Patent Application: US-82478204-A

Abstract:
the invention relates to the process for generating infectious newcastle disease virus entirely from cloned full - length cdna and to the use of vaccines and diagnostic assays generated with and derived from said process . the process offers the possibility to modify the ndv genome by means of genetic modification and allows the introduction of mutations , deletions and / or insertions . the process can be used to modify the virulence of ndv , thereby generating new attenuated live vaccines with enhanced properties . the process can be used to modify the antigenic make - up of ndv , thus allowing the generation of live ndv marker vaccines which can be serologically distinguished from ndv field strains .

Description:
standard cloning procedures were carried out according to sambrook et al . ( 1989 ) unless stated otherwise . all constructions involving dna fragments which were generated by means of the polymerase chain reaction ( pcr ) were verified by sequence analysis . in the primer sequences given below , the underlined nucleotides correspond to ndv sequences and the position within the ndv genome is indicated . the nucleotide sequence of restriction sites which were used for cloning are indicated in boldface . cer cells ( smith et al ., 1976 ) were grown in gmem / emem ( 1 : 1 ) containing 5 % fetal calf serum and 2 % of an antibiotic mix that contained 1000 u / ml penicillin , 1000 μg / ml streptomycin , 20 μg / ml fungizone , 500 μg / ml polymixin b , and 10 mg / ml kanamycin . qt35 cells ( moscovici et al ., 1977 ; cho , 1982 ) were grown in medium supplied by gibcobrl / life technologies ( cat . no . 041 - 91536 ; proprietary composition of fort dodge labs , ft . dodge , iowa ) supplemented with 5 % fcs and 2 % antibiotic mix . qm5 cells ( antin and ordahl , 1991 ) were grown in m199 medium supplemented with 10 % tryptose phosphate broth , 10 % fcs and 2 % antibiotic mix . ndv strain lasota was obtained from atcc ( atcc vr - 699 ) and was passaged two times in embryonated eggs . before we started with the construction and cloning of cdna , the virus was plaque purified by three rounds of plaque purification on primary chicken embryo fibroblasts ( cef ). to this end , the virus was titrated on cef cells cultured in gmem / emem ( 1 : 1 ) containing 5 % fetal calf serum , 2 % antibiotic mix , 5 % allantoic fluid , 30 mm mgcl 2 , 200 μg / ml deae - dextran ( sigma ) and 0 . 8 % agar nobel ( difco ). virus from the third round of plaque purification ( designated clone e13 - 1 ) was grown in embryonated eggs and four days after inoculation the allantoic fluid was harvested and stored in aliquots at − 70 ° c . the fowl pox recombinant virus fpeflt7pol ( britton et al ., 1996 ; hereafter called fpv - t7 ), which expresses t7 rna polymerase , was a kind gift of dr . michael skinner and was grown on qt35 cells . all manipulations were carried out in rnase - free glassware or plastics and all solutions were made up with rnase - free water which was treated with 1 % diethyl - pyrocarbonate ( depc ) and sterilized by autoclaving . virus was pelleted from allantoic fluid by centrifugation at 21 , 000 rpm for 70 minutes in a beckman sw40 rotor at 4 ° c . the pellet was resuspended in homogenization buffer ( 50 mm tris - hcl ph 7 . 5 , 50 mm nacl , 5 mm edta , 0 . 5 % sds ) and treated with proteinase k ( 200 μg / ml ) for 90 minutes at 37 ° c . during constant agitation . the lysate was extracted two times with an equal volume of phenol / chloroform ( 1 : 1 ) ph 5 . 4 and once with an equal volume of chloroform . the viral rna was precipitated from the aqueous phase by the addition of 0 . 1 volume of 3m naoac ph 5 . 3 and 2 . 5 volumes of 100 % ethanol . the precipitate was collected by centrifugation , washed once with 70 % ethanol , resuspended in water , and stored in aliquots at − 70 ° c . viral rna ( 1 . 5 μg ) was mixed with 500 ng of primer in a volume of 12 μl and incubated for 10 minutes at 70 ° c . four μl of 5 × rt buffer ( 250 mm tris - hcl , ph 8 . 3 , 375 mm kcl , 15 mm mgcl 2 ; gibcobrl / life technologies ), 2 μl 0 . 1 m dtt and 2 μl 10 mm dntp &# 39 ; s ( 2 . 5 mm each ) was added and the mixture was incubated for 2 minutes at 42 ° c . reverse transcription was performed in a final volume of 20 μl by the addition of 200 units of reverse transcriptase ( superscript ii ; gibcobrl / life technologies ) followed by incubation for 60 minutes at 42 ° c . all pcr reactions which were used to determine the 3 ′ and 5 ′ end of the ndv genome ( see below ) were carried out by using taq dna polymerase ( perkin elmer ). for the cloning of individual ndv genes or large subgenomic cdna &# 39 ; s , either the proofreading dna polymerase pwo , or mixtures of taq and pwo ( expand high fidelity kit or expand long template kit ) were used according to the instructions of the supplier ( boehringer mannheim ). all samples were incubated for 2 minutes at 94 ° c . before the start of the indicated number of pcr cycles . after the indicated number of pcr cycles , the samples were incubated at the elongation temperature for at least 3 × the duration of the elongation time of the pcr cycle . pcr fragments were purified directly by using the high pure pcr product purification kit ( boehringer mannheim ) or after agarose gel electrophoresis by using the qiaexii extraction kit ( qiagen ) essentially as described by the suppliers . all sequences were determined by using the prism ready reaction dye deoxy terminator cycle sequencing kit ( perkin elmer ). reaction mixtures ( 5 μl ) were subjected to 25 cycles of linear amplification ( 10 seconds at 94 ° c ., 5 seconds at 50 ° c ., and 4 minutes at 60 ° c .) in a geneamp2400 thermocycler . subsequently , the reaction mixtures were precipitated with ethanol , washed once with 70 % ethanol , resuspended in 15 μl tsr buffer ( perkin elmer ) and heated for 2 minutes at 94 ° c . before being loaded on an applied biosystems ab310 automatic sequencer . the nucleotide sequences of the primers which were used to sequence the complete genome of ndv strain lasota were either derived from published sequences or from sequences established during this sequencing project . the primers are shown in table 1 . cloning and sequencing of the 3 ′ and 5 ′ termini of the genome of ndv strain lasota the nucleotide sequence of the 3 ′ and 5 ′ termini of the ndv genome were determined by using race procedures ( rapid amplification of cdna ends ). ndv rna was used in a reverse transcription reaction in a final volume of 20 μl by using primer p360 ( 5 ′- ggcgatgtaatcagcctagtgctt - 3 ′ ( seq id no : 47 ); nt 14756 - 14779 ) which was derived from the published sequence of the l gene of ndv ( yusoff et al ., 1987 ). the single - stranded cdna ( 2 . 5 μl of the rt mixture ) was added to 8 pmol anchor primer alg3 ( 5 ′- cacgaattcactatcgattctggatccttc - 3 ′ ( seq id no : 83 )) and ligated overnight at room temperature in 20 μl of a reaction mixture containing 50 mm tris - hcl , ph 8 . 0 , 10 mm mgcl 2 , 10 μl / ml bsa , 25 % peg , 1 mm hcc , 20 um atp and 10 units of t4 rna ligase ( new england biolabs ) as described by tessier et al . ( 1986 ). one μl of the ligation reaction was used as template in a pcr reaction by using primers p375 ( 5 ′- caatgaat tcaaaggatattacagtaact - 3 ′ ( seq id no : 84 ); nt 14964 - 14983 ) and alg4 ( 5 ′- gaaggatccagaatcgatag - 3 ′ ( seq id no : 85 )). the latter primer is complementary to anchor primer alg3 ( seq id no : 83 ). the pcr conditions ( 40 cycles ) were as follows : 1 minute at 94 ° c ., 1 minute at 55 ° c ., and 2 minutes at 72 ° c . the pcr products were purified and cloned in t - vector pbluescriptii - tsk ( ichihara and kurosawa , 1993 ). alternatively , the purified pcr products were treated with klenow dna polymerase i to create blunt ends and cloned in the hindii site of plasmid pgem4z ( promega ). thirteen independent clones ( 8 × pbluescriptii - tsk and 5 × pgem4z ) were sequenced to determine the nucleotide sequence of the 5 ′ end of the genome of ndv strain lasota . the nucleotide sequence of the 3 ′ end was determined by two independent methods . in method i , primer alg3 ( seq id no : 83 ) was ligated to the 3 ′ end of the viral rna by using t4 rna ligase as described by schütze et al . ( 1995 ). the reaction mixture ( final volume 10 μl ) contained 2 . 5 μg ndv rna , 100 pmol alg3 ( seq id no : 83 ), 1 μl 10 × t4 rna ligase buffer ( 500 mm tris - hcl , ph 7 . 8 , 100 mm mgcl 2 , 100 mm dtt , 10 mm atp ), 1 μl dmso , 1 μl 10 um hexamine - cobalt chloride , 1 μl rnasin ( promega ) and 10 units of t4 rna ligase ( new england biolabs ). the mixture was incubated overnight at room temperature and 5 μl of the ligation reaction was used as template in a reverse transcription reaction by using alg4 ( seq id no : 85 ) as primer . one μl of the rt - reaction was used in a pcr reaction by using primers alg4 ( seq id no : 85 ) and p376 ( 5 ′- gagcc tta aggagctg c tcgtact g atc - 3 ′ ( seq id no : 86 ); nt 137 - 164 ) which was derived from the published sequence of the 3 ′ end of ndv ( ishida et al ., 1986 ). the pcr conditions were as described above for the 5 ′- race . in method ii , the 3 ′ and 5 ′ ends of the viral ndv rna were ligated to each other by using t4 rna ligase using the same conditions as described above for method i . five μl of the ligation mixture was used as template in a reverse transcription reaction by using primer p360 ( seq id no : 47 ). one μl of the rt - reaction was used in a pcr reaction by using primers p375 ( seq id no : 84 ) and p376 ( seq id no : 86 ) and pcr conditions described above for the 5 ′- race . the pcr products were treated with klenow dna polymerase i to create blunt ends and cloned in the hindii site of plasmid pgem4z ( promega ). ten independent clones ( 4 from method i and 6 from method ii ) were sequenced to determine the nucleotide sequence of the 3 ′ end of the genome of ndv strain lasota . a low - copy - number transcription vector was constructed by using plasmid pok12 ( vieira and messing , 1991 ) as the basic replicon . plasmid pok12 was digested with pvuii and the dna fragment containing the replication origin and the kanamycin - resistance gene was isolated . this dna fragment was ligated to an eco47iii - aflii fragment ( the aflii site was made blunt by using klenow dna polymerase i ) from transcription vector 2 . 0 ( a generous gift of dr . andrew ball ; pattnaik et al ., 1992 ). from the resulting plasmid an xbai - nhei fragment was deleted to eliminate as much unique restriction sites as possible . the resulting plasmid was designated poltv5 ( fig1 ). transcription vector poltv5 contains the t7 dna - dependent rna polymerase promoter followed by unique stui and smai restriction sites , the autocatalytic ribozyme from hepatitis delta virus ( hdv ) and the transcription termination signal from bacteriophage t7 . dna fragments cloned between the stui and smai restriction sites can be transcribed either in vitro or in vivo by using t7 rna polymerase . after transcription , the 5 ′ end of the resulting transcripts contains two g residues encoded by the plasmid . due to the autocatalytic action of the hdv ribozyme , the 3 ′ end of the transcripts corresponds to the exact terminal nucleotide of the cloned dna fragment ( pattnaik et al ., 1992 ). in order to examine the requirements for replication and transcription of ndv , minigenome plasmids were constructed which contained the 3 ′- and 5 ′- terminal regions of ndv flanking a reporter gene that replaced all ndv genes ( fig2 ). dna fragments corresponding to the 3 ′- and 5 ′- terminal regions of ndv were generated by means of pcr by using pwo dna polymerase ( 30 cycles ; 15 seconds at 94 ° c ., 30 seconds at 50 ° c ., and 30 seconds at 72 ° c .) and using plasmids containing the 3 ′- and 5 ′- race fragments as templates ( see above ). the 3 ′- region ( nt 1 - 119 ) was generated by using primers 3uit ( 5 ′- accaaacagagaatccgtgagttacga - 3 ′ ( seq id no : 1 ); nt 1 - 27 ) and seap3 ( 5 ′- atcgatactggtcagcatgc tggcagaaggctttctcg - 3 ′ ( seq id no : 87 ); nt 102 - 119 ). the 5 ′- region ( nt 14973 - 15186 ) was generated by using primers seap5 ( 5 ′- gcatgctgaccagtatcgat attacagtaactgtgact - 3 ′ ( seq id no : 88 ); nt 14973 - 14990 ) and 5ndv ( 5 ′- accaaacaaagatttggtgaatgacga - 3 ′ ( seq id no : 79 ); nt 15158 - 15186 ). the two dna fragments were joined in an overlap pcr ( the overlap is shown in italics in the primer sequences shown above ) by using primers 3uit ( seq id no : 1 ) and 5ndv ( seq id no : 79 ). the resulting dna fragment , which is a fusion of the 3 ′ and 5 ′ ends of ndv separated by 20 nucleotides , was phosphorylated by treatment with t4 polynucleotide kinase and cloned in both orientations in transcription plasmid poltv5 ( fig1 ) which was cleaved with stui and smai and dephosphorylated with calf intestinal phosphatase ( boehringer mannheim ). finally , the seap gene ( encoding secreted alkaline phosphatase ) was recovered from plasmid pseap - basic ( clontech ) by digestion with sphi and clai , and cloned between the sphi and clai sites between the 3 ′ and 5 ′ ends of ndv . the resulting plasmids were designated poltv535 and poltv553 , respectively . in vivo or in vitro transcription using t7 rna polymerase of plasmid poltv535 gives rise to antigenomic rna ([+]- rna ), whereas transcription of plasmid poltv553 gives rise to genomic rna ([−]- rna ). plasmids poltv535n0 to - n5 and poltv553n0 to - n5 were generated by inserting self - complementary oligonucleotides in the clai site located between the seap gene and the 5 ′ end of ndv in poltv535 and poltv553 , respectively ( see fig2 ). the oligonucleotides used were : n0 , 5 ′- cgcgagctcg - 3 ′ ( seq id no : 89 ); n1 , 5 ′- cgcgagsctcg - 3 ′ ( seq id no : 90 ); n2 , 5 ′- cgcgagcgctcg - 3 ′ ( seq id no : 91 ); n3 , 5 ′- cgcgagcwgctcg - 3 ′ ( seq id no : 92 ); n4 , 5 ′- cgcgagcatgctcg - 3 ′ ( seq id no : 93 ); n5 , 5 ′- cgcgagcastgctcg - 3 ′ ( seq id no : 94 ) ( w = a or t ; s = c or g ). to generate in vitro or in vivo transcripts containing the authentic 5 ′- and 3 ′- terminal ends of ndv , the t7 promoter in plasmids poltv535 and poltv553 was modified such that transcription would start at the first nucleotide of the 3 ′- or 5 ′- terminal ends of ndv . primers were designed which contained , 1 ) a bgli - restriction site , 2 ) the sequence of the t7 promoter ( shown in italics ) which was modified such that the two g residues at the end of the t7 promoter were replaced by an a residue , and 3 ) the 3 ′ ( nt 1 - 21 ) or 5 ′ end ( nt 15164 - 15186 ) of ndv . primers bgl3f2 ( 5 ′- gatatggccattcaggcttaatacgactcactata accaaacagagaatccgtgag - 3 ′ ( seq id no : 95 )) and seap3 ( seq id no : 87 ) ( see above ) were used to generate a dna fragment containing the modified t7 promoter and the entire 3 ′ end of ndv up to the start of the seap gene in poltv535 . similarly , a dna fragment was generated containing the modified t7 promoter and the entire 5 ′ end of ndv up to the end of the seap gene in poltv553 by using primers bgl5f2 ( 5 ′- gatatggccattcaggcttaatacgactcactata accaaacaaagatttggtgaatg - 3 ′ ( seq id no : 95 )) and seap5 ( seq id no : 88 ). the resulting fragments were digested with bgli and sphi ( 3 ′ end ) or bgli and clai ( 5 ′ end ), respectively , and used to replace the bgli - sphi fragment in poltv535 , or the bgli - clai fragment in poltv553 . the resulting plasmids were designated poltv735 and poltv753 , respectively . plasmids poltv735n3 and poltv753n3 were generated by inserting a self - complementary oligonucleotide ( 5 ′- cgcgagcwgctcg - 3 ′ ( seq id no : 92 ); w = a or t ) in the clai site located between the seap gene and the 5 ′ end of ndv in poltv735 and poltv753 , respectively . plasmid pcineoseap was constructed by cloning an xhoi - clai fragment ( clai site was made blunt by using klenow dna polymerase i ) containing the seap gene from plasmid pseap - basic ( clontech ) between the xhoi and smai sites of the eukaryotic - expression vector pcineo ( promega ). the latter plasmid contains the human cytomegalovirus ( hcmv ) promoter in addition to the bacteriophage t7 promoter . in order to examine and quantify seap expression by transcripts generated from the t7 promoter only , another plasmid was constructed which lacked the hcmv promoter . to this end , the hcmv promoter was deleted from pcineo by partial digestion with hindiii followed by complete digestion with bglii . the dna fragment ( nt 756 - 5469 according to numbering of clontech ) from which the hcmv promoter was deleted was isolated , treated with t4 dna polymerase to generate blunt ends and recircularized by using t4 dna ligase . the resulting plasmid was designated pcineod . finally , the seap gene was recovered from pseap - bacis as a mlui - acci fragment and cloned in pcineod between the mlui and clai sites . the resulting plasmid was designated pcineod seap . cells were seeded in 24 - well culture dishes , grown overnight to 60 - 80 % confluency , and infected at an m . o . i . of 1 with fpv - t7 for 1 hour at 37 ° c . the cells were transfected with 0 . 5 μg minigenome plasmid dna by using 3 μl of lipofectamine ™ and optimem essentially as described by the supplier ( gibcobrl / life technologies ). after incubation for 4 hours ( cer cells ) or 16 hours ( qm5 cells ) at 37 ° c . the cells were either infected with ndv ( dutch virulent isolate no . 152608 ; 200 μl per well ) for 1 hour at a m . o . i . of 5 , or left uninfected . the inoculum was aspirated and replaced by 1 ml of complete medium and the cells were further incubated at 37 ° c . for co - transfections , cells were grown in 6 - well culture dishes and infected with fpv - t7 as described above . the cells were co - transfected with 0 . 25 μg minigenome plasmid dna , 0 . 4 μg pcineonp , 0 . 2 μg pcineop and 0 . 2 μg pcineol ( c ) or pcineo by using either 8 μl of lipofectamine or 9 μl of fugene ™ 6 ( boehringer mannheim ). in order to generate infectious virus , the minigenome plasmid was replaced by a transcription plasmid that contained the full - length ndv cdna . the amount of seap which was secreted into the medium of transfected cells was measured in disposable 96 - well plates by using the phospha - light ™ chemiluminescent reporter assay for secreted alkaline phosphatase kit essentially as described by the supplier ( tropix ). chemiluminescense was quantified by using a liquid scintillation counter ( wallac 1450 microbeta plus ). cloning and sequencing of cdna &# 39 ; s spanning the entire genome of ndv strain lasota to clone and sequence the entire genome of ndv strain lasota , large subgenomic cdna clones were generated by means of rt - pcr and cloned in pgem - t . first strand cdna synthesis was performed by using primer 3uit ( seq id no : 1 ) as described above , and 1 μl of the rtreaction was used in a pcr reaction by using the expand long template pcr kit ( boehringer mannheim ). the pcr consisted of 5 cycles of 10 seconds at 94 ° c ., 30 seconds at 58 ° c ., and 6 minutes at 68 ° c ., followed by 10 cycles of 10 seconds at 94 ° c ., 30 seconds at 58 ° c ., and 6 minutes at 68 ° c ., in which the elongation time at 68 ° c . was increased by 20 seconds per cycle . the pcr fragments were cloned in pgem - t by using the pgem - t cloning kit essentially as described by the supplier ( promega ). ligation mixtures were transformed into e . coli strain sure ii ( stratagene ). two independent rt - pcr reactions ( a and b ) were performed and each yielded a similar set of cdna clones . the nucleotide sequence of the subgenomic cdna clones was determined by using ndv - specific primers ( table 1 ) and by primers flanking the inserts . after comparison of the nucleotide sequence of the a and b series of clones , remaining ambiguities were resolved by sequencing relevant regions of a third independent series of cdna &# 39 ; s ( c series ). the nucleotide sequence of ndv strain lasota is shown in seq id no : 134 . nucleotide sequence ( seq id no : 134 ) of the genome of ndv strain lasota and deduced amino acid sequence of the ndv genes . the sequence shown corresponds to the antigenomic strand and is shown in the 5 ′ to 3 ′ direction in the form of ssdna . the sequence shown in this figure is that of the consensus sequence which was determined by completely sequencing two independent sets of overlapping subgenomic cdna &# 39 ; s which span the entire ndv genome . remaining ambiguities ( probably as a result of pcr errors ) were resolved by sequencing relevant regions of a third independent set of clones . the sequence of the full length cdna clone pndfl + which was assembled from overlapping subgenomic cdna clones ( see fig3 ), differs from that of the consensus ndv sequence at the following positions ( consensus sequence between parentheses ): nt 1755 , g ( a ); nt 3766 , a ( g ); nt 5109 , g ( a ); nt 6999 , t ( c ); nt 7056 , g ( a ); nt 9337 , g ( a ); nt 9486 , a ( t ); nt 10195 , t ( c ); nt 13075 , a ( g ). these differences results in 3 amino acid changes ( consensus sequence between parentheses ): f protein , r 189 ( q ); hn protein s 200 ( p ) l - protein n 369 ( i ). the full - length ndv cdna was assembled in transcription plasmid poltv5 by using poltv535 as the starting plasmid . the dna fragments were joined at overlaps by using common restriction enzymes as detailed in portion b of fig3 . in a series of cloning steps , a plasmid ( designated p535 - di ) was constructed containing nucleotides 1 - 3521 and 12355 - 15186 separated by a clai site that was generated by joining the clai sites at position 3521 and 12355 . in another series of cloning steps , a plasmid ( designated pgem - b ) was constructed which contained part of the ndv genome including nucleotides 3521 - 12355 ( clai fragment ). to facilitate cloning , the latter clai fragment was tagged with the chloramphenicol - resistance ( cm ) gene from plasmid pacyc184 ( chang and cohen , 1978 ). to this end , the cm gene was recovered from pacyc184 by means of pcr by using primers cat - f ( 5 ′- gcgtacgtctagactggtgtccctgttgataccgg - 3 ′ ( seq id no : 96 ) and cat - r ( 5 ′- gctctagacgtacgaccctgccctgaaccgacg - 3 ′ ( seq id no : 97 ). the pcr was carried out with pwo dna polymerase and consisted of 30 cycles of 30 seconds at 94 ° c ., 45 seconds at 60 ° c ., and 60 seconds at 72 ° c . the resulting pcr fragment was digested with bsiwi and cloned in the unique bsiwi site of pgem - b , yielding pgem - b ( cat ). the clai fragment from pgem - b ( cat ) was cloned in the unique clai site of p535 - di , yielding pndfl ( cat ). finally , the cm gene was removed from this plasmid by digestion with bsiwi followed by religation and transformation of e . coli strain dh5a . the resulting plasmid was designated pndfl + and contains the entire ndv cdna sequence cloned between the t7 promoter and the hdv ribozyme in transcription plasmid poltv5 . dna fragments containing each of the ndv lasota genes were generated by means of rt - pcr and cloned in pcineo . after cloning , all fragments were sequenced by using primers flanking the inserts and by gene - specific primers . np gene : primer 386 ( 5 ′- gagcaatcgaagtcgtacgggtagaaggtg - 3 ′ ( seq id no : 98 ); nt 40 - 69 ) was used for reverse transcription . primers 365 ( 5 ′- gtgtgaattc cgagtgcgagcccgaag - 3 ′ ( seq id no : 99 ); nt 77 - 94 ) and 892 ( 5 ′- ttgcatgcctgca ggtcagtacccccagtc - 3 ′ ( seq id no : 100 ); nt 1577 - 1593 ) were used for pcr by using pwo dna polymerase . the following pcr profile ( 30 cycles ) was used ; 30 seconds at 95 ° c ., 40 seconds at 65 ° c ., and 45 seconds at 72 ° c . the resulting dna fragment was digested with ecori and cloned in pcineo between the ecori and smai sites . expression of np was verified in an immunoperoxidase monolayer assay ( ipma ) as described by peeters et al . ( 1992 ) by using monoclonal antibody 38 ( russell et al ., 1983 ). p gene : primer prt1 ( 5 ′- caaagaattc agaaaaaagtacgggtagaa - 3 ′( seq id no : 8 ); nt 1794 - 1814 ) was used for reverse transcription . primers prt1 ( seq id no : 8 ) and p2 ( 5 ′- gcagtctaga ttagccattcactgcaaggcgc - 3 ′ ( seq id no : 101 ); nt 3053 - 3071 ) were used for pcr by using pwo dna polymerase . the following pcr profile ( 30 cycles ) was used ; 30 seconds at 95 ° c ., 40 seconds at 65 ° c ., and 60 seconds at 72 ° c . the resulting dna fragment was digested with ecori and xbai and cloned in pcineo between the ecori and xbai sites . expression of p was verified in an ipma by using monoclonal antibody 688 ( russell et al ., 1983 ). m gene : primer 3uit ( 5 ′- accaaacagagaatccgtgagttacga - 3 ′ ( seq id no : 1 ); nt 1 - 27 ) was used for reverse transcription . primers ndv5m ( 5 ′- gggtgctagc ggagtgccccaattgtgccaa - 3 ′ ( seq id no : 102 ); nt 3268 - 3288 ) and ndv3m ( 5 ′- tctccccggg gcagcttatttcttaaaaggat - 3 ′ ( seq id no : 56 ); nt 4368 - 4389 ) were used for pcr by using the expand high fidelity kit . the pcr consisted of 10 cycles of 15 seconds at 95 ° c ., 30 seconds at 55 ° c ., and 2 minutes at 68 ° c ., followed by 15 cycles in which the elongation time at 68 ° c . was increased for 20 seconds per cycle . the resulting dna fragment was treated with t4 dna polymerase to create blunt ends , digested with nhei , and cloned in pcineo between the nhei and smai sites . expression of the m protein was verified in an ipma by using monoclonal antibody 424 ( russell et al ., 1983 ). f gene : primer 3uit ( seq id no : 1 ) ( see above ) was used for reverse transcription . primers ndv5f ( 5 ′- acgggctagc gattctggatcccggttgg - 3 ′ ( seq id no : 15 ); nt 4508 - 4526 ) and ndv3 f ( 5 ′- actaccc gggaaaccttcgttcctcat - 3 ′ ( seq id no : 60 ); nt 6212 - 31 ) were used for pcr by using the expand high fidelity kit using the conditions described above for the m gene . the resulting dna fragment was treated with t4 dna polymerase to create blunt ends , digested with nhei , and cloned in pcineo between the nhei and smai sites . expression of the f protein was verified in an ipma by using monoclonal antibody 8e12a8c3 ( id - dlo , department of avian virology ). hn gene : primer 3uit ( seq id no : 1 ) was used for reverse transcription . primers ndv5hn ( 5 ′- gtaggctagc aagagaggccgcccctcaat - 3 ′ ( seq id no : 22 ); nt 6335 - 6354 ) and ndv3hn ( 5 ′- cgagcccggg ccggcattcggtttgattcttg - 3 ′ ( seq id no : 104 ); nt 8205 - 8227 ) were used for pcr by using the expand high fidelity kit using the conditions described above for the m gene . the resulting dna fragment was treated with t4 dna polymerase to create blunt ends and after digestion with xmai it was cloned in pcineo between the blunted ( klenow dna polymerase ) nhei site and the xmai site . expression of the hn protein was verified in an ipma by using monoclonal antibody 86 ( russell et al ., 1983 ). l gene : the l gene was recovered from cdna clone pgem - l7a ( portion a of fig3 ) by digestion with sacii and sali . before digestion with sali , the sacii site was made blunt by treatment with t4 dna polymerase . the resulting fragment was cloned in pcineo between the blunted ( klenow dna polymerase ) nhei site and the sali site . the 5 ′ untranslated region between the t7 promoter and the atg start codon of the l gene contained 2 out - of - frame atg codons which might interfere with correct expression of the l protein . therefore , a new plasmid was constructed in which the first atg was missing and in which the second atg was changed to aag by means of pcr mutagenesis , as follows . primers 5le ( e ) 5 ′- caatg ga at t caaggcaaaacagctca a ggtaaataatacggg - 3 ′ ( seq id no : 104 ); nt 8332 - 8374 ) and 3le ( b ) 5 ′- gtgaatct agaatgccggatccgtacgaatgc - 3 ′ ( seq id no : 105 ); nt 8847 - 8870 ) were used in a pcr reaction using plasmid pgem - l7a ( fig3 ) as a template . the pcr was carried out by using pwo dna polymerase and consisted of 30 cycles of 30 seconds at 94 ° c ., 45 seconds at 60 ° c ., and 60 seconds at 72 ° c . the resulting dna fragment was digested with ecori and xbai and cloned in pcineo between the ecori and xbai sites , generating plasmid pcineol ( n ). subsequently , the bsiwi - sali fragment from pgem - l7a , which contains the remaining part of the l gene ( nt 8852 - 15046 ), was cloned in pcineol ( n ) between the bsiwi and sali sites , generating plasmid pcineol ( c ). since antibodies against the l - protein are not available , expression of l could not be checked by immunochemistry . to show unambiguously that infectious virus can be generated from cloned full - length cdna , a genetic tag was introduced in the f gene by means of pcr mutagenesis . to this end , the f gene was cloned by using two overlapping pcr fragments . the first pcr fragment was generated by using primers ndv5f ( seq id no : 15 ) ( see above ) and primer f5r ( 5 ′- aa a gcgcc g ctgtctcc t ccctccagatgtagtcac - 3 ′ ( seq id no : 106 ); nt 4859 - 4894 ). the residues shown in bold are changes which were introduced in the primer in order to change the amino acid sequence of the proteolytic cleavage site between f1 and f2 from that of the ndv lasota strain ( ggrqgr | l ) ( seq id no : 135 ) to that of the consensus cleavage site for virulent ndv strains ( grrqrr | f ) ( seq id no : 102 ) the second pcr fragment was generated by using primers f3f ( 5 ′- gg a ggagacag c ggcgc t ttataggcgccattattgg - 3 ′ ( seq id no : 107 ); nt 4875 - 4911 ) and iv09 ( 5 ′- ctctgtcgac acagactaccagaactttcac - 3 ′ ( seq id no : 108 ); nt 6246 - 6266 ). the pcr was performed with pwo dna polymerase and consisted of 25 cycles of 15 seconds at 94 ° c ., 30 seconds at 55 ° c ., and 2 minutes at 72 ° c . the two overlapping pcr fragments ( the overlap is shown in italics in the primer sequences ) were joined in a second pcr by using primers ndv5f ( seq id no : 15 ) and iv09 ( seq id no : 108 ) and by using the same pcr conditions . the resulting fragment , which contains the entire orf of the f gene and which encodes a virulent consensus cleavage site , was digested with nhei and sali and cloned in pcineo between the nhei and sali sites , yielding pcineof wt . the stui - noti fragment ( nt 4646 - 4952 ) from pcineof wt was used to replace the corresponding fragment in plasmid p535 - s which had been constructed by inserting the clai - scai ( nt 3521 - 10311 ) from pgem - b in p535 - di between the clai and scai sites ( see portion c of fig3 ). the resulting plasmid was designated p535 - s [ f wt c ]. a pcr fragment containing the cm - resistance gene from pacyc184 ( see above ) was cloned as an xbai fragment into the unique xbai site ( position 6172 in the ndv sequence ) of plasmid p535 - s [ f wt c ], yielding plasmid p535 - s [ f wt c ] cm . subsequently , the cm - tagged apai - spei fragment ( nt 2285 - 8094 ) of this plasmid was used to replace the corresponding fragment of the full - length cdna clone pndfl +. finally , the cm gene was removed from this plasmid by digestion with xbai followed by recircularization using t4 dna ligase . the resulting plasmid , which contains the genetically tagged full - length ndv cdna , was designated pndfl +[ f wt ]. generation of stably transformed cell lines that express individual ndv genes plasmids pcineonp , pcineop , pcineom , pcineof , pcineof wt , and pcineohn were used for the generation of stably transformed cell lines that express these proteins individually . the day before transfection , cer cells were seeded in 6 cm culture dishes and incubated overnight to give a confluency of 60 - 80 %. the cells were transfected with 2 μg of plasmid dna by using 12 μl of lipofectamine and optimem essentially as described by the supplier ( gibcobrl / life technologies ). after 48 hours the cells were trypsinized and dilutions were seeded in 10 cm culture dishes in medium containing 500 μg / ml of g418 ( boehringer mannheim ). every 3 days the medium was replaced by fresh medium containing increasing ( in steps of 100 μg / ml ) amounts of g418 until a concentration of 800 μg / ml was reached . cells were kept in medium containing 800 μg / ml g418 and three weeks after transfection individual colonies were picked and transferred to 96 - well culture dishes . the cloned cell lines were examined for the expression of the respective ndv gene by using an ipma as described above for transient - expression studies . cell lines that constitutively expressed np , p , m , or f could be identified and isolated . we were unable , however , to generate cell lines that expressed the hn protein . perhaps constitutive expression of hn is toxic to the cells . generation of stably transformed cell lines that express t7 rna polymerase the gene encoding t7 rna polymerase was recovered from plasmid prt7nt ( rene van gennip , id - dlo , department of mammalian virology ) by digestion with ecori and sali . the resulting fragment contains the t7 rna polymerase gene located behind the baculovirus p10 promoter . the dna fragment was cloned in plasmid pcineo0 between the ecori and sali sites , generating plasmid pcineo107 . plasmid pcineo0 lacks the t7 promoter and was derived from pcineo by cleavage with nhei followed by partial cleavage with scai , filling in the sticky ends with klenow dna polymerase and recircularization by using t4 dna ligase . the baculovirus sequences were removed from pcineo107 by digestion with ecori and paci , followed by t4 dna polymerase treatment to generate blunt ends and recircularization . the resulting plasmid was designated pcineo007 . expression of t7 dna polymerase was verified by co - transfection of cells with pcineo007 and pprh01 . the latter plasmid contains the e2 protein of classical swine fever virus cloned behind a t7 promoter and containing an internal ribosome entry site ( rene van gennip , personal communication ). expression of e2 was determined in an ipma by using monoclonal antibody v4 ( wensvoort et al ., 1986 ). stably transformed cer cell lines expressing t7 rna polymerase were generated and isolated as described above except that 10 cm culture dishes were used and the cells were transfected with 5 μg of pcineo007 dna and 25 μl of lipofectamine . to examine individual cell lines for the expression t7 rna polymerase , they were transfected with plasmid pprh01 and expression of e2 ( which is dependent on t7 rna polymerase ) was determined in an ipma by using monoclonal antibody v4 . several cell lines which expressed t7 rna polymerase were identified . one cell line , designated cer - c9 , was used for subsequent experiments . primer 3uit ( seq id no : 1 ) was used to synthesize single - stranded cdna of ndv and avian - paramyxovirus serotype - 2 and - 4 ( apmv2 and apmv4 ) as described above . all subsequent pcr reactions were performed by using 25 cycles of 15 seconds at 94 ° c ., 30 seconds at 55 ° c . and 2 minutes at 72 ° c . the entire coding region of the hn gene of apmv2 was recovered by means of pcr by using primers iv03 ( 5 ′- gggg ga a ttc cccattcaatgaagggtctac - 3 ′ ( seq id no : 110 )) and iv05 ( 5 ′- gatccccggg tcttaaaccaggcttcgcaatg - 3 ′ ( seq id no : 111 )) which were derived from the sequence of the hn gene of apmv2 ( genbank accession number d14030 ). the entire coding region of the hn gene of apmv4 was recovered by means of pcr by using primers iv06 ( 5 ′- gggggaattc tggtagggtggggaaggtagc - 3 ′ ( seq id no : 112 )) and iv08 ( 5 ′- attgcccggg gggtaactaatcaggatctcag - 3 ′ ( seq id no : 113 )) which were derived from the sequence of the hn gene of apmv4 ( genbank accession number d14031 ). the resulting pcr fragments were digested ( either directly or after subcloning in pgem - t ), with ecori and xmai and cloned in pcineo between the ecori and xmai sites . the resulting plasmids were designated pcineohn2 and pcineohn4 , respectively . hybrids between the hn gene of ndv strain lasota and the hn genes of apmv2 and - 4 were constructed by means of overlap pcr as follows . the n - terminal part ( aa 1 - 141 ) of the hn gene of ndv strain lasota was amplified with pwo dna polymerase by using primers iv01b ( 5 ′- gtag gaattc aagagaggccgcccctcaat - 3 ′ ( seq id no : 114 ); nt 6325 - 6354 ) and iv10 ( 5 ′- aatgagttctttgcctatcccccc - 3 ′ ( seq id no : 115 ); nt 6811 - 6834 ). the c - terminal part of the hn gene of apmv2 ( aa 142 - 580 ) was amplified with pwo dna polymerase by using primers iv11b ( 5 ′- ggggggataggcaaagaactcatt caaggagatgcatctgcaggc - 3 ′ ( seq id no : 116 ) and iv05 ( seq id no : 111 ). the resulting pcr fragments were joined in an overlap pcr ( overlap shown in italics ) by using primers iv01b ( seq id no : 114 ) and iv05 ( seq id no : 111 ) and by using the expand high fidelity enzyme mix . the resulting pcr fragment was digested ( either directly or after subcloning in pgem - t ) with ecori and xmai and cloned in pcineo between the ecori and xmai sites . the resulting plasmid which contains a hybrid hn gene consisting of aa 1 - 141 of ndv and aa 142 - 580 of apmv2 was designated pcineohn1 / 2 141 . the c - terminal part of the hn gene of apmv4 ( aa 143 - 569 ) was amplified by using primers iv 14b ( 5 ′- ggggggataggcaaagaactcatt gtagatgatgcatctgcaggcctaaatttcc - 3 ′ ( seq id no : 117 ) and iv08 ( seq id no : 113 ). this fragment was joined with the n - terminal part of the hn gene of ndv ( see above ) in an overlap pcr by using primers iv01b ( seq id no : 114 ) and iv08 ( seq id no : 113 ). the resulting pcr fragment was digested ( either directly or after subcloning in pgem - t ) with ecori and xmai and cloned in pcineo between the ecori and xmai sites . the resulting plasmid which contains a hybrid hn gene consisting of aa 1 - 141 of ndv and aa 143 - 569 of apmv4 was designated pcineohn1 / 4 141 . in analogy to the constructions described above , hybrid hn genes were constructed which consisted of aa 1 - 143 of ndv and aa 144 - 580 of apmv2 , or aa 1 - 143 of ndv and aa 145 - 569 of apmv4 . for these constructions pcr fragments were obtained by using the following pairs of primers ; ndv aa 1 - 143 , primer iv01b ( seq id no : 114 ) and iv13 ( 5 ′- atctacaatgagttctttgcctatc - 3 ′ ( seq id no : 118 ); nt 6816 - 6840 ); apmv2 aa 144 - 580 , primer iv14b ( 5 ′- ggggggataggcaaagaactcattgtagat gatgcatctgcaggcctaaatttcc - 3 ′ ( seq id no : 117 ) and iv05 ( seq id no : 111 ); apmv4 ( seq id no : 128 ) aa 145 - 569 , primer iv15b ( 5 ′- ggggggataggcaaagaactcattgtagat caaacagctgactacacagcag - 3 ′ ( seq id no : 119 ) and iv08 ( seq id no : 113 ). the pcr fragments were digested ( either directly or after subcloning in pgem - t ) with ecori and xmai and cloned in pcineo between the ecori and xmai sites . the resulting plasmids were designated pcineo1 / 2 143 and pcineo1 / 4 143 , respectively . to examine expression of the hn proteins , cer cells or qm5 cells were infected with fpv - t7 for 1 hour at a m . o . i . of 1 , transfected with plasmids pcineohn , pcineohn2 , pcineohn4 , pchneohn1 / 2 141 , pcineohn1 / 2 143 , pcineohn1 / 4 143 and pcineohn1 / 4 143 and 24 hours after transfection the monolayers were overlayed with a 1 % suspension of chicken erythrocytes in pbs for 45 minutes at room temperature . subsequently , the monolayers were carefully washed three times with pbs and adhesion of erythrocytes to transfected cells was examined microscopically . to examine induction of cell fusion after co - expression of the hn and f protein , cer cells or qm5 cells were co - transfected with pcineof wt together with either pcineo - hn1 pcineohn2 , pchneohn4 , pcineohn1 / 2 141 , pcineohn1 / 4 141 , pcineohn1 / 2 143 or pcineohn1 / 4 143 . after incubation for 2 to 3 days , the monolayers were washed with pbs , stained for 15 minutes with a giemsa solution ( 1 : 30 dilution in water ), and examined microscopically . cloning of hybrid hn genes in full length genomic ndv cdna a synthetic linker , designated hn12 , was inserted between the noti and spei sites of pgem - t ( promega ) by using oligonucleotides hn12a ( 5 ′- ggccgcatattctagagttaacgactta - 3 ′ ( seq id no : 120 ) and hn12b ( 5 ′- ctagtaagtcgttaactctagaatatgc - 3 ′ ( seq id no : 121 )). a synthetic linker , designated hn14 , was inserted between the noti and spei sites of pgem - t by using oligonucleotides hn14a ( 5 ′- ggccgcatattctagagttaacga - 3 ′ ( seq id no : 122 ) and hn14b ( 5 ′- ctagtcgttaactctagaatatgc - 3 ′ ( seq id no : 123 ). the resulting plasmids were designated pgem - hn12 and pgem - hn14 , respectively . these plasmids were digested with noti and xbai and used to clone the noti - spei fragment ( nt 3390 - 7488 ) from plasmid p535 - s [ f wt c ] cm . the resulting plasmids were designated pgem - hn1 / 2ns and pgem - hn1 / 4ns , respectively . the hn genes of these plasmids were replaced by the hybrid hn genes from plasmids pcineohn1 / 2 143 and pcineohn1 / 4 143 , respectively ( see section : cloning and expression of hn genes and hybrid hn genes ). to this end , pcineohn1 / 2 143 and pcineohn1 / 4 143 were digested with nhei and smai and the resulting fragments ( containing the hybrid hn1 / 2 143 and hybrid hn1 / 4 143 genes ) were cloned between the nhei and hpai site of plasmids pgem - hn1 / 2ns and pgem - hn1 / 4ns , resulting in pgem + hn12 and pgem + hn14 , respectively . the latter plasmids were used to introduce the hybrid hn genes into the full length genomic cdna clone of ndv . to this end , plasmids pgem + hn12 and pgem + hn14 were digested with noti and spei and the fragment containing either the hn12 or hn14 gene was used to replace the corresponding fragment of pndfl +, yielding pndfl + hn1 / 2 143 cm and pndfl + hn1 / 4 143 cm , respectively . the cm gene was removed from these plasmids by digestion with xbai followed by recircularization using t4 dna ligase . in order to comply with the “ rule - of - six ,” a linker was inserted into the unique spei site of these plasmids by using self - complementary oligonucleotides . linker h2 ( 5 ′- ctagcgagcgctcg - 3 ′ ( seq id no : 124 ) was inserted in plasmid pndfl + hn1 / 2 143 and linker h3 ( 5 ′- ctagcgagcwgctcg - 3 ′ ( seq id no : 125 ) was inserted in pndfl + hn1 / 4 143 , yielding plasmids pndfl + hn1 / 2 143 ( h2 ) and pndfl + hn1 / 4 143 ( h3 ), respectively . elimination of a specific epitope in the hn protein of ndv lasota a specific epitope , i . e ., amino acids 346 to 354 ( pdeqdyqir ) ( seq id no : 126 ), in the hn protein of ndv lasota that is recognized by mab 4de ( long et al ., 1986 ; meulemans et al ., 1986 ), was eliminated by replacing this sequence by the corresponding sequence of the hn proteins of either apmv - 2 ( nrtdiqqti ) ( seq id no : 127 ) or apmv - 4 ( pdplqdqil ) ( seq id no : 128 ). to this end , plasmid pcineohn ( see section : cloning and expression of individual ndv genes ) was used as template to create overlapping pcr fragments . for the apmv - 2 ( seq id no : 127 ) sequence the first pcr fragment was generated by using primers iv01 ( 5 ′- gtagacgcgtaagagaggccgcccctcaat - 3 ′ ( seq id no : 129 ) and primer 3hn2 ( 5 ′- gatagtttgctgtatatcagtccgattgcatgtgtcattgtatcgcttgtatatcac - 3 ′ ( seq id no : 130 ). the second pcr was generated by using the primers 5hn2 ( 5 ′- aatcggactgatatacagcaaactatcatggccaagtcttcgtataagcctggagcc - 3 ′ ( seq id no : 131 ) and ndv3 - hn ( 5 ′- cgagcccgggccggcattcggtttgattcttg - 3 ′ ( seq id no : 103 )). the resulting fragments were combined and used as template for a third pcr by using the primers iv01 b ( 5 ′- gtaggaattcaagagaggccgcccctcaat - 3 ′ ( seq id no : 114 )) and primer ndv3 - hn ( seq id no : 103 ). for the apmv - 4 ( seq id no : 128 ) sequence the first pcr fragment was generated by using primers iv01 ( seq id no : 129 ) and primer 3hn4 ( 5 ′- taagatctgatcttgcagcgggtcagggcatgtgtcattgtatcgcttgtatatcac - 3 ′ ( seq id no : 115 )). the second pcr was generated by using the primers 5hn4 ( 5 ′- cctgaccgctgcaagatcagatcttaatggccaagtcttcgtataagcctggagcc - 3 ′ ( seq id no : 116 )) and ndv3 - hn ( seq id no : 104 ). the resulting fragments were combined and used as template for a third pcr by using the primers iv01b ( seq id no : 114 ) and ndv3 - hn ( seq id no : 104 ). primers 3hn2 / 5hn2 and 3hn4 / 5hn4 are partly complementary and contain the genetic codes for the hn2 sequence ( nrtdiqqti ) ( seq id no : 127 ) and hn4 sequence ( pdplqdqil ) ( seq id no : 128 ), respectively . the pcr reactions were performed by using the expand long template pcr kit ( boehringer mannheim ). the pcr consisted of 30 cycles of 10 seconds 94 ° c ., 30 seconds 58 ° c . and 2 minutes at 68 ° c ., followed by 1 cycle of 4 minutes 68 ° c . the pcr fragments were digested with econi and bsu36i , and cloned between the econi and bsu36i sites of pcineohn . the resulting plasmids were designated pcineohn1 ( hn2e ) and pcineohn1 ( hn4e ), respectively . transient expression studied indicated that the modified hn proteins were correctly expressed and transported to the cell surface as judged from hemadsorbtion studies using chicken erythrocytes . furthermore , mab 6d4 which is directed against a linear epitope of hn of ndv and which consists of ( or at least includes ) amino acids 346 - 354 , did not react with the modified hn proteins . plasmids pcineohn1 ( hn2e ) and pcineohn1 ( hn4e ) were digested with nari and spei and the fragments containing the modified hn genes were cloned between the nari and spei sites of pgem - hn1 / 2ns and pgem - hn1 / 4ns , respectively . the resulting plasmids , designated pgem - hn1 ( hn2e ) and pgem - hn1 ( hn4e ), were digested with noti and spei , and used to replace the noti - spei fragment in pndfl +. the resulting plasmids were designated pndfl - hn ( hn2e ) cm and pndfl - hn ( hn4e ) cm , respectively . the cm gene was removed from these plasmids by digestion with xbai followed by religation . the resulting plasmids were designated pndfl - hn ( hn2e ) and pndfl - hn ( hn4e ), respectively . nucleotide sequence of the 3 ′- and 5 ′- terminal ends of the genome of ndv strain lasota the sequence of a putative 3 ′ end of the ndv genome has been published ( ishida et al ., 1986 ) albeit from another ndv strain ( d26 ) than the one used here ( lasota ). yusoff et al . ( 1987 ) have published a sequence of the l gene and a relatively large non - coding region behind the l gene of ndv strain beaudette c . however , as shown herein , this sequence did not include the full terminal 5 ′ end of the viral genome which makes it impossible to generate infectious copy ndv . the 3 ′- and 5 ′- terminal ends of the genome of negative - strand rna viruses fulfill an essential function in replication and transcription ( lamb and kolakofsky , 1996 ). thus , in order to generate a full - length ndv cdna which can be used to generate infectious virus by means of reverse genetics ( conzelmann , 1996 ), it is absolutely essential to include the correct 3 ′ and 5 ′ ends of the viral genome . therefore , we determined the exact nucleotide sequence of both the 3 ′ and 5 ′ ends of the genomic rna of ndv strain lasota by using 3 ′- and 5 ′- race procedures ( rapid amplification of cdna ends ). the 5 ′ end was recovered by means of pcr after ligation of a single - stranded anchor primer ( alg3 ) ( seq id no : 83 ) to single - stranded cdna which was generated by reverse transcription of the 5 ′ end of the genomic rna . by using a primer ( alg4 ) ( seq id no : 85 ) that is complementary to the anchor primer and an ndv - specific primer , pcr products were generated which contained the 5 ′ end . to clone the 3 ′ end of ndv , the single - stranded anchor primer alg3 ( seq id no : 83 ) was ligated to the 3 ′ end of viral rna by using t4 rna ligase and amplified by means of pcr by using primer alg4 ( seq id no : 128 ) and an ndv - specific primer ( method i ). alternatively , the 3 ′ and 5 ′ ends of the ndv rna were ligated to each other by using t4 rna ligase and the resulting concatenated rna was used for rt - pcr by using ndv - specific primers that flanked the ligation point ( method ii ). the 3 ′- and 5 ′- race products were cloned in t - vector pbluescriptii - tsk ( ichihara and kurosawa , 1993 ) or in pgem4z and several independent clones were isolated and sequenced . the results are compiled in table 2 . to enable the direct comparison of the 3 ′- and 5 ′- terminal ends , the sequences are shown in the form of dna and the 3 ′ end of the genomic strand is represented as the 5 ′ end of the antigenomic strand . at the genomic rna level the sequence of the 3 ′ end reads 3 ′- ugguuugucucuuag ( seq id no : 132 ) whereas the sequence of the 5 ′ end reads uuuagaaacaaacca - 5 ′ ( seq id no : 133 ). the sequence of the 3 ′ end is almost similar to the published 3 ′- terminal sequence of ndv strain d26 ( ishida et al ., 1986 ). however , the sequence of the 5 ′ end showed that ndv strain lasota contains 64 additional nucleotides in comparison with the published sequence of the l gene of ndv strain beaudette c ( yusoff et al ., 1987 ) ( fig5 .) to determine whether the 3 ′ and 5 ′ ends of ndv are functional in replication and transcription , minigenomes were constructed which consisted of the 3 ′ end of ndv ( nt 1 - 119 ), a reporter gene encoding secreted alkaline phosphatase ( seap ), and the 5 ′ end of ndv ( nt 14973 - 15186 ) ( fig2 ). these minigenomes were cloned in both orientations in transcription vector poltv5 , generating plasmids poltv535 and poltv553 , respectively ( for details of the construction see materials and methods ). plasmid poltv5 ( fig1 ) contains the t7 rna polymerase promoter followed by unique stui and smai restriction sites , the autocatalytic ribozyme from hepatitis delta virus ( hdv ) and the transcription termination signal from bacteriophage t7 ( pattnaik et al ., 1992 ). in vivo or in vitro transcription using t7 rna polymerase of plasmid poltv535 gives rise to antigenomic rna ( or [(+]- rna ), whereas transcription of plasmid poltv553 gives rise to genomic rna ( or [(−]- rna ) ( fig4 ). to examine whether the minigenome rna &# 39 ; s generated by plasmids poltv535 and poltv553 could be replicated and expressed by using ndv as helper virus , we used cer cells which expressed t7 rna polymerase either constitutively ( cer - c9 cells , see materials and methods ), or after infection with fowl pox recombinant fpeflt7pol ( britton et al ., 1995 ; hereafter called fpv - t7 ) that expresses t7 rna polymerase . cer - c9 cells and fpv - t7 infected cer cells were transfected with the minigenome plasmids poltv535 or poltv553 and after incubation for 3 hours at 37 ° c . the cells were either infected with ndv for 1 hour , or left uninfected . approximately 24 hours after transfection , a sample was taken from the medium and assayed for seap activity . the results showed the seap expression as very high in fpv - t7 infected cells which were transfected with poltv535 . this is not surprising since transcription by t7 rna polymerase generates antigenomic [+]- rna which is capped by fowl pox enzymes and which is efficiently translated by the host cell . in cells transfected with poltv553 , transcription by t7 rna polymerase generates genomic [−]- rna which must be converted , into [+]- rna by helper virus in order to be translated into seap protein ( cf . fig4 ). in both cases , no increase in seap expression could be observed in ndv infected cells in comparison to non - infected cells . on the contrary , seap - expression in ndv infected cells was consistently approximately two times lower than in uninfected cells ( results not shown ). for poltv535 - transfected cells this may be explained by the already very high level of seap expression by transcripts generated by t7 rna polymerase . however , in poltv553 - transfected cells , where efficient expression of seap is dependent on the conversion of genomic [−]- rna into antigenomic [+]- rna or mrna by the viral polymerase complex , we would have expected an increase in seap expression after ndv infection . we could think of two reasons why the minigenomes could not be expressed and replicated by ndv . first , the size of the minigenome rna &# 39 ; s does not conform to the so - called “ rule - of - six ” ( calain and roux , 1993 ; kolakofsky et al ., 1998 ). according to this rule , paramyxovirus genomes are only replicated efficiently when they are a multiple 6 nt in length . second , the two extra g residues which are present at the 5 ′ end of the minigenome rna &# 39 ; s might interfere with correct replication and / or transcription by the viral polymerase complex . to find out whether replication of the genomes was dependent on the rule - of - six , we inserted a series of short self - complementary oligonucleotides which increased 1 nt in size in the unique clai site in plasmids poltv535 and poltv553 ( fig2 ). the resulting plasmids ( poltv535n0 to - n5 and poltv553n0 to - n5 ) differ in size from 1 to 6 nt and therefore one of them should generate a minigenome rna which conforms to the rule - of - six . the plasmids were used to transfect cer cells or fpv - t7 infected cer - c9 cells as described above . the results showed that only plasmids poltv535n3 and poltv553n3 gave rise to an enhanced seap activity after ndv infection . the length of the minigenome rna &# 39 ; s generated from these plasmids by t7 rna polymerase were calculated to be 6n + 2 . since two extra g residues are present at the 5 ′ end of the minigenome rna &# 39 ; s , these results suggest that only the size of the rna sequence which is located between the authentic 3 ′ and 5 ′ ends of the minigenome rna &# 39 ; s is relevant for the rule - of - six . this was verified by constructing minigenome plasmids in which the transcription start of t7 rna polymerase was changed so that the first nucleotide which was incorporated into rna was the first nucleotide of the 3 ′ or 5 ′ end of ndv ( see materials and methods ). transfection of these plasmids indicated that only minigenome rna &# 39 ; s generated by plasmids poltv735n3 and poltv753n3 are replicated by helper virus ( results not shown ). these findings again indicate that replication of ndv is strictly dependent on the rule - of - six . furthermore , these findings indicate that the presence of two extra g residues at the 5 ′ end of the minigenome rna &# 39 ; s does not interfere with correct replication . similar results have been obtained with minigenome plasmids ( or di plasmids ) from other paramyxoviridae ( pattnaik et al ., 1992 ; harty and palese , 1995 ). to determine whether minigenome rna &# 39 ; s could be packaged by ndv helper virus , the medium of the transfected cells was transferred to fresh monolayers and after 1 hour of adsorption , the monolayers were washed three times with pbs and further incubated in complete medium . after 24 hours of incubation , the seap activity in the medium was measured . the results showed that seap activity was present only in cells which had been treated with the medium from cells transfected with minigenome plasmid poltv553n3 ( table 4 ). this finding indicates that minigenome rna &# 39 ; s can be packaged into ndv envelopes and that these particles are able to infect cells . furthermore , these results show that packaging is dependent on replication which indicates that only rna molecules which are complexed with the viral np , p and l proteins are packaged into virus - like particles . replication of ndv minigenomes by plasmids expressing the np , p , and l proteins to determine whether the minigenome rna &# 39 ; s could also be replicated by plasmids encoding the essential np , p , and l proteins , we performed co - transfection experiments in cells infected with fpv - t7 . cells were transfected with a combination of plasmids consisting of the minigenome plasmid and plasmids pcineonp , - p , and - l ( c ), respectively . as a negative control , pcineol ( c ), which encodes the essential l protein , was replaced by the vector plasmid pcineo . the results ( table 5 ) indicated that indeed plasmids encoding np , p , and l are able to replicate minigenome rna &# 39 ; s . the results furthermore show that , similar to minigenome replication by helper virus , also replication by the np , p , and l proteins is dependent on the rule - of - six . sub - genomic cdna fragments spanning the entire ndv genome were constructed by means of rt - pcr ( fig3 ). to keep the number of pcr errors to a minimum , a proofreading enzyme - mix ( expand long template ; boehringer mannheim ) was used in combination with a limited number of pcr cycles ( 15 cycles ). primer 3uit ( seq id no : 1 ) which is complementary to the 3 ′ end of ndv rna was used for reverse transcription , and gene - specific primers were used for pcr . to identify possible pcr errors , three independent rt reactions were performed and used to generate three independent sets of subgenomic cdna &# 39 ; s . the cdna &# 39 ; s , which varied in size from approximately 4 to 7 kb , were cloned in pgem - t . the nucleotide sequence of two sets of cdna &# 39 ; s was determined by using primers which were either deduced from published ndv sequences , or by primers derived from the ndv sequence that was deduced during this sequencing project ( table 1 ). remaining ambiguities were resolved by sequencing the relevant regions of the third set of cdna clones . the genome of ndv strain lasota consists of 15186 nt ( seq id no : 134 ), which makes it the smallest of all paramyxovirus genomes from which the entire sequence has been established to date ( kolakofsky et al ., 1998 ). construction of a full - length ndv cdna clone in transcription plasmid poltv5 to construct a full - length cdna clone of ndv strain lasota , overlapping cdna clones spanning the entire ndv genome were joined at shared restriction sites according to the strategy shown in fig3 . the entire ndv cdna was assembled in the minigenome plasmid poltv535 ( see above ) which is derived from transcription plasmid poltv5 . as can be seen in portion b of fig3 the last step in the assembly of the complete ndv cdna was the cloning of an approximately 8 . 8 kb clai ( nt 3521 - 12355 ) fragment from pgem - b into p535 - di which contained the ndv sequences flanking the clai site at either side ( i . e ., nt 1 - 3521 and 12355 - 15186 , respectively ). this step proved to be quite difficult since we repeatedly failed in generating the correct clones . therefore , the clai fragment of pgem - b was tagged with the chloramphenicol - resistance ( cm ) gene from plasmid pacyc184 . the clai fragment harboring the cm gene was isolated and cloned in the clai site of p535 - di and transformants were selected for resistance against both cm . since transformants grew poorly , the antibiotic selection was reduced to 15 μg / ml cm and 10 μg / ml km and the incubation temperature was reduced from 37 ° c . to 32 ° c . finally , the cm gene was removed from this plasmid by digestion with bsiwi followed by recircularization by using t4 dna ligase . after transformation of e . coli , cells harboring the desired plasmid were identified phenotypically by screening for km - resistance and cm - sensitivity . the resulting plasmid which consisted of the full - length ndv cdna cloned between the smai and stui sites of transcription plasmid poltv5 was designated pndfl +. to generate infectious ndv entirely from cloned cdna , plasmid pndfl + was used in co - transfection experiments with pcineonp , - p , and - l ( c ), as described above for the minigenome plasmids . transfection of cer and cef cells was monitored by using minigenome plasmid poltv553n3 and by measuring seap expression . as a negative control , pcineol ( c ) was replaced by pcineo . after co - transfection , the cells were incubated for 3 to 6 days in medium containing 5 % allantoic fluid . the addition of allantoic fluid is necessary because cer or cef cells lack the appropriate proteases which are required to cleave the f protein of ndv strain lasota . cleavage of the f protein is absolutely required for cell - to - cell spread and for the generation of infectious virus . after 3 days of incubation , we performed an immunological staining of the fixed monolayers by using a monoclonal antibody against the f protein . the results showed that cells that were stained with the antibody were only present in monolayers which had been co - transfected with pndfl (+), pcineonp , - p , and - l ( c ). these results indicated that genome replication and expression was occurring in these cells . no staining cells were observed when pcineol ( c ) was replaced by pcineo in the co - transfection experiments . to recover infectious virus , the supernatant of transfected cef monolayers was injected into the allantoic cavity of embryonated eggs . four days later the allantoic fluid was harvested , analyzed in a hemagglutination assay , and passaged further in eggs . the results showed that only the supernatant of cells transfected with a combination of pndfl + and pcineonp , - p , and - l ( c ) yielded a positive reaction in the hemagglutination assay . allantoic fluid which showed a positive hemagglutination reaction was subsequently analyzed in a hemagglutination - inhibition assay by using monoclonal antibodies 7b7 , 8c11 , 5a1 , 7d4 , and 4d6 ( long et al ., 1986 ) which can be used to differentiate between different ndv strains . the results of this assay indicated that the ndv strain which was recovered from the inoculated eggs showed the same reactivity as the original lasota strain . the virus which was recovered from the inoculated eggs was designated ndfl to distinguish it from the original lasota strain . to show unambiguously that the co - transfection system could be used to recover infectious virus from cloned full - length ndv cdna , a genetic tag was introduced in plasmid pndfl (+). to this end , the amino acid sequence of the protease cleavage site in the fo protein was changed from that of the lasota strain ( ggrqgr | l ) ( seq id no : 134 ) to the consensus sequence of virulent ndv strains ( grrqrr | f ) ( seq id no : 135 ) by means of pcr mutagenesis ( for details see materials and methods ). the resulting plasmid , pndfl +[ f wt ], was used to generate virus by using the co - transfection system described above . infectious virus , designated ndfl [ f wt ], was recovered from the allantoic fluid of embryonated eggs which had been inoculated with the medium of co - transfected cef cells . in an hi test , all mabs including 7d4 , which is specific for the lasota strain , showed the same reactivity with the newly generated virus as with the original lasota strain . the nucleotide sequence of the region encoding the protease cleavage site of the f protein was determined by means of rt - pcr . the results showed that the nucleotide sequence contained the exact nucleotide changes which were introduced in the mutagenic primer which was used to modify the original lasota sequence . this finding shows that the virus was derived from plasmid pndfl +[ f wt ] and demonstrates that ( genetically modified ) ndv can be generated entirely from cloned full - length ndv cdna . the protease cleavage site of the fo protein of ndv is a key determinant for virulence it is generally assumed that the amino acid sequence of the protease cleavage site of the fo protein is a key determinant for virulence of different ndv strains . the generation of a genetically modified lasota strain in which the amino acid sequence of the protease cleavage site was changed from a lentogenic ( non - virulent ) to that of a velogenic ( virulent ) ndv strain offered the unique opportunity to test this assumption . therefore , we determined the intracerebral pathogenicity index ( icpi ) of the newly generated virus ndfl [ f wt ] and compared it with that of strain ndfl and of the original lasota strain ( clone e13 - 1 ). the results showed that the icpi of strain ndfl [ f wt ] was 1 . 3 which is far above the value for strains ndfl ( icpi = 0 . 0 ) and clone e13 - 1 ( icpi = 0 . 3 ). these results show that , as expected , the virulence of ndv is largely determined by the amino acid sequence of the protease cleavage site of the fo protein . the envelope glycoproteins f and hn of ndv are the most immunogenic proteins of the virus . after infection , both the f and hn protein elicit a strong neutralizing antibody response . the induction of such a neutralizing antibody response is the basis of successful vaccination by non - virulent ndv strains ( such as the widely used lasota strain ). however , the antibody response against ndv vaccine strains cannot be distinguished from the antibody response against virulent ndv field strains . thus , infections with virulent field virus cannot be traced by serological methods . this situation is undesirable since field virus infections are masked by vaccination and clinical signs which are caused by field strains may be overlooked or are even attributed to the vaccine . since successful differentiation between vaccination and infection is essential for eradication of ndv , we set out to develop genetically modified ndv strains which can be used for vaccination and which can be serologically distinguished from ndv field strains ( so called marker vaccines ). in order to develop an ndv marker vaccine , the virus has to be genetically modified such that one or several immunodominant epitopes of one of the ( major ) antigens are either deleted or modified . deletion of part ( s ) of an essential protein may lead to the loss of the biological function of that protein . therefore , we chose to modify one of the immunodominant envelope proteins of ndv in such a way that the biological function of the protein was retained whereas the antibody repertoire against the modified protein differed from that against the original protein . for reasons specified below , we chose for one embodiment of the invention to modify the hn protein of ndv . infection of ndv is initiated by fusion of the virion envelope with the plasma membrane of the host cell . for this process , both the f protein and the hn protein are required . it has been shown that the f and hn proteins physically interact and that this interaction is required for membrane fusion ( deng et al ., 1995 ). furthermore , it has been shown that the interaction is type specific , i . e ., the f and hn proteins must be derived from the same virus in order to shown fusion activity . the interacting domain of the hn protein of ndv has been localized to the so - called stalk - or stem - region of the protein , comprising the first 92 amino acid residues of the ectodomain of the hn protein ( deng et al ., 1995 ). hybrid hn proteins consisting of aa 1 - 141 of ndv and aa 141 - 572 of human parainfluenza virus type - 3 ( hpiv3 ) were shown to retain fusion activity when co - expressed with the ndv f protein . these finding suggests that genetically modified ndv strains which harbor a hybrid hn protein which consists of the stem region of ndv followed by the globular head of the hn protein of a different avian - paramyxovirus serotype may be viable . furthermore , such strains would elicit an anti - hn antibody response which is different from that of ndv . since the neutralizing antibody response against the f protein is sufficient to allow efficient protection against challenge virus infection , such genetically modified ndv strains meet the two essential requirements of a marker vaccine , i . e ., protection against disease and serological differentiation . hybrid hn genes were constructed which consisted of a fusion of either aa 1 - 141 of ndv and aa 142 - 580 of avian - paramyxovirus type - 2 ( apmv2 ) ( designated hn1 / 2 141 ) or aa 1 - 143 of ndv and aa 144 - 580 of apmv2 ( designated hn1 / 2 143 ). similarly , hybrid hn genes were constructed which consisted either of aa 1 - 141 of ndv and aa 143 - 569 of ampv4 ( designated hn1 / 14 141 ) or aa 1 - 143 of ndv and aa 145 - 569 of apmv4 ( designated hn1 / 4 143 ). the hybrid genes were cloned in the eukaryotic - expression vector pcineo and used in co - transfection experiments with a plasmid harboring the ndv f protein . to this end , the f protein was modified such that the amino acid sequence of the proteolytic cleavage site between f2 and f1 was changed from the lasota sequence to that of the consensus sequence of virulent ndv strains ( f wt , see materials and methods section ). co - transfection experiments in cer cells and qm5 cells indicated that both hn1 / 2 141 and hn1 / 2 143 as well as hn1 / 4 141 and hn1 / 4 143 induced cell fusion when co - expressed with the f wt protein . these results indicated that the complexes between the hybrid hn proteins and the f protein were biologically active . the hybrid hn proteins hn1 / 2 143 and hn1 / 4 143 were used to replace the original hn gene in the full - length cdna clone pndfl +, yielding pndfl - hn1 / 2 143 and pndfl - hn1 / 4 143 . the latter two plasmids were subsequently used for the generation of infectious virus by using the co - transfection system described above . viable recombinant viruses ( designated ndfl - hn1 / 2 143 and ndfl - hn1 / 4 143 ) could be isolated from the allantoic fluid of embryonated eggs which had been inoculated with the supernatant of transfected monolayers . the presence of the hybrid hn gene in each of two recombinants was verified by means of rt - pcr . hemagglutination - inhibition tests showed that monoclonal antibodies and polyvalent antisera against ndv were unable to inhibit agglutination of chicken erythrocytes by the recombinant viruses ndfl - hn1 / 2 143 and ndfl - hn1 / 4 143 . these results indicate that strains ndfl - hn1 / 2 143 and ndfl - hn1 / 4 143 may be used as vaccines that can be serologically distinguished from classical ndv vaccines . to examine whether foreign genes can be inserted into the ndv genome , we constructed a recombinant virus that carried the seap reporter gene . the seap gene was derived from plasmid poltv535 and was modified to include the typical transcriptional stop and start boxes of ndv . a dna fragment containing the seap gene followed by the transcriptional stop and start boxes was inserted into the xmni site ( nt 109 ) in plasmid pndfl +[( f wt ]. infectious virus , designated ndfl - ap , was generated by means of the co - transfection system , and the presence of the seap gene was verified by means of rt - pcr . cells infected with strain ndfl - ap expressed very high levels of the seap protein . by using the specific activity of the seap protein , we calculated that x % of the proteins expressed in cells infected with ndfl - ap consisted of seap protein . these results show that heterologous genes can be expressed to very high levels from recombinant ndv . generation of an ndv deletion mutant on a trans - complementing cell line in order to abrogate expression of the m protein of ndv , a large part of the m gene was deleted by digestion of pndfl +[ f wt ] with bsaai ( nt 3087 ) followed by partial digestion with hindiii ( nt 4252 ). after filling in the hindiii end with klenow dna polymerase , the fragment was recircularized by using t4 dna ligase and used to transform e . coli . the resulting plasmid , designated pndfl +[ f wt ] dm , was used to generate virus by means of the co - transfection system in trans - complementing cer - m cells that expressed the ndv m protein . the supernatant of transfected monolayers was passaged three times on cer - m cells and analyzed for the presence of virus . virus was obtained as evidenced by the fact that the culture supernatant of the third passage yielded positive results in hemagglutination ( ha ) and hemagglutination - inhibition ( hi ) tests . the virus was designated ndfl - dm . when ndfl - dm was used to infect monolayers of cef cells , the virus was still able to spread by cell - to - cell transmission as seen in an ipma by using a monoclonal antibody against the f protein . as expected , expression of the m protein could not be demonstrated in an ipma by using monoclonal antibodies against the m protein . when the supernatant was used to infect either cef cells or cer - m cells , we were unable to show the presence of replicating virus in these monolayers by means of ipma . this finding indicates that infectious virus could not be generated in non - complementing cef cells . this finding was confirmed by the observation that inoculation of embryonated eggs with supernatant from infected cef cells did not result in the generation of progeny virus when tested in ha or hi tests . the need for better ndv vaccines , and especially the need for ndv marker vaccines , prompted us to develop a reverse genetics system which would allow the genetic modification of ndv . in this document we describe the generation of infectious ndv entirely from cloned full - length cdna . we show that the virulence of ndv can be dramatically changed by modifying only 3 nucleotides which determine the specificity of the protease cleavage site of the f protein . in this case the protease cleavage site was changed from that of the lasota strain to that of the consensus cleavage site of virulent ndv strains . by generating this genetically modified ndv strain we deliver the formal proof that the cleavability of the f protein is the key determinant ( but not the only determinant ) for virulence of ndv . by using the same reverse genetics approach , the cleavage site can be modified , at will , to any other amino acid sequence . this may lead to the generation of a series of ndv strains which display a spectrum of virulence levels . as previously mentioned , it has been shown that , besides the cleavability of the f and hn proteins , other viral factors may contribute to pathogenicity . alterations in transcription and translation can modulate growth and cell - to - cell spread of the virus and / or cytopathogenicity . the availability of an infectious cdna of ndv allows for the systematic modification of sequences which are involved in transcription and replication . this may lead to the design of new ndv vaccines which sport optimal immunogenicity to virtually non - existing virulence . safety is one of the most important properties of live vaccines . however , for many live vaccines , including ndv , immunogenicity is often inversely related to virulence . therefore , further attenuation of live vaccines without losing immunogenicity is one of the most desired alterations for which genetic modification could be used . in this respect it is worthwhile mentioning that it has been shown that elimination of expression of the v protein of sendai virus resulted in a markedly reduced in vivo pathogenicity for mice ( kato et al ., 1997 ). similar to sendai virus , ndv also generates a v protein by a mechanism known as rna editing ( steward et al ., 1993 ). it is predictable that elimination of expression of the v protein of ndv may also result in an attenuated phenotype in vivo . apart from changing the virulence of ndv , we show that it is possible to modify the antigenic make - up of ndv in such a way that strains can be generated which can be serologically discriminated from ndv field strains . these , so called , marker vaccines are an invaluable tool to assess the prevalence of ndv in commercial flocks around the world . furthermore , the large - scale application of such marker vaccines may ultimately lead to the complete eradication of ndv by a process of intensive screening and stamping out of infected flocks . in this document we show that foreign genes can be inserted into the genome of ndv . these foreign genes can be expressed to very high levels in infected cells . this shows that ndv can be used as a vaccine vector for the expression of antigens from other ( poultry ) pathogens . several properties make ndv an ideal vaccine vector for vaccination against respiratory or intestinal diseases . 1 ) ndv can be easily cultured to very high titers in embryonated eggs . 2 ) mass culture of ndv in embryonated eggs is relatively cheap . 3 ) ndv vaccines are relatively stable and can be simply administered by mass application methods such as addition to drinking water or by spraying or aerosol formation . 4 ) the natural route of infection of ndv is by the respiratory and / or intestinal tracts which are also the major natural routes of infection of many other poultry pathogens . 5 ) ndv can induce local immunity despite the presence of circulating maternal antibody . finally , we show that viable ndv deletion mutants can be generated by using trans - complementing cell lines . an ndv deletion mutant was generated which is unable to express the matrix ( m ) protein which is involved in budding of ndv at the inner cell membrane . we show that a phenotypically complemented ndv strain that is unable to express the m protein is still able to infect cells and spread by means of cell - to - cell transmission . however , the mutant virus is unable to generate infectious progeny on non - complementing cells . this finding shows that phenotypically complemented ndv deletion mutants may be used as safe self - restricted vaccines which are unable to spread into the environment . such a non - transmissible vaccine combines the most important advantage of live vaccines , i . e ., efficacy , with the most important advantage of killed vaccines , i . e ., safety . [ 0221 ] table 2 sequence of 3 ′- and 5 ′- terminal ends of the genome of ndv strain la sota a . sequence of 3 ′- terminal end ( shown as 5 ′ end of antigenomic dna strand ) method i . clone sequence 04 accaaacagagaatc ( seq id no : 80 ) 05 accaaacagagaatc ( seq id no : 80 ) 13 accaaacagagaatc ( seq id no : 80 ) 21 accaaacagagaatc ( seq id no : 80 ) method ii . clone sequence 26 accaaacagagaatc ( seq id no : 80 ) 28 accaaacagagaatc ( seq id no : 80 ) 30 accaaacagagaatc ( seq id no : 80 ) 31 gccaaacagagaatc ( seq id no : 81 ) 32 accaaacagagaatc ( seq id no : 80 ) 33 accaaacagagaatc ( seq id no : 80 ) consensus accaaacagagaatc ( seq id no : 80 ) b . sequence of 5 ′- terminal end ( shown as dna ) pbluescriptii - tsk clones clone sequence r3101 - 13 accaaacaaagattt ( seq id no : 82 ) r3101 - 14 accaaacaaagattt ( seq id no : 82 ) r3101 - 15 accaaacaaagattt ( seq id no : 82 ) r2601 - 17 accaaacaaagattt ( seq id no : 82 ) r2601 - 18 accaaacaaagattt ( seq id no : 82 ) r2601 - 19 accaaacaaagattt ( seq id no : 82 ) r2601 - 20 aacaaggtgaagata ( seq id no : 149 ) r2601 - 21 accaaacaaagattt ( seq id no : 82 ) pgem4z clones clone sequence r3101 - 16 accaaacaaagattt ( seq id no : 82 ) r3101 - 17 accaaacaaagattt ( seq id no : 82 ) r3101 - 18 accaaacaaagattt ( seq id no : 82 ) r3101 - 19 accaaacaaagattt ( seq id no : 82 ) r3101 - 22 accaaacaaagattt ( seq id no : 82 ) consensus accaaacaaagattt ( seq id no : 82 ) [ 0222 ] table 3 minigenome replication by ndv helper virus plasmid + ndv − ndv ratio a . seap activity ( cps ) after transfection of cer - c9 cells with the poltv535 and poltv553 - series of plasmids . poltv535n0 3 . 5 × 10 4 7 . 1 × 10 4 0 . 49 poltv535n1 5 . 9 12 . 1 0 . 49 poltv535n2 2 . 4 6 . 2 0 . 39 poltv535n3 7 . 6 5 . 2 1 . 46 poltv535n4 1 . 8 4 . 1 0 . 44 poltv535n5 1 . 5 3 . 0 0 . 50 poltv553n0 5 . 5 × 10 3 9 . 6 × 10 3 0 . 57 poltv553n1 9 . 6 27 . 6 0 . 35 poltv553n2 2 . 4 3 . 5 0 . 68 poltv553n3 15 . 1 9 . 5 1 . 59 poltv553n4 3 . 4 7 . 9 0 . 43 poltv553n5 2 . 9 4 . 8 0 . 60 b . seap activity ( cps ) after transfection of fpv - t7 infected cer cells with the poltv553 - series of plasmids . poltv553n0 7 . 2 × 10 4 8 . 3 × 10 4 0 . 86 poltv553n1 8 . 4 12 . 0 0 . 70 poltv553n2 8 . 9 12 . 6 0 . 71 poltv553n3 27 . 4 8 . 6 3 . 19 poltv553n4 9 . 7 10 . 4 0 . 93 poltv553n5 8 . 5 8 . 1 1 . 05 [ 0223 ] table 4 transfer of seap activity ( cps ) after treatment of cer cells with the supernatant of fpv - t7 infected cer cells which had been transfected with the poltv553 - series of plasmids and which had been superinfected with ndv ( see table 3 ). plasmid poltv553n0 2 . 4 × 10 3 poltv553n1 6 . 2 poltv553n2 2 . 0 poltv553n3 20 . 6 poltv553n4 2 . 0 poltv553n5 2 . 1 [ 0224 ] table 5 seap activity ( cps ) after co - transfection of cer cells with the poltv553 - series of plasmids and plasmids pcineonp , pcineop and pcineol ( c ) ( or pcineo as a negative control ). plasmid ratio np , p & amp ; l np , p & amp ; pcineo poltv553n0 3 . 1 × 10 4 2 . 7 × 10 3 11 . 7 poltv553n1 4 . 1 5 . 2 7 . 9 poltv553n2 3 . 1 3 . 1 10 . 0 poltv553n3 35 . 9 3 . 6 100 . 8 poltv553n4 1 . 9 4 . 6 4 . 1 poltv553n5 1 . 0 4 . 1 2 . 5 alexander , d . j . 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