu xaa met phe trp tyr gln thr met glu his ser gly gln ala ala trp lys gln val val gly glu ala thr leu thr lys leu ser gly leu asp ile val thr his phe gln his leu ala ala val glu ala asp ser cys arg phe leu ser gln tyr asn thr thr leu asp arg val glu leu ile phe pro thr pro gly thr arg pro lys leu thr asp phe arg gln trp leu ile ser val val leu trp leu arg ile pro ala leu arg tyr val phe gly phe his cys phe trp phe pro leu ala his gly asn thr ser phe glu leu thr ile asn tyr thr ile cys met pro cys ser thr ser gln ala ala arg gln arg leu glu pro gly arg asn met trp cys lys ile gly his asp leu ser phe ser tyr ala ala gln phe his pro glu leu phe gly ile gly asn val ser arg val phe val asp lys arg his gln phe ile cys ser trp ser phe arg thr ser ile val ser asp leu thr gly ser gln val ser glu ala phe ala cys lys pro cys phe ser thr his leu ser asp ile glu thr asn thr thr ala ala ala gly phe met val leu gln asp ile asn cys phe arg pro his gly val ser ala ala gln glu lys ile ser phe gly lys ser ser gln cys arg glu ala val gly thr pro gln tyr ile thr ile thr ala asn val thr asp glu ser tyr leu tyr val ser ala cys val asn phe thr asp tyr val ala his val thr gln phe leu thr pro ser ala met arg trp ala thr thr ile ala cys leu met arg cys ser his lys leu gly arg phe leu thr pro his ser cys ile cys glu leu asn gly thr asp trp leu ser ser his phe gly trp ala val glu thr phe val leu tyr pro val ala thr his ile leu ser asn phe ile val asp asp arg gly arg val his arg trp lys ser pro thr ile lys his val val leu glu gly val lys ala gln pro leu thr met gly gly leu asp asp phe cys asn asp pro ile ala ala gln lys phe leu asn cys ser phe thr phe gly tyr met thr tyr val his phe leu trp gly val tyr ser phe thr glu ser trp lys phe ile thr ser asn arg ala tyr ala val arg lys pro gly leu thr ser val asn gly gly lys val ser phe gln val glu phe met leu pro val ala his thr