Patent Application: US-71453403-A

Abstract:
the invention relates to the field of coronaviruses and diagnosis , therapeutic use , and vaccines derived therefrom . the invention provides replicative coronaviruses and virus - like particles from which large parts of their genome are deleted without abolishing their replicative capacities . the deletion preferably results in at least a functional deletion in that the corresponding gene is not or is only partly expressed wherein the resulting gene product is dysfunctional or at least functionally distinct from a corresponding wild - type gene product . one result seen with vlps provided with deletions as provided herein is that the deleted vlp , albeit capable of replication in vitro and in vivo , are generally well attenuated , in that they do not cause disease in the target host , making them very suitable for therapeutic use , such as a delivery vehicle for genes and other cargo , and for use as a vaccine , being attenuated while carrying important immunogenic determinants that help elicit an immune response .

Description:
general aim : establish whether deletion from the coronaviral ( i . e . mhv - a59 ) genome of genes or gene clusters not belonging to the genes specifying the polymerase functions ( orf1a / 1b ) or the structural proteins n , m , e , and s , is tolerated and yields viable viruses even if these gene sequences are removed altogether ; establish whether such deletions have an attenuating effect on the virus when inoculated into mice . specific aim : generate mhv - a59 deletion mutants lacking genes 2a + he ( mhv -□ δ2ahe ) genes 4a + 4b + 5a ( mhv -□ δ45a ), and genes 2a + he + 4a + 4b + 5a ( mhv - min ). approach : targeted rna recombination ( 3 , 9 , 10 ) using fmhv , the mhv - a59 derivative infecting feline ( fcwf ) cells not murine ( lr7 ) cells ( 7 ), and synthetic donor rnas carrying the intended deletions ( fig1 a , top ). procedure : transcription vectors for the production of synthetic donor rnas were constructed from the plasmid pmh54 ( 7 ), which encodes a run - off transcript consisting of the 5 ′ end of the mhv - a59 genome ( 467 nt ) fused to codon 28 of the he gene and running to the 3 ′ end of the genome ( fig1 ). plasmid pmh54 was used to reconstruct the recombinant wt - mhv , an fmhv derivative again infecting murine cells . transcription vector pxh □ δ45a lacks the orfs 4a , 4b , and 5a . for the construction of this plasmid a pcr product was obtained from plasmid pb59 ( 2 ) by using primer 1089 ( 5 ′- acctgcaggactaatctaaactttattctttttagggccacga - 3 ′ ( seq id no : _ ), which encodes a psti / sse8387i restriction site , an intergenic sequence ( igs ) and which is complementary to the sequence just upstream of the e or 5b gene , and primer 1092 ( 5 ′- ccttaaggaattgaactgc - 3 ′ ( seq id no : _ ) which is complementary to the 5 ′ end of the m protein coding region . the pcr product was cloned into pgem - t easy ( promega ) according to the manufacturer &# 39 ; s instructions , yielding pxh0803 . the pcr product was subsequently excised with psti and ecorv and cloned into pmh54 treated with sse8387i and ecorv , resulting in pxh □ δ45a . transcription vector pxh □ δ2ahe lacks orfs 2a and he and contains approximately 1200 bp of the 3 ′ end of the polymerase gene fused to the s gene . to construct this plasmid a pcr product was obtained by splicing overlap extension pcr . one pcr product was obtained from plasmid p96 ( 1 ) using primer 1128 ( 5 ′- acggtccgactgcgcgcttgaacacgttg - 3 ′ ( seq id no : _ ) which encodes a rsrii restriction site and is complementary to the region 1200 bp upstream of the orf1b stop codon , and primer 1130 ( 5 ′- catgcaagctttatttgacatttactaggct - 3 ′ ( seq id no : _ ) which is complementary to the 3 ′ end of the polymerase coding region and the igs region upstream of the s gene . the other pcr product was obtained from pmh54 using primer 1129 ( 5 ′- gtcaaataaagcttgcatgaggcataatctaaac - 3 ′) ( seq id no : _ ) which is complementary to primer 1130 , and primer 1127 ( 5 ′- ccagtaagcaataatgtgg - 3 ′) ( seq id no : _ ) which is complementary to the 5 ′ end of the s gene . the pcr products were purified and mixed and then amplified with primers 1128 and 1127 . the pcr product obtained in the second round of pcr was cloned into pgem - t easy , yielding pxh1802 . the pcr product was excised with rsrii and avrii and cloned into pmh54 treated with the same enzymes , resulting in pxhδ □ 2ahe . transcription vector pxhmin has the orf1b 3 ′ end fused to the s gene and the deletion of orf4a , 4b and 5a . this vector was constructed by cloning the fragment excised with psti and ecorv from pxh0803 into pxh □ 2ahe treated with sse83871 and ecorv . the composition of all pcr - generated segments was confirmed by dna sequencing . to generate the deletion mutant viruses , donor rnas were transcribed from the ( paci - linearized ) pmh54 - derived plasmids and transfected by electroporation into feline fcwf cells that had been infected with fmhv . these infected and transfected cells were then plated onto a monolayer of mouse lr7 ( 7 ) cells . after 24 h of incubation at 37 ° c ., progeny viruses were harvested by taking off the cell culture supernatant and candidate recombinants were selected by two rounds of plaque purification on lr7 cells . result : with each of the synthetic donor rnas used , clear plaques were obtained . conclusion : recombinant viruses had been obtained that had regained the ability to grow on murine cells . aim : confirming by rt - pcr the genetic make - up of the recombinant viruses obtained . procedure and results : cloned recombinant viruses , one from each recombination experiment , were produced on lr7 cells , viral rna was isolated and rt - pcr was done using standard methods on genomic rna as shown in fig2 . to confirm the deletion of the orfs 4 and 5a , the rt step was performed with primer 1092 ( 5 ′- ccttaaggaattgaactgc - 3 ′), which is complementary to the 5 ′ end of the m gene , while the pcr was performed with primer 1261 ( 5 ′- gctgcttactcctatcatac - 3 ′) ( seq id no : _ ) and primer 990 ( 5 ′- cctgatttatctctcgatttc - 3 ′) ( seq id no : _ ) which are complementary with the 3 ′ end of the e and s gene , respectively . in the case of the recombinant mhv - wt an rt - pcr product corresponding in size with the expected 1328 bp was observed ( fig2 , top ). as expected , a pcr product of the same length was observed for mhv - δ □ 2ahe . in contrast , both for mhv -□ δ45a and mhv - min much smaller rt - pcr products were detected . the smaller size of the rt - pcr products corresponded with the deletion of 736 bp . the deletion of orfs 2a and he was analyzed in a similar way . the rt step was performed with primer 1127 ( 5 ′- ccagtaagcaataatgtgg - 3 ′) which is complementary to the 5 ′ end of the s gene . the pcr was performed with primer 1173 ( 5 ′- gacttagtcctctccttga - 3 ′) ( seq id no : _ ) and primer 1260 ( 5 ′- cttcaacggtctcagtgc - 3 ′) ( seq id no : _ ), which are complementary to the 3 ′ end of the 1b gene and the 5 ′ end of the s gene , respectively . both for mhv - wt and mhv -□ δ45a pcr products were detected , which were much bigger than the pcr products detected for mhv - δ2ahe and mhv - min ( fig2 , bottom ). the difference in size corresponded with the deletion of 2164 bp . finally , the newly generated junctions , present in the genomes of the deletion mutant viruses ( fig1 a [ triangles ] and 1b [ sequence ]), were analyzed by sequencing of the rt - pcr products . to this end the pcr products were cloned into the pgem - t easy vector ( promega ). the sequences obtained were in perfect agreement with the predictions . aim : confirming the patterns of rnas synthesized in cells infected by the mutant viruses . procedure : infected 17cl1 cells were metabolically labeled with [ 33 p ] orthophosphate in the presence of actinomycin d essentially as described ( 4 , 10 ). samples of total cytoplasmic rna , purified using ultraspec reagent ( biotecx ), were denatured with formaldehyde and formamide , separated by electrophoresis through 1 % agarose containing formaldehyde , and visualized by fluorography ( fig3 ; note that the subgenomic rna species in this figure are designated by their composition , rather than as rna2 through rna7 , since the numerical designations would be ambiguous for the deletion mutants ). result : for the recombinant mhv - wt the rna pattern and the relative molar amounts of the six subgenomic ( sg ) rna species and the genomic ( g ) rna were very similar to those reported previously for mhv ( 10 )( 4 )( 5 )( 8 ), with one notable exception . the 4 - 5a / e - m - n sgrna , which is usually denoted rna4 , was far more abundant than previously observed for this species in wild - type mhv ( 10 )( 4 )( 5 )( 8 ). this was presumably due to the three nucleotide changes at positions 13 , 15 , and 18 upstream of the consensus transcription regulatory signal , ( 5 ′ aaucuaaac3 ′) that precedes gene 4 ( fig1 ) which were introduced into the transcription vector pmh54 to create the sse8387i site downstream of the s gene ( 7 ). for the deletion mutants , all variant sgrnas had mobilities that corresponded to their predicted sizes ( fig3 and table 3 ), and no prominent extra species were observed . the relative molar amounts of the mutant sgrna species were quite similar to those of their wild - type counterparts originating from the corresponding transcription regulatory signals . conclusion : the results confirm the genotypes of the recombinant viruses and demonstrate their expected phenotypes at the rna level . procedure and results : confluent lr7 cell monolayers grown in 35 - mm dishes were infected with each recombinant virus ( 8 pfu / cell ) and viral infectivity in culture media at different times post - infection ( p . i .) was determined by titration on lr7 cells . tcid 50 ( 50 % tissue culture infective doses ) values were calculated and plotted ( fig4 ). the recombinant viruses did not differ appreciably with respect to the induction of extensive syncytia or cytopathic effects or in their plaque size . however , mhv -□ δ45a and mhv - min differed from mhv - wt and mhv -□ δ2ahe in their one - step growth kinetics ( fig4 ). the two viruses displayed approximately 10 - fold lower titers at all time points . conclusion : all deletion viruses multiply well in vitro although the deletion of genes 4 and 5a had a slightly negative effect . procedure and results : the recombinant viruses were characterized in their natural host , the mouse . as a first step , we determined the virulence of the recombinant viruses . an ld 50 ( 50 % lethal dose ) assay was carried out by inoculating mhv - negative , c57b1 / 6 mice mice intracranially with four 10 - fold serial dilutions ( 5 × 10 5 - 5 × 10 2 ) of recombinant viruses . viruses were diluted using pbs containing 0 . 75 % bovine serum albumin . a volume of 25 μl was used for injection into the left cerebral hemisphere . five animals per dilution per virus were analyzed . ld 50 values were calculated by the reed - muench method based on death by 21 days post - infection . clearly , deletion mutant viruses were attenuated when compared to the recombinant mhv - wt . while the mhv - wt virus had an ld50 of 1 . 8 × 10 4 no ld 50 could be derived for the deletion mutants . although the animals inoculated with the higher doses showed some signs of illness , none of the animals infected with any of the deletion mutant viruses died up to input of 50 , 000 pfu / mouse . this implies that the ld 50 for these viruses exceeds a value of 50 , 000 and may well be above 100 , 000 . fig2 illustrates the kinetics of mortality of mice infected with the highest inoculation dose , 2 . 5 × 10 5 plaque forming units ( pfu ), of wt and deletion viruses . while all the animals inoculated with wild type virus had died by seven days post infection , the deletion viruses were highly attenuated , displaying no death and less severe clinical symptoms . despite the observation of no mortality , all mice infected with the □ 45a and □ 2ahe viruses showed clinical signs of hunched posture , disheveled appearance and waddling gait during the first week post infection ; these symptoms were less severe and observed in fewer mice infected with mhv - min . conclusion : viruses with deletions of the sequences specifying the genes 2a + he or genes 4a + 4b + 5a or of the combination of all these genes exhibit a significantly attenuated phenotype in mice . in other words , the non - essential genes of coronaviruses are not crucial for in vitro growth but determine viral virulence . the attenuation acquired by their deletion thus provides excellent viral vaccines and therapeutic vectors . general aim : establish whether the invariable order of the genes specifying the polymerase functions ( orf1a / 1b ) and the structural proteins s , e , m , and n in the coronaviral genome is essential for the viability of these viruses or whether rearrangement of this order is tolerated . specific aim : generate mhv - a59 mutants in which the relative positions of structural protein genes in the mhv - a59 genome are changed by moving the m and / or e gene . approach : targeted rna recombination using fmhv and synthetic donor rnas carrying the intended rearrangements ( fig5 , top ). procedure : transcription vectors for the production of donor rna for targeted recombination were constructed from plasmids pmh54 and pxh □ 2ahe ( described above ). in order to generate transcription vector pxhsm45n ( fig5 , lower left part ), a pcr product was generated by splicing overlap extension ( soe )- pcr that contained the 3 ′ end of the m gene and the 5 ′ end of the n gene and in which a ecorv restriction site was introduced between the m gene and igs just upstream of the n gene . to generate this pcr fragment , outside primer 1c ( 5 ′- gtgtatagatatgaaaggtaccgtg - 3 ′) ( seq id no : _corresponding to the region of the m gene that contains the unique kpni site , and outside primer 1097 ( 5 ′- cgaaccagatcggctagcag - 3 ′) ( seq id no : _ , corresponding to the region of the n gene that contains the unique nhei site , were used . primer 1095 ( 5 ′- agattagatatcttaggttctcaacaatgcgg - 3 ′) ( seq id no : _and primer 1096 ( 5 ′- gaacctaagatatctaatctaaactttaaggatg - 3 ′) ( seq id no : _ ) were used as inside primers . they correspond to the sequence between the m and the n gene and introduce the ecorv restriction site . the resulting pcr product was cloned into pgem - t easy ( promega ) yielding vector pxh0302 . as a next step in the construction of pxhsm45n , pmh54 was treated with the restriction enzymes sse83871 and ecorv and the resulting fragment was cloned into the ecorv site of pxh0302 after being blunted by t4 dna polymerase treatment , yielding vector pxh0902 . after excision of the sse8387i - ecorv fragment of pmh54 , the remaining vector was also blunted by t4 dna polymerase treatment and religated resulting in plasmid pxh1401 . finally , plasmid pxh0902 was treated with restriction enzymes nhei and bsshii and the resulting fragment was cloned into pxh1401 treated with the same enzymes , yielding pxhsm45n . for the construction of pxhmsmn , first the orfs 4 , 5 and m were removed from pmh54 by restriction of this vector with enzymes sse83871 and bsshii , followed by treatment with t4 dna polymerase and religation of the remaining vector , which yielded pxh □ 45m5 ′. next , the fragment resulting from treatment of pxh0302 with enzymes nhei and bsshii was cloned into pmh54 treated with the same enzymes , resulting in pxhmen . subsequently , the fragment obtained after restriction of pxhmen with ecorv was cloned into pxh1802 ( described above ), which was digested with hindiii and treated with klenow fragment of dna polymerase i , yielding pxh0305b . the fragment obtained by restriction of pmh54 with enzymes mlui and ecorv was cloned into pb59 ( 2 ) treated with the same enzymes , which resulted in vector pxh2801 . next , the fragment resulting from the treatment of pxh2801 with restriction enzymes kpni and psti was treated with t4 dna polymerase and cloned into pxh1802 treated with restriction enzyme hindiii and with klenow fragment of dna polymerase i , yielding pxh0806 . subsequently , the fragment obtained by digestion of pxh0305b with spei and aflii was cloned into pxh0806 treated with the same enzymes , resulting in pxh1506 . finally , pxhsmn was obtained by cloning the fragment resulting from restriction of pxh1506 with rsrii and avrii into pxh □ 45m5 ′ treated with the same enzymes . for the construction of transcription vector pxh1bms , vector pxhmen was restricted with ecorv . the resulting fragment was removed and the vector was religated yielding pxh □ m . next , the fragment obtained by digestion of pxh0305b with rsrii and avrii was cloned into pxh □ m treated with the same enzymes , yielding pxh1bms . all constructs were confirmed by restriction and / or sequence analysis . they are depicted schematically in fig5 ( left ). all new junctions generated , including the introduction of the sse8387i site downstream of the s gene in pmh54 ( 7 ), are indicated with arrowheads , while their sequences are shown in table 1 . recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the fmhv genome as described above . after 2 rounds of plaque purification on lr7 cells the viruses were analyzed by reverse transcriptase - pcr on genomic rna and found to contain the genomes with the expected organization . conclusion : the strict gene order of the coronaviruses is not an essential prerequisite for viability . aim : confirming the patterns of rnas synthesized in cells infected by the mutant viruses . procedure : infected 17cl1 cells were metabolically labeled with [ 33 p } orthophosphate in the presence of actinomycin d essentially as described ( 4 , 10 ). samples of total cytoplasmic rna , purified using ultraspec reagent ( biotecx ), were denatured with formaldehyde and formamide , separated by electrophoresis through 1 % agarose containing formaldehyde , and visualized by fluorography ( fig2 a ). result : coronaviruses express their genome via the generation of a 3 ′ co - terminal nested set of sg rnas . recombinant viruses with a rearranged genome organization are therefore predicted to synthesize patterns of viral rnas that are distinctly different from that of the parent virus . for the reconstructed wild - type virus ( mhv - wt ) and for mhv -□ 2ahe , the rna patterns and the amounts of the genomic ( g ) and sg rna species appeared to be similar to those observed previously ( fig3 ). note that mhv -□ 2ahe — as well as its derivatives mhv - msmn and mhv - 1bms — does not synthesize the sg rna species encoding the 2a protein . for the mhv mutants with the rearranged genomes , all variant sg rnas had mobilities that corresponded to their predicted sizes ( fig2 a and table 4 ), and no obvious additional species were observed . mhv - sm45n grew very poorly , and was therefore labeled only weakly . all sg rnas could be detected except the one from which the m protein should be translated . the low abundance of this sg rna , the reason of which is unknown , is likely to be the cause of the impaired growth of this virus . overall , the patterns of viral rnas synthesized by the cells infected with the recombinant viruses nicely reflect the changes made to the coronavirus genome organization . conclusion : the results confirm the genotypes of the recombinant viruses and demonstrate their expected phenotypes at the rna level . procedure and results : confluent lr7 cell monolayers grown in 35 - mm dishes were infected with each recombinant virus ( 8 pfu / cell ) and viral infectivity in culture media at different times post - infection was determined by titration on lr7 cells . tcid 50 values were calculated and plotted ( fig6 ). all viruses except the mutant mhv - sm45n were analyzed in this way . the infectious titer of this latter virus was too low ( approximately 1000 times lower than the wt recombinant mhv ) to perform a one - step growth curve . although mhv - 1bms appeared to induce syncytia somewhat slower than the wt recombinant , all viruses replicated approximately to the same extent in the one - step growth curve . conclusion : except for mutant virus mhv - sm45n , the gene rearrangement had no dramatic effect on their in vitro growth characteristics . aim : establishing whether viruses with rearranged gene order are able to replicate in mice . procedure and results : eight weeks old , mhv - negative , female balb / c mice were used in the experiment . viruses were diluted in pbs and a total volume 100 μl ( 10 6 tcid 50 ) was used for injection in the peritoneal cavity . four animals per virus were inoculated . mice were sacrificed and the livers were removed at day 4 post - infection . the livers were placed in 1 . 5 ml dmem , weighed and then frozen at − 80 ° c . until titred for virus . virus titers were determined by plaque - assay on lr7 cell monolayers following homogenization of the organs . the replication of mhv - wt , mhv -□ 2ahe and mhv - msmn was studied in their natural host , the mouse . while the 50 % lethal dose of mhv - wt in mice was previously determined at 2 . 7 × 10 4 pfu , mhv -□ 2ahe was not virulent enough for a 50 % lethal dose value determination ( section i . 1 . e .). therefore , we now decided to analyze the in vivo replication of the recombinant viruses . mice inoculated intraperitoneally with 1 × 10 6 tcid 50 were euthanized at day 4 post - infection and the viral replication in the liver was determined . the results are shown in fig2 b . mhv - u □ 2ahe and mhv - msmn replicated in the liver to a similar extent albeit much lower than mhv - wt . deletion of orfs 2a and he generated a recombinant virus ( mhv -□ 2ahe ) that was attenuated in the natural host , as shown in section i . 1 . e ., while additional rearrangement of the coronavirus gene order ( mhv - msmn ) did not result in a more attenuated phenotype in this assay . conclusion : viruses with a rearranged gene order , which lack the typical coronavirus genome organization , and viruses that lack orfs 2a and he are able to replicate in their natural host , the mouse . aim : establish whether foreign genes can be inserted at different positions in the viral genome , either as an additional gene or replacing deleted non - essential genes or in combination with a rearranged gene order ; establish whether these genes are expressed and whether they are stably maintained during in vitro passage of the virus . procedure : several viruses were constructed containing a foreign reporter gene in their genome at different positions ( see fig7 a ). two reporter genes were used , encoding renilla luciferase ( rl ) and firefly luciferase ( fl ). for both genes , a plasmid was constructed in which the gene was preceded by the mhv intergenic sequence ( igs ). from this construct the expression cassette ( gene plus igs ) could then be transferred into the different transcription vectors . as a first step , the mhv igs was cloned in front of the rl gene . to this end , primer 1286 ( 5 ′- ggatactaatctaaactttag - 3 ′) ( seq id no : _ ) and 1287 ( 5 ′- ctagctaaagtttagattagatatcctgca - 3 ′) ( seq id no : _were annealed to each other and cloned into prl - null ( promega ) treated with nhei and psti , resulting in pxh1909 . for the construction of the transcription vector containing the rl gene between genes 2a and s ( pxh22arls ) the following steps were taken . vector p 96 ( 1 ) was restricted with hindiii and mlui and the resulting fragment was cloned into pxh1802 ( described above ) treated with hindiii and bsshii , yielding pxh2103 . next , the rl expression cassette was removed from pxh1909 by restriction with ecorv and xbai , treated with klenow fragment of dna polymerase i and cloned into pxh2103 digested with hindiii and treated with klenow fragment , resulting in pxh2509a . finally pxh22arls was constructed by cloning the fragment resulting from digestion of pxh2509a with rsrii and avrii into pmh54 treated with the same enzymes . for the construction of pxh2erlm the same expression cassette , that was used to make pxh2509a , was cloned into pmh54 digested with ecorv . this same expression cassette was also cloned into pxhmen ( described above ) treated with ecorv , yielding pxh2erln . subsequently , pxh2mrln was constructed by cloning the fragment resulting from restriction of pxhmen with ecorv into pxh2erln treated with the same enzyme . pxhmsmnrl was constructed by cloning the renilla expression cassette into pxhmsmn ( described above ) treated with ecorv . for the construction of pxheflm , the fl gene was first cloned behind the same igs as was used for the rl expression cassette . to this end , the luciferase gene was removed from psp - luc +( promega ) by restriction with avrii and xbai and cloned into pxh1909 treated with nhei and xbai , yielding pxh2711 . subsequently , the fl expression cassette was cut out of pxh2711 by restriction with ecorv and xbai , treated with klenow fragment of dna polymerase i , and cloned into pmh54 restricted by ecorv , resulting in pxheflm . plasmid pxhminfl was constructed by cloning the fl expression cassette into pxhmin ( described above ) digested with ecorv . this plasmid lacks orfs 2a / he / 4a / 4b / 5a and contains the fl gene between genes e and m . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the fmhv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . results of recombinant viruses containing the rl gene are shown in fig7 b and 27 . to confirm the insertion of this gene an rt step was performed on genomic rna with primer 1412 ( 5 ′- ctgcggaccagttatcatc - 3 ′) ( seq id no : _ ) which is complementary to the 5 ′ end of the rl gene . subsequently , a pcr was performed with primer 1091 ( 5 ′- gttacaaacctgaatctcatcttaattctggtcg - 3 ′) ( seq id no : _ ) and primer 1413 ( 5 ′- catccgtttcctttgttctgg - 3 ′) ( seq id no : _ ) for mhv - erlm and mhv - mrln or with primer 1173 ( 5 ′- gacttagtcctctccttgattg3 ′) ( seq id no : _ ) and primer 1413 for mhv - 2arls . primer 1091 and primer 1173 correspond with the 3 ′ end of gene 4b and gene 1b , respectively , while primer 1413 corresponds with the 5 ′ end of the rl gene . as positive controls , the appropriate transcription vectors were taken along . in all cases , pcr fragments were obtained of the same size as the positive controls , while the water control was negative . the observed ( and predicted ) fragment sizes were approx . 1 , 000 bp for mhv - 2arls , approx . 670 bp for mbv - erlm , and approx . 1 , 300 bp for mhv - mrln ( 7b ). for mhv - msmnrl a pcr was performed with primer 1091 and primer 1413 and with primer 1173 and primer 1413 . as positive controls , the appropriate transcription vector was taken along . in all cases , pcr fragments were obtained of the same size as the positive controls . the observed ( and predicted ) fragment sizes were approx . 670 bp for the pcr reaction with primers 1091 and 1413 , and approx . 1 , 200 bp for the pcr reaction with primers 1173 and 1413 ( 7b ) ( note that in fig7 b and 27 for each type of virus 2 independently obtained viral clones were analyzed and included ). the results confirmed the insertion of the rl gene into the mhv genome at the correct position . results of recombinant viruses containing the fl gene are shown in fig2 . to confirm the insertion of this gene an rt step was performed on genomic rna with primer 1475 ( 5 ′- gcctaatgcagttgctctcc - 3 ′) ( seq id no : _ ) which is complementary to the 5 ′ end of the fl gene . subsequently , a pcr was performed with primer 935 ( 5 ′- gttttagcacagggtgtggctcatg - 3 ′) ( seq id no : _ ), which corresponds with the 3 ′ end of the s gene , and primer1474 ( 5 ′- ccatcttccagcggatag - 3 ′) ( seq id no : _ ), which corresponds with the 5 ′ end of the fl gene . as positive controls , the appropriate transcription vectors were taken along . in all cases , pcr fragments were obtained of the same size as the positive controls , while the water control was negative . the observed ( and predicted ) fragment sizes were approx . 1 , 200 bp for mhv - eflm , and approx . 500 bp for mhv - minfl ( note that in fig2 for each type of virus 2 independently obtained viral clones were analyzed and included ). conclusion : insertion of genetic modules into the coronaviral genome is tolerated at all positions tested ; all the intended viruses were viable . aim : confirming the patterns of rnas synthesized in cells infected by the mutant viruses . procedure : infected 17cl1 cells were metabolically labeled with [ 33 p ] orthophosphate in the presence of actinomycin d essentially as described ( 4 , 10 ). samples of total cytoplasmic rna , purified using ultraspec reagent ( biotecx ), were denatured with formaldehyde and formamide , separated by electrophoresis through 1 % agarose containing formaldehyde , and visualized by fluorography ( fig2 - 31 ; note that the subgenomic rna species in this figure are designated by their composition , rather than as rna2 through rna7 , since the numerical designations would be ambiguous for the mutants ). result : for all the mhv mutants with the luciferase genes , all variant sg rnas had mobilities that corresponded to their predicted sizes ( fig2 - 31 ). for the viruses containing the renilla luciferase gene no obvious additional species were observed . for the viruses containing the firefly luciferase gene two additional rna species were observed , which correspond in size with transcription of sgrnas from sequences in the firefly luciferase gene that are similar to the mhv igs . overall , the patterns of viral rnas synthesized by the cells infected with the recombinant viruses nicely reflect the insertion of the luciferase expression cassettes into the coronavirus genome . conclusion : the results confirm the genotypes of the recombinant viruses and demonstrate their expected phenotypes at the rna level . aim : compare the growth characteristics of the viruses with wild - type virus and with each other . procedure and results : after two rounds of plaque purification virus stocks were prepared , titrated and used for high m . o . i ( m . o . i . of 8 ) infection of lr7 cells after which the viral infectivities in the culture media were monitored . the results are represented by the growth curves shown in fig8 a and 8b . and by the results shown in fig3 . of mhv - eflm two viral clones , independently obtained from the recombination experiment described under i . 3 . a , were analyzed . in fig3 , the tcid 50 values obtained at 8 - 9 hours post - infection are shown . obviously , the growth characteristics of the recombinant viruses shown in fig8 a and 8b are essentially indistinguishable ; all viruses grew to titers that were comparable to that of recombinant wild - type virus . mhv - msmnrl reached somewhat lower titers ( fig3 ). conclusion : the inserted expression cassettes hardly affected the in vitro growth characteristics of the recombinant viruses . procedure and results : confluent monolayer cultures of lr7 cells were infected at a m . o . i . of 8 and the production of luciferase activity in the cells was monitored over time . rl expression in cells was measured by using the dual - luciferase reporter assay system ( promega ) according to the manufacturer &# 39 ; s instructions . similarly , fl expression was measured by using the luciferase assay system ( promega ) according to the manufacturer &# 39 ; s instructions . rl and fl activity was measured in relative light units ( rlu ) using a luminometer ( lumac biocounter m2500 or turner designs model td - 20 / 20 ). the results are shown graphically in fig9 a , 9b and 33 . all recombinant viruses except for the recombinant wild - type virus expressed high levels of luciferase , indicating that both the renilla and the firefly luciferase gene cassettes were functional at each genomic position tested . for most viruses the highest expression level was reached at 9 h post - infection . the expression level of firefly luciferase at this time - point was determined at 1 . 6 ug / 10 6 cells . mhv - minfl expressed levels of luciferase activity that were comparable to those produced by the other recombinant viruses containing the fl gene , while expression of the renilla luciferase gene in cells infected with mhv - msmnrl was approx . 10 - fold lower than in cells infected with mhv - erlm . this difference corresponds with the difference found in replication between mhv - msmnrl and mhv - erlm . conclusion : foreign genes can be expressed by coronaviruses both by the additional insertion of such a gene , by using the genetic space created by deletion of nonessential genes , or in combination with a rearranged genome organization . aim : evaluate the stability of the inserted luciferase genes during viral passage . procedure and results : the recombinant viruses mhv - erlm and mhv - eflm were passaged 8 times over lr7 cells at low m . o . i . (& lt ; 0 . 05 ). after each passage the viral infectivity ( tcid50 ) in the culture medium at 9 h post - infection was determined . subsequently , the firefly and renilla luciferase activity was determined in a parallel expression experiment ( m . o . i .= 5 ) at 8 hr post - infection ( fig1 ). both of mhv - erlm and of mhv - eflm two independently obtained clones a and b were analyzed . while the renilla luciferase expression was consistently stable for at least 8 passages , that of the firefly luciferase was stable only for 5 passages . after passage 5 of mhv - eflm , the expression level clearly decreased for both independent clones . after 8 passages 10 viral clones were isolated by plaque assay both of mhv - erlm and of mhv - eflm and tested for luciferase expression . while for mhv - erlm all 10 clones were positive , 9 of the mhv - eflm clones no longer showed clearly detectable luciferase expression . conclusion : a foreign gene can be stably maintained in the coronaviral genome for at least 8 passages in vitro . aim : establish whether the inserted luciferase gene is expressed in the natural host , the mouse . procedure and results : eight weeks old , mhv - negative , female balb / c mice were used in the experiment . mice were inoculated intranasally with 10 6 tcid 50 mhv - eflm . four animals per virus were used . mice were sacrificed and the livers and brains were removed at day 4 post - infection . organs were quick - frozen in liquid nitrogen and homogenized in cell culture lysis reagent provided with the luciferase assay system ( promega ). fl activity was measured according to the manufacturer &# 39 ; s instructions using a luminometer ( lumac biocounter m2500 ). clearly , as shown in fig3 , luciferase activity could be detected both in liver and brain . as an alternative way to evaluate whether the foreign gene was also expressed in vivo , i . e . in animals , a mouse was inoculated intraperitoneally with 10 6 tcid 50 mhv - eflm . four days later the mouse was sedated and luciferain was administered subcutaneously . the luciferase expression was evaluated 5 min later by real time recording of the emission of light from the body of the sedated mouse using a sensitive screen coupled to a ccd camera . light emanating from the liver area of the mouse was clearly observed . conclusion : a foreign gene can be expressed by coronaviruses in their natural host . i . 3 . g . generation of mhv expressing two foreign genes from one genome aim : establish whether two foreign genes can be expressed from a single genome . procedure and results : pxh2arlseflm was generated by cloning the fl expression cassette into pxh2arls digested with ecorv . after confirmation of the construct by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the fmhv genome as described above . the resulting virus , mhv - rlfl , was genetically confirmed by rt - pcr analysis . both renilla luciferase activity and firefly luciferase activity could be detected in individual plaques . after generation of a high titer stock , confluent monolayer cultures of lr7 cells were infected at a m . o . i . of 8 and the production of luciferase activity in the cells was monitored over time . rl expression in cells was measured by using the renilla assay system ( promega ) according to the manufacturer &# 39 ; s instructions . similarly , fl expression was measured by using the luciferase assay system ( promega ) according to the manufacturer &# 39 ; s instructions at 8 hr post - infection . rl and fl activity was measured in relative light units ( rlu ) using a luminometer ( lumac biocounter m2500 ). the results show that mhv - rlfl replicated to the same extent as mhv - 2arls and mhveflm ( fig4 a ). mhv - rlfl expressed both renilla luciferase and firefly luciferase , mhv - 2arls expressed renilla luciferase only , while mhv - eflm expressed firefly luciferase only ( fig4 b ). conclusion : two foreign genes can be expressed from a single coronavirus genome . aim : establish whether recombinant mhv can be generated that expresses and incorporates chimaeric spike - gfp proteins . procedure and results : the gfp gene was cloned in frame with the s gene in pmh54 . after confirmation of the construct by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the fmhv genome as described above . the resulting virus , mhv - sgfp , was genetically confirmed by rt - pcr analysis . plaques were microscopically analyzed and showed expressing of gfp as evidenced by the green fluorescence . immunoprecipitation analysis indicated that hybrid proteins were generated that could be precipitated with specific antibodies to mhv - s and gfp . conclusion : mhv can be generated that expresses a chimaeric s - gfp gene in stead of a wild - type s gene . this mutant virus is viable and could be propagated in cell culture indicating that these hybrid proteins are incorporated into the viral particle . i . 4 . inhibition of infection and of cell fusion by spike protein derived peptide general aim : inhibit coronaviral infection and the spread of an ongoing infection by interfering with membrane fusion using peptides . specific aim : produce a peptide constituting a sequence derived from the membrane - proximal heptad repeat region ( hr2 ) of the mhv - a59 s protein and demonstrate its inhibitory effect on mhv - a59 entry into lr7 cells and on cell - cell fusion in an infected culture of these cells . a pcr fragment from a template plasmid ptums ( 13 ) containing the mhv - a59 spike gene was obtained , corresponding to amino acid residues 1216 - 1254 ( hr2 ) of the s protein ( fig2 ). the forward primer used was : 5 ′ gcggatccatcgaaggtcgtgatttatctctcgatttc 3 ′. this primer introduced an upstream bamhi site and a sequence encoding a factor xa cleavage site immediately downstream the bamhi site into the amplified fragment . the reverse primer ( 5 ′ cgaattcattccttgaggttgatgtag 3 ′) ( seq id no : _ ) contained a downstream ecori site as well as a stop codon preceding the ecori site . the pcr fragment was cloned into the bamhi - ecori site of the pgex - 2t bacterial expression vector . freshly transformed bl21 cells ( novagen ) were grown in 2yt medium to log phase ( od 600 of 1 . 0 ) and subsequently expression was induced by adding iptg ( gibcobrl ) to a final concentration of 0 . 4 mm . two hours after the start of induction the cells were pelleted , resuspended in { fraction ( 1 / 25 )} of culture volume 10 mm tris ph ( 8 . 0 ), 10 mm edta , 1 mm pmsf and sonicated on ice ( 5 times for 2 min with 1 - min intervals ). cell lysates were centrifuged at 20 , 000 × g for 60 min at 4 ° c . then , 2 ml glutathione - sepharose 4b ( 50 % v / v in pbs ) was added per 50 ml of supernatant and the suspension was incubated overnight ( o / n ) at 4 ° c . under rotation . beads were washed three times with 50 ml pbs and resuspended in a final volume of 1 ml pbs . peptides were cleaved from the gst moiety on the beads using 20 u of thrombin by incubation for 4 hours at room temperature ( rt ). peptides in the supernatant were hplc purified on a phenyl column with a linear gradient of acetonitrile containing 0 . 1 % trifluoroacetic acid . peptide containing fractions were vacuum - dried o / n and dissolved in water . peptide concentration was determined by measuring the absorbance at a 280 nm or by bca protein analysis ( micro bca ™ assay kit , pierce ). the potency of the hr2 peptide in inhibiting viral infection was determined using the recombinant mhv - eflm expressing the firefly luciferase . confluent monolayers of lr7 cells in 96 wells plates were inoculated at 37 ° c . in dmem at a multiplicity of infection of 5 for 1 h in the presence of varying concentrations of peptide ranging from 0 . 4 - 50μm . after 1 h , cells were washed with dmem and medium was replaced by peptide - free dmem . at 5 h post infection cells were lysed for 15 minutes at rt in 50 μl lysis buffer , according to the manufacturer &# 39 ; s protocol ( luciferase assay system , promega ). upon mixing of 10 μl cell lysate with 40 μl substrate , luciferase activity was measured immediately using a wallace betaluminometer . the 50 % effective inhibitory concentration ( ec 50 value ) was calculated by fitting the inhibition data to an equilibrium binding equation : % luciferase activity = 100 /( 1 +( c / ec 50 ). the ability of the peptide hr2 to inhibit spike mediated cell - cell fusion was determined using a plaque assay . monolayers of lr7 cells in 6 wells plates were inoculated with 50 pfu of mhv - a59 in dmem at 37 ° c . after one hour the cells were washed with dmem and an agar overlay was added containing the hr2 peptide at 50 , 10 , 2 , 0 . 4 and 0 . 08 μm concentrations . plaques were counted at 48 h p . i ., after staining and fixing with 0 . 9 % formaldehyde / 0 . 75 % crystal violet . results : the potency of the hr2 peptide to inhibit virus entry was tested using a virus expressing a luciferase reporter gene as this allows extremely sensitive detection of infection . inoculations of cells with the virus were carried out in the presence of different concentrations of hr2 peptide . after 1 h of inoculation , cells were washed and incubated further in culture medium in the absence of peptide . at 4 h p . i ., before syncytium formation normally takes place , cells were lysed and tested for luciferase activity ( fig1 ). in this figure the normalized luciferase activity , representing the success of infection , was plotted against the peptide concentration present during inoculation . the hr2 peptide blocked viral entry very efficiently , infection being inhibited virtually completely at the concentration of 50 μm . the effective concentration ( ec 50 ) at which 50 % of viral infection was inhibited was 0 . 15 μm . the ability of the hr2 peptide to inhibit cell - cell fusion mediated by the spike protein was examined by using a plaque assay . after inoculation of ( parallel cultures of ) cells in the absence of peptide , an overlay was applied containing different concentrations of hr2 peptide . the formation of plaques appeared to be completely abolished in the presence of hr2 peptide concentrations of up to 0 . 4 μm ( fig1 ). at 0 . 08 μm of the hr2 peptide only tiny plaques could be observed . the specificity of the inhibition was demonstrated in all these assays by testing in parallel the effect of other peptides ( fig2 ) prepared identically and used at the same concentrations , including for instance the peptide corresponding to amino acids 1003 - 1048 of the mhv - a59 s protein ( shown as “ control peptide ” in fig1 ). only the hr2 peptide was effective . conclusion : the hr2 peptide is a potent inhibitor both of virus entry into cells and of mhv - a59 spike mediated cell - cell fusion . ii . 1 . generation of mfipv , a feline coronavirus growing on murine cells general aim : to set up a targeted rna recombination system for feline coronavirus fipv 79 - 1146 similar to the one described above for the genetic manipulation of the murine coronavirus mhv - a59 . specific aim : generate mfipv , a fipv derivative in which the spike protein ectodomain has been replaced genetically by that of the mhv - a59 s protein , thereby shifting the tropism of the chimaeric virus to murine instead of feline cells . aim : prepare a plasmid construct from which synthetic donor rna can be transcribed for targeted rna recombination and which consists of sequences derived from the 5 ′ end of the fipv genome fused to sequences derived from the 3 ′ part of the viral genome , i . e . all sequences downstream of ( and including ) the 3 ′- terminal end of the orfib . also : prepare a derivative of this plasmid in which the sequence encoding the spike ectodomain has been replaced by that encoding the corresponding domain of the mhv - a59 spike protein . procedure and results : using standard dna cloning techniques the vector pbrdi1 was constructed which contains a cdna copy of the 5 ′- most 702 bases ligated to the 3 ′- most 9 . 262 bases of the fipv 79 - 1146 genome ( fig1 a ). the ligation was done in such a way that the orf 1a ( pol 1a ) gene fragment was fused in frame at its 3 ′- end to the 5 ′- end of the orf 1b ( pol 1b ) gene fragment ( fig1 b ). furthermore , at this point a unique saci restriction site was introduced ( fig1 b ). the feline coronavirus sequence was placed under the control of a bacteriophage t7 polymerase promoter sequence followed by a triple g ( fig1 a ) to drive efficient in vitro rna transcription using the t7 polymerase . at the 3 ′- end , the sequence was terminated by a polya tail of 15 a &# 39 ; s followed by a unique noti restriction site ( fig1 c ) to facilitate run - off transcription . the t7 promoter sequence is preceded by a unique xhoi restriction site ( fig1 a ) such that the feline coronavirus cdna could be cloned as a xhoi - noti restriction fragment of 10 . 015 bp into the backbone vector pbrxn ( 11 ) resulting in pbrdi1 ( fig1 a ). plasmid pbrdi1 was subsequently used to prepare pbrdi2 in which the fipv s gene was replaced by a chimaeric spike gene ( ms ) composed of a part encoding the ectodomain of the mhv spike protein and a part encoding the transmembrane and endodomain of the fipv spike protein . to introduce this hybrid gene into pbrdi1 , first the 3 ′ end of the feline pol1b gene was fused to the 5 ′ end of the murine spike gene ( see fig1 d for sequence at junctions ). this fusion product was cloned into ptmfs ( 12 ) as a saci - stui fragment , resulting in ptmfs1 ( fig1 b ). the hybrid gene was then isolated from ptmfs1 as a saci - alfii fragment and used to replace the fipv spike gene from pbrdi1 resulting in pbrdi2 ( fig1 b ). the sequences of pbrdi1 and pbrdi2 are shown in addendum 1 and 2 , respectively . procedure and results : capped , run - off donor rna transcripts were synthesized from noti - linearized pbrdi2 using a t7 rna polymerase kit ( ambion ) according to the instructions of the manufacturer . the transcripts were introduced into feline fcwf cells ( 80 cm 2 culture flask ) that had been infected before with fipv 79 - 1146 ( m . o . i . of 1 ), by electroporation ( gene pulser electroporation apparatus , biorad , 2 consecutive pulses ; 0 . 3 kv / 960 microf ). the electroporated cells were cocultured in a 25 cm 2 flask with murine lr7 cells ( 50 % confluency ) to allow recombination of the synthetic and genomic rna ( fig1 ). after 24 h of incubation at 37 ° c . massive syncytia could be observed of both murine lr7 cells and feline fcwf cells . candidate mfipv recombinants were selected by taking off the culture supernatant and passaging the virus by three consecutive end - point dilutions on lr7 cells . the resulting virus was unable to infect and cause cytopathic effect on fcwf cells . conclusion : a virus with the intended murine cell tropism was obtained . aim : confirm the identity of mfipv at the level of viral protein synthesis . procedure and results : murine lr7 cells were infected with mfipv and the proteins were labeled with 35 s - labeled amino acids for 2 h starting at 5 h p . i . as controls , we infected lr7 and fcwf cells with mhv and fipv , respectively , and labeled them similarly from 5 - 7 h p . i . after the labeling , cell lysates were prepared and immunoprecipitations were carried out in the presence of detergent as described ( 12 ). the following antibodies were used ( for references , see 12 ): g73 ( αfipv ), an ascitis fluid obtained from an fipv - infected cat ( provided by h . vennema ); k134 ( αmhv ), a rabbit serum raised against purified mhv - a59 ; wa3 . 10 ( αs m ), a mab against an epitope present in the mhv - a59 s ectodomain ; 23f4 . 5 ( αs f ), a mab against an epitope in the fipv s ectodomain . the immunoprecipitated proteins were taken up in electrophoresis sample buffer and heated for 2 min at 95 ° c . except for one protein sample ( lane 4 in fig1 ) which was kept at room temperature to prevent aggregation of the mhv m protein . the proteins were analyzed by electrophoresis in sds - 12 . 5 % polyacrylamide gel . the electrophoretic patterns are shown in fig1 . as expected , the anti - fipv antibodies precipitated the fipv proteins s , m and n from the lysate of fipv - infected cells ( lane 10 ), but none of the mhv proteins from lysate of mhv - infected cells ( lane 1 ). the 23f4 . 5 mab precipitated the feline s of fipv ( lane 11 ) but not the murine s of mhv ( lane 2 ), as expected . also , the anti - mhv serum precipitated the mhv proteins s , n and m ( lane 3 and 4 ) but not the fipv proteins ( lane 12 ), and the wa3 . 10 mab precipitated the mhv s protein ( lane 5 ) but not the fipv s protein ( lane 13 ). when looking at the proteins precipitated from the mfipv - infected cell lysates , it is clear that the anti - fipv serum g73 precipitated the m and n proteins , but not the s protein ( lane 6 ). s protein was also not precipitated by the 23f4 . 5 mab ( lane 7 ). the mfipv s protein was , however , precipitated by the anti - mhv serum ( lane 8 ) as well as by the mab wa3 . 10 ( lane 9 ). conclusion : cells infected by mfipv express the predicted viral proteins , particularly the hybrid s protein with the mhv - derived ectodomain . aim : compare titers obtained for mfipv with those for fipv and mhv - a59 . procedure and results : infection and titration experiments revealed that the recombinant virus mfipv was no longer able to infect feline fcwf cells but did grow efficiently in murine lr7 cells showing similar growth characteristics as mhv - a59 . this is demonstrated , for instance , by a comparison of their one - step growth curves in these cells shown in fig1 . in this experiment cells were infected with mfipv and mhv - a59 ( m . o . i . of 5 each ) after which samples from the culture fluid were taken at various time points and titrated on lr7 cells by end - point dilution . also , mfipv induced extensive syncytia and cytopathic effects upon infection of these cells . stocks of the recombinant mfipv grown in lr7 cells reached titers of the same order of magnitude as fipv did in fcwf cells ( 5 . 10 7 pfu / ml ). this was , however , an order of magnitude lower than was observed for mhv - a59 in lr7 cells . conclusion : the replacement of the spike protein ectodomain has resulted in a recombinant mfipv that replicates well in its “ new ” host cells and has apparently not lost much of its biological fitness . general aim : delete gene sequences from the fipv genome that are non - essential for viral replication in vitro to obtain deletion viruses that are attenuated in feline animals and are therefore viral ( vector ) vaccine candidates . aim : construct the plasmids for the synthesis of donor rna transcripts lacking the genes 3abc and / or 7ab for targeted recombination with mfipv rna ( fig1 , top part ). procedure and results : deletions of genes 3abc and 7ab were introduced into the plasmid pbrdi1 using soe - pcr . for the primers used , see table 2 and fig1 , left side . to delete the 3abc cluster , combinations of primers 1 and 4 and of 2 and 3 were used to generate fragments of 375 bp ( a ) and 1012 bp ( b ), respectively ( fig1 , left side ). fragments a and b were fused using the overlap between both fragments through primers 3 and 4 , and amplified using primers 1 and 2 resulting in a 1366 bp fragment ( c ). fragment c was digested with aflii and snabi and cloned into aflii and snabi - digested pbrdi1 , resulting in pbrdi1δ3abc ( fig1 ). to delete the 7ab genes , combinations of primers 5 and 8 and of primers 6 and 7 were used to generate fragments of 1215 bp ( d ) and of 324 bp ( e ), respectively ( fig1 ). fragments d and e were fused using the overlap between both fragments through primers 7 and 8 , and amplified using primers 5 and 6 resulting in a 1524 bp fragment ( f ). fragment f was digested with mlui and noti and cloned into mlui and noti - digested pbrdi1 , resulting in pbrdi1δ7ab ( fig1 ). the correctness of the sequences of fragments c and f was confirmed by dna sequencing . to construct pbrdi1δ3abc + δ7ab , the 1524 bp mlui / noti fragment of pbrdi1δ7ab was introduced into mlui / noti - digested pbrdi1δ3abc ( fig1 ). conclusion : the pbrdi1 - derived donor rna constructs lacking the gene clusters 3abc and / or 7ab were obtained . aim : generate the recombinant fipvs that lack the sequences for the 3abc genes , for the 7ab genes , and for both these gene clusters . procedure and results : capped , run - off donor transcripts were synthesized from noti - linearized pbrdi1 , pbrdi1δ3abc , pbrdi1δ7ab and pbrdi1δ3abc + δ7ab , respectively , using a t7 rna polymerase kit ( ambion ) as specified by the manufacturer . the donor transcripts were introduced into murine lr7 cells ( 80 cm 2 flask ), that had been infected before with mfipv ( m . o . i . of 0 . 4 ), by electroporation ( gene pulser electroporation apparatus , biorad , 2 consecutive pulses ; 0 . 85 kv / 50 microf ). the electroporated cells were cocultured in a 25 cm 2 culture flask with feline fcwf cells ( 50 % confluency ). after 24 h incubation at 37 ° c . massive syncytia could be detected in both the murine lr7 cells and the feline fcwf cells . candidate deletion viruses released into the mixed cell culture supernatant were purified by two rounds of plaque purification on fcwf cells . conclusion : viruses that had acquired the ability to grow in feline fcwf cells had been obtained from the recombination experiment with mfipv . procedure and results : to evaluate whether the intended deletions indeed occurred in the various fipv deletion mutants , rt - pcr on the genomic viral rna &# 39 ; s was performed focusing on the 3abc and the 7ab region . the primers used and dna sizes expected are indicated in table 2 and fig1 ( right side ). the results of the rt - pcr analyses are shown in fig1 . fig1 a reveals that the recombinant wild - type virus ( r - wtfipv ) and fipvδ7ab are each carrying the 3abc region whereas this region is lacking in the viruses fipvδ3abc and fipvδ3abc + δ7ab , as judged by the sizes of the amplified fragments . fig1 b demonstrates that r - wtfipv and fipvδ3abc are still carrying the 7ab region whereas the viruses fipvδ7ab and fipvδ3abc + δ7ab are lacking this region . the 397 bp and 646 bp fragments , indicative of a 3abc and 7ab deletion , respectively , were cloned and sequenced which confirmed the expected dna sequences . aim : check cell tropism of the deletion viruses and evaluate their in vitro growth . results : all 4 recombinant viruses were inoculated onto mouse lr7 cells but failed to produce any cytopathic effects . they did , however , grow efficiently in feline fcwf cells , as expected . the viruses r - wtfipv , fipvδ3abc and fipvδ7ab reached titers that were similar to those obtained with the parent fipv strain 79 - 1146 on these cells . the titers were all in the order of 5 . 10 7 pfu / ml ( fig4 ). however , the double mutant fipvδ3abc + δ7ab grew less efficient ; titers obtained with this virus were generally 1 to 2 log units lower ( fig4 ). conclusion : the sequences comprising the gene clusters 3abc and 7ab are not essential for the viability of fipv . apparently , neither the nucleotide sequences nor the proteins encoded by the genes are essential for the replication of the virus . procedure and results : the recombinant deletion viruses were characterized in their natural host , the cat . to this purpose , 24 spf cats ( 5 months old ) were placed into 5 groups and inoculated oronasally ( 100 pfu ) with fipv strain 79 - 1146 ( n = 4 ), r - wtfipv ( n = 5 ), fipvδ3abc ( n = 5 ), fipvδ7ab ( n = 5 ) and fipvδ3abc + δ7ab ( n =), respectively , and followed for ( at least ) 3 months . clinical disease signs were scored as shown in table 5 . inoculation of the cats with the deletion variants did not induce clinical signs of disease . rather , all cats remained totally healthy throughout the experiment ( table 6 and fig3 ). in contrast , infection with the wild type controls fipv 79 - 1146 and r - wtfipv induced a rapid onset of clinical disease characterized by depression , anorexia , jaundice , weight loss and leukopenia ( table 6 ). three out of four and five out of five cats inoculated with fipv 79 - 1146 and r - wtfipv , respectively , had to be euthanized due to advanced symptoms of fip between day 14 and 42 after infection ( fig3 ). conclusions : 1 ) fipv 79 - 1146 and its wild type recombinant equivalent r - wtfipv are equally virulent ; and 2 ) viruses with deletions of the sequences specifying the genes 3abc or 7ab or of the combination of some or all these genes exhibit a significantly attenuated phenotype in cats . procedure and results : fipv - specific antibody responses of the cats inoculated with the deletion viruses were characterized . to this purpose , blood samples were obtained at days 0 , 21 and 90 post infection , and heat - inactivated sera were prepared and incubated with fipv 79 - 1146 ( 10 . 000 pfu ) after which the fipv - neutralizing activity was determined using fcwf cells in a 96 - well microplate assay . titers were expressed as the reciprocal of the lowest dilution that no longer inhibited viral cytopathic effects . as expected , at day 0 none of the cat sera showed a significant fipv - neutralizing activity . at day 21 , all cats had sero - converted and showed high titers of neutralizing antibodies . the titers observed in cats inoculated with fipv 79 - 1146 , r - wtfipv , fipvδ3abc and fipvδ7ab were comparable whereas the titers observed in fipvδ3abc + δ7ab infected cats were approximately 50 fold lower ( fig3 ). overall , the titers remnained high for at least 90 days ( end of experiment ). conclusion : despite the absence of clinical disease signs , high titers of neutralizing antibodies are observed in cats that had been inoculated with fipv deletion variants . this strongly suggests that the deletion viruses are viable and replicate in the cat leading to a strong immune response in the form of an antibody response . aim : to study whether previous inoculation with the attenuated deletion variants protects the cats against a fipv 79 - 1146 challenge . procedure and results : the cats previously inoculated with the attenuated deletion variants were challenged oranasally with fipv 79 - 1146 ( 100 pfu ) at day 90 . as a control group , 4 untreated cats of similar age were challenged identically . the control group showed a rapid onset of clinical disease characterized by depression , anorexia , jaundice , weight loss and leukopenia ( day 7 ). a similar rapid onset of symptoms was observed with 3 out 5 cats previously inoculated with fipvδ3abc + δ7ab , whereas all cats previously infected with fipvδ3abc or fipvδ7ab remained generally healthy and without typical fip symptoms throughout the experiment ( table 7 ), although 2 out 5 cats previously inoculated with fipvδ3abc showed temporary weight loss . due to advanced symptoms of fip , one cat out of the control group had to be euthanized at day 32 , whereas the other cats in the control group recovered from their initial fip symptoms . a lethality score of 25 % is lower than observed in the previous experiments . this is supposedly due to the advanced age of the cats which is known to lead to reduced susceptibility for fipv . the three fipvδ3abc + δ7ab vaccinated cats with initial symptoms remained ill and were euthanized between days 11 and 56 ( fig3 ). apparently , the combined deletion of the two gene clusters 3abc and 7ab which we found to reduce the fitness of fipvδ3abc + δ7ab — as judged by its decreased in vitro growth — also affects the replication rate of the virus in the cats , as testified by the lower levels of neutralizing antibodies induced . thus , the virus is over - attenuated and only capable of partially protecting against a fipv challenge . full protection would require a higher or repeated dose . conclusion : prior vaccination with fipvδ3abc or fipvδ7ab protects cats against disease caused by a fipv challenge . aim : establish whether foreign genes can be inserted at different positions in the viral genome , either as an additional gene or replacing deleted non - essential genes ; and establish whether these genes are expressed and whether they are stably maintained during in vitro passage of the virus . procedure : a virus was constructed containing a foreign reporter gene in its genome at the position of the 3abc genes . the reporter gene , renilla luciferase ( rl ), was placed under the transcriptional control of the igs preceeding gene 3a . to delete the 3abc cluster and introduce the rl gene into the plasmid pbrdi1 , combinations of primers 1244 ( 5 ′- gccattctcattgataac - 3 ′) ( seq id no : _ ) and 1514 ( 5 ′- ctgagtctagagtagctagctaatgactaataagtttag - 3 ′) ( seq id no : _ ) and of 1245 ( 5 ′- gcttctgttgagtaatcacc - 3 ′) ( seq id no : _ ) and 1513 ( 5 ′- gctagctactctagactcaggcggttctaaac - 3 ′) ( seq id no : _ ) were used to generate fragments of 336 bp ( a ) and 1068 bp ( b ) via pcr , respectively . in primer 1513 and 1514 , the underlined and bold sequences represent a nhei and xbai restriction site , respectively . fragments a and b were fused using the overlap between both fragments through primers 1514 and 1513 and amplified using primers 1244 and 1245 , using soe - pcr , resulting in a 1384 bp fragment ( c ). fragment c was cloned into the pgem - t easy vector ( promega ), resulting in pgem - c . the rl gene derived from prl - null ( promega ) was introduced as a nhei / xbai fragment into nhei and xbai digested pgem - c , resulting in pgem - c + luc . fragment c + luc was introduced as a aflii / snabi fragment into aflii and snabi - digested pbrdi1 , resulting in pbrdi1δ3abc + luc . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . conclusion : the foreign reporter gene renilla luciferase can be placed into the genome of the type i coronavirus fipv . the intended recombinant viruses are viable . procedure and results : after two rounds of plaque purification virus stocks of 2 independently obtained recombinants under ii . 3 . a were prepared , titrated and used for high m . o . i ( m . o . i . of 8 ) infection of fcwf cells after which the viral infectivities in the culture media were monitored . the results are represented by the growth curves shown in fig3 . both independently obtained recombinants grew to a 1 to 2 log lower titer as compared to that of recombinant wild - type virus . conclusion : the recombinant viruses with inserted expression cassette grew normally in vitro but their yields were affected . procedure and results : confluent monolayer cultures of fcwf cells were infected at a m . o . i . of 8 and the production of luciferase activity in the cells was monitored over time . rl expression in cells was measured by using the dual - luciferase reporter assay system ( promega ) according to the manufacturer &# 39 ; s instructions . rl was measured in relative light units ( rlu ) using a luminometer ( lumac biocounter m2500 or turner designs model td - 20 / 20 ). the results are shown graphically in fig3 . in contrast to the recombinant wild - type virus , the two recombinant luciferase gene containing viruses expressed high levels of luciferase activity , indicating that the renilla luciferase gene is functional . expression started as early as 2 h post - infection . the highest expression level was reached at 9 h post - infection . conclusion : foreign genes can be expressed by coronaviruses by using the genetic space created by deletion of nonessential genes . aim : development of multivalent vaccines based on a live attenuated fipv strain as a vector . approach : genes ( or gene fragments ) from other feline and canine pathogens encoding antigens known to induce protective immunity against these pathogens were selected . expression cassettes of these genes were introduced into the fipv genome in combination with attenuating deletions of non - essential genes . the expression cassettes contain the fipv trs in front of ( parts of ) the gene to be expressed . ii . 4 . a . construction of a multivalent feline leukemia virus ( felv ) vaccine based on fipv vector aim : development of felv vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related felv genes gag and env were placed into expression cassettes and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the felv gag and env genes was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related felv genes gag and env can be introduced into and expressed by a live attenuated fipv strain . these recombinants can therefore function as a multivalent vaccine against a feline leukemia virus and fipv infection . ii . 4 . b . construction of a multivalent feline immunodeficiency virus ( fiv ) vaccine based on a fjpv vector aim : development of fiv vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related fiv genes gag and env were placed into expression cassettes and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the fiv gag and env genes was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related fiv genes gag and env can be introduced into and expressed by a live attenuated fipv strain . these recombinant viruses can therefore function as a multivalent vaccine against a feline immunodeficiency virus and fipv infection . ii . 4 . c . construction of a multivalent feline calicivirus ( fcv ) vaccine based on a fipv vector aim : development of fcv vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related fcv capsid gene was placed into an expression cassette and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the fcv capsid gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related fcv capsid gene can be introduced into and expressed by a live attenuated fipv strain . these recombinant viruses can therefore function as a multivalent vaccine against a feline calicivirus and fipv infection . ii . 4 . d . construction of a multivalent feline panleucopenia virus ( fpv ) vaccine based on a fipv vector aim : development of fpv vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related r3 gene , encoding the vp1 and vip2 capsid proteins was placed into an expression cassette and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of ( parts of ) the r3 gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related r3 gene can be introduced into and expressed by a live attenuated fipv strain . these recombinant viruses can therefore function as a multivalent vaccine against a feline panleucopenia virus and fipv infection . ii . 4 . e . construction of a multivalent feline herpes virus ( fhv ) vaccine based on a fipv vector aim : development of fhv vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related gb , gc , gd , ge , gh , gi , gk , gl , gm , gm , icp0 , icp1 and icp4 feline herpes virus genes were placed into an expression cassette and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the inserted fhp genes was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related gb , gc , gd , ge , gh , gi , gk , gl , gm , icp0 , icp1 and icp4 feline herpes virus genes can be introduced and expressed in a live attenuated fipv strain . these recombinant viruses can therefore function as a multivalent vaccine against feline herpes virus and fipv . ii . 4 . f . construction of a multivalent fipv serotype i and ii vaccine based on a fipv serotype ii vector . aim : development of a vaccine protecting both against serotype i and against ii feline coronaviruses , based on a live attenuated fipv serotype ii strain as a vector . procedure : ( parts of ) the protection related spike gene of a serotype i feline coronavirus of which the region encoding the signal sequence was deleted was placed into an expression cassette and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . for example : in one case a spike gene construct was used from which the region encoding the signal sequence had been deleted ; in another case a truncated spike gene was used , from which the region encoding the transmembrane domain + endodomain had been deleted . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the inserted the serotype i spike gene construct was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related spike gene of feline coronavirus serotype i can be introduced and expressed in a live attenuated fipv serotype ii strain and can therefore function as a multivalent vaccine against both serotype i and ii feline coronaviruses . aim : development of a fipv vaccine in which deletion of non - essential genes is combined with a rearranged gene order by moving the n gene upstream of the s gene in the fipv genome . procedure and results : capped , run - off donor transcripts were synthesized from noti - linearized pbrdi1δ3abc , pbrdi1δ7ab and pbrdi1δ3abc + δ7ab vectors in which the n gene , together with its trs , was inserted at a position upstream of the s . the donor transcripts were introduced into murine lr7 cells ( 80 cm 2 flask ), that had been infected before with mfipv ( m . o . i . of 0 . 4 ), by electroporation ( gene pulser electroporation apparatus , biorad , 2 consecutive pulses ; 0 . 85 kv / 50 microf ). the electroporated cells were co - cultured in a 25 cm 2 culture flask with feline fcwf cells ( 50 % confluency ). after 24 h incubation at 37 ° c . massive syncytia could be detected in the cell cultures . deletion mutant viruses with rearranged genomes released into the mixed cell culture supernatant were purified by two rounds of plaque purification on fcwf cells . conclusion : deletion mutant fipvs with rearranged gene order could be generated . these viruses can function as live attenuated fipv vaccines . by virtue of their rearranged gene order these viruses represent safer vaccines because of their reduced ability to generate viable progeny by recombination with viruses circulating in the field . general aim : development of vaccines based on a live attenuated fipv serotype ii strain as a vector against canine pathogens . approach : since type ii feline coronaviruses express canine coronavirus - like spike proteins ( 2a ), such feline coronavirus vectors can be used as vaccines in canines . therefore , the live attenuated fipv vaccine that we developed can also be used for vaccination of canines against canine coronavirus ( ccv ). furthermore , multivalent vaccines can be generated by introducing expression cassettes of protection related genes of other canine pathogens into the fipv genome in combination with attenuating deletions of non - essential genes and with genome rearrangements . the expression cassettes contain the fipv trs in front of ( parts of ) the gene to be expressed . aim : development of canine distemper vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related h and f genes were placed into an expression cassette and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of ( parts of ) the h and f genes was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related h and f genes of canine distemper virus can be introduced into and expressed by a live attenuated fipv strain . these recombinant viruses can therefore function as a vaccine against canine distemper virus and ccv infection . aim : development of canine parvovirus vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related r3 gene , encoding the vp1 and vip2 capsid proteins were placed into an expression cassette and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of ( parts of ) the r3 gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related r3 gene of canine parvovirus can be introduced into and expressed by a live attenuated fipv strain . these recombinant viruses can therefore function as a vaccine against canine parvovirus disease and ccv infection . aim : development of canine adenovirus 1 vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related structural protein genes of canine adenovirus 1 were placed into an expression cassette and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of ( parts of ) the protection related structural protein genes of canine adenovirus 1 was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related structural protein genes of canine adenovirus 1 can be introduced into and expressed by a live attenuated fipv strain . these recombinant viruses can therefore function as a vaccine against infectious canine hepatitis and ccv infection . aim : development of canine herpes virus 1 vaccine based on a live attenuated fipv strain as a vector . procedure : ( parts of ) the protection related gb , gc , gd , ge , gh , gi , gk , gl , gm , gm , icp0 , icp1 and icp4 canine herpes virus genes were placed into an expression cassette and subsequently introduced into pbrdi1δ3abc and pbrdi1δ7ab , respectively . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by rna - rna recombination between transcription vector run - off transcripts and the mfipv genome as described above . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the inserted genes was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related gb , gc , gd , ge , gh , gt , gk , gl , gm , icp0 , icp1 and icp4 canine herpes virus genes can be introduced and expressed in a live attenuated fipv strain . these recombinant viruses can therefore function as a multivalent vaccine against canine herpes virus 1 and ccv . approach : to this aim recombinant tgev deletion mutant viruses were generated that lack the non - essential genes 3ab and / or 7 . procedure : recombinant viruses were generated as described by almazan et al . ( 2000 ). from pbac - tgev fl , an infectious tgev cdna clone placed behind a cmv promoter in pbelobac11 , the 3ab and 7 genes were deleted via standard cloning techniques , leaving the surrounding open reading frames and transcription regulatory sequences intact . epithelial swine testis ( st ) cells were transfected with this pbac - tgev fl derivative which lacks the 3ab and 7 genes ( pbac - tgev fl ). recombinant tgev viruses were plaque purified and characterized by rt - pcr for the absence of the 3ab and / or 7 genes . large amounts of the recombinant tgev deletion viruses were generated by infecting st cells . conclusion : recombinant tgev was generated that lacked the genes 3ab and 7 . these viruses can function as a live attenuated vaccine against tgev . aim : development of multivalent vaccines based on a live attenuated tgev as a vector . approach : expression cassettes of protection related genes of porcine pathogens were introduced into the tgev genome in combination with attenuating deletions of non - essential genes and genome rearrangements . the expression cassettes contain the tgev trs in front of ( parts of ) the gene to be expressed . iii . 2 . a . construction of a multivalent porcine parvovirus ( ppv ) vaccine based on a tgev vector aim : development of a vaccine based on a live attenuated tgev strain as a vector . procedure : ( parts of ) the protection related r3 gene , encoding the vp1 and vp2 capsid proteins , was placed into an expression cassette and subsequently introduced into a pbac - tgev fl derivative which lacks genes 3ab and 7 . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by transfecting st cells with this pbac - tgev fl deletion derivative which contains ( parts of ) the protection related r3 gene . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the inserted r3 gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related r3 gene can be introduced into and expressed by a live attenuated tgev strain . these recombinant viruses can therefore function as a multivalent vaccine against a porcine parvovirus and tgev infection . iii . 2 . b . construction of a multivalent swine influenza virus vaccine based on a tgev vector aim : development of a swine influenza virus vaccine based on a live attenuated tgev strain as a vector . procedure : ( parts of ) the protection related hemagglutinin ( ha ) and neuraminidase ( na ) genes were placed into an expression cassette and subsequently introduced into a pbac - tgev fl derivative which lacks the genes 3ab and 7 . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by transfecting st cells with this pbac - tgev fl deletion derivative which contains ( parts of ) the protection related hemagglutinin ( ha ) and neuraminidase ( na ) genes . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the inserted hemagglutinin ( ha ) and neuraminidase ( na ) genes was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related hemagglutinin ( ha ) and neuraminidase ( na ) genes can be introduced into and expressed by a live attenuated tgev strain . these recombinant viruses can therefore function as a multivalent vaccine against a swine influenza virus and tgev infection . iii . 2 . c . construction of a multivalent african swine fever virus vaccine based on a tgev vector aim : development of an african swine fever virus vaccine based on a live attenuated tgev strain as a vector . procedure : ( parts of ) the protection related structural protein encoding genes were placed into an expression cassette and subsequently introduced into a pbac - tgev fl derivative which lacks the genes 3ab and 7 . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by transfecting st cells with this pbac - tgev fl deletion derivative which contains ( parts of ) the protection related structural - protein encoding genes . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the structural - protein encoding genes was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related structural protein encoding genes can be introduced into and expressed by a live attenuated tgev strain . these recombinant viruses can therefore function as a multivalent vaccine against an african swine fever virus and tgev infection . iii . 2 . d . construction of a multivalent porcine circovirus type 2 vaccine based on a tgev vector aim : development of a porcine circovirus type 2 vaccine based on a live attenuated tgev strain as a vector . procedure : ( parts of ) the protection related c1 , c2 , v1 and v2 genes of the porcine circovirus type 2 were placed into an expression cassette and subsequently introduced into a pbac - tgev fl derivative which lacks the genes 3ab and 7 . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by transfecting st cells with this pbac - tgev fl deletion derivative which contains ( parts of ) the protection related c1 , c2 , v1 and v2 genes of the porcine circovirus type 2 . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the c1 , c2 , v1 and v2 genes of the porcine circovirus type 2 was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related c1 , c2 , v1 and v2 genes of the porcine circovirus type 2 can be introduced into and expressed by a live attenuated tgev strain . these recombinant viruses can therefore function as a multivalent vaccine against a porcine circovirus type 2 , and tgev infection . iii . 2 . e . construction of a multivalent porcine respiratory and reproductive syndrome virus vaccine based on a tgev vector aim : development of a porcine respiratory and reproductive syndrome virus vaccine based on a live attenuated tgev strain as a vector . procedure : ( parts of ) the protection related orf2 to orf7 of the porcine respiratory and reproductive syndrome virus were placed into an expression cassette and subsequently introduced into a pbac - tgev fl derivative which lacks the genes 3ab and 7 . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by transfecting st cells with this pbac - tgev fl deletion derivative which contains ( parts of ) the protection related orf2 to orf7 of the porcine respiratory and reproductive syndrome virus . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of orf2 to orf7 of the porcine respiratory and reproductive syndrome virus was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related orf2 to orf7 of the porcine respiratory and reproductive syndrome virus can be introduced into and expressed by a live attenuated tgev strain . these recombinant viruses can therefore function as a multivalent vaccine against a porcine respiratory and reproductive syndrome virus and tgev infection . iii . 2 . e . construction of a multivalent foot - and - mouth disease virus vaccine based on a tgev vector aim : development of a food - and - mouth disease virus vaccine based on a live attenuated tgev strain as a vector . procedure : ( parts of ) the protection related sequences encoding vp1 to vp4 of the foot - and - mouth disease virus were placed into an expression cassette and subsequently introduced into a pbac - tgev fl derivative which lacks the genes 3ab and 7 . after confirmation of all constructs by restriction and sequence analysis , recombinant viruses were generated by transfecting st cells with this pbac - tgev fl deletion derivative which contains ( parts of ) the protection related sequences encoding vp1 to vp4 of the food - and - mouth disease virus . the resulting viruses were genetically confirmed by rt - pcr analysis . expression of the sequences encoding vp1 to vp4 of the foot - and - mouth disease virus was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related sequences encoding vp1 to vp4 of the foot - and - mouth disease virus can be introduced into and expressed by a live attenuated tgev strain . these recombinant viruses can therefore function as a multivalent vaccine against a foot - and - mouth disease virus and tgev infection . aim : development of a tgev vaccine in which deletion of non - essential genes is combined with a rearranged gene order by moving the n gene upstream of the s gene in the tgev genome . procedure and results : recombinant viruses were generated as described by almazan et al . ( 2000 ). from pbac - tgev fl , an infectious tgev cdna clone placed behind a cmv promoter in pbelobac11 , the 3ab and 7 genes were deleted via standard cloning techniques , leaving the surrounding open reading frames and transcription regulatory sequences intact . in addition the n gene was cloned to a position in the genome upstream of the s gene . epithelial swine testis ( st ) cells were transfected with this pbac - tgev fl derivative which lacks the 3ab and 7 genes and has a rearranged genome . recombinant tgev viruses were plaque purified and characterized by rt - pcr for the absence of the 3ab and / or 7 genes . large amounts of the tgev deletion recombinants were generated by infecting st cells . conclusion : recombinant tgev was generated that lacked the genes 3ab and 7 , and which has a rearranged gene order . these viruses function as a live attenuated vaccine against tgev , as well as against other porcine pathogens when genes encoding relevant antigens are appropriately incorporated . approach : recombinant ibv deletion mutant viruses were generated that lack the non - essential genes 3a / b and / or 5a / b . in order to acquire vaccine viruses that protect against multiple ibv serotypes , spike gene constructs derived from such different viruses were incorporated . procedure : recombinant viruses were generated as described by casais et al . ( 2001 ). infectious ibv cdna clones were assembled in the vaccinia virus genome by using sequential in vitro ligation of cdna fragments derived from pfrag1 , pfrag2 , and pfrag3 derived plasmids , followed by direct cloning into the genome of vaccinia virus vnoti / tk as described ( 1a ). genes 3a / b and 5a / b were removed from pfrag3 , by pcr mutagenesis leaving the surrounding open reading frames and transcription regulatory sequences intact , resulting in pfrag3δ . the s gene in pfrag3δ could be replaced by s genes derived from different ibv serotypes by using rt - pcr . recombinant vaccinia viruses were generated by recombination between the fowl pox virus hp1 . 441 and the vnoti / tk - ibv in vitro ligation mixture . recombinant viruses were plaque purified and characterized by pcr and southern blot analysis . finally , infectious ibv lacking genes 3a / b and / or 5a / b was generated as follows : chick kidney ( ck ) cells were infected with recombinant fowl pox virus expressing t7 rna polymerase . subsequently cells were transfected with dna isolated from the recombinant vaccinia virus containing the ibv cdna clone lacking genes 3a / b and 5a / b . at 3 days post - infection the culture medium was collected and used to infect ck cells to generate large amounts of recombinant ibv . the recombinant viruses were genetically confirmed by rt - pcr analysis . conclusion : recombinant ibv was generated that lacked the genes 3a / b and / or 5a / b . these viruses can function as a live attenuated vaccine against ibv . by replacing the s gene , recombinant ibvs could be obtained that function as live attenuated vaccines against different ibv serotypes . aim : development of multivalent vaccine based on a live attenuated ibv strain as a vector . approach : expression cassettes of protection related genes of chicken pathogens were introduced into the ibv genome in combination with attenuating deletion of non - essential genes . the expression cassettes contain the ibv trs in front of ( parts of ) the gene to be expressed . iv . 2 . ai . construction of a multivalent vaccine based on an ibv vector that protects against more than one ibv serotype . aim : development of a ibv vaccine based on a live attenuated ibv strain that lacks non - essential genes and protects against more than one serotype . procedure : ( parts of ) the protection related ibv s gene from a different ibv serotype were placed into expression cassettes , and subsequently introduced into pfrag3δ . the largest construct contained a complete extra copy of the s gene except for the sequence encoding the signal peptide ; another construct had a carboxy - terminally truncated s gene lacking the code for the transmembrane domain and endodomain . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . the proper expression of the heterologous s gene constructs was verified by immunoprecipitation analysis with type - specific antisera using radiolabeled recombinant virus infected cell lysates . conclusion : ( parts of ) the protection related ibv s gene from a different ibv serotype can be introduced in and expressed from a live attenuated ibv strain . these recombinant viruses can therefore function as multivalent vaccines to protect against infection by more than one ibv serotype . iv . 2 . aii . construction of a multivalent vaccine based on an ibv vector that protects against newcastle disease . aim : development of a newcastle disease virus ( avian paramyxovirus type i ; apmv - 1 ) vaccine based on a live attenuated ibv strain that lacks non - essential genes . procedure : ( parts of ) the protection related apmv - 1 genes hn and f were placed in expression cassettes , and subsequently introduced into pfrag3δ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related apmv - 1 genes hn and f can be introduced in and expressed from a live attenuated ibv strain . the recombinant viruses can therefore function as a multivalent vaccine to protect against apmv - 1 and ibv infections . iv . 2 . b . construction of a multivalent vaccine based on an ibv vector that protects against avian influenza . aim : development of an avian influenza virus vaccine based on a live attenuated ibv strain that lacks non - essential genes . procedure : ( parts of ) the protection related avian influenza genes h and n were placed into expression cassettes , and subsequently introduced into pfrag3δ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related avian influenza genes h and n can be introduced in and expressed from a live attenuated ibv strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against avian influenza and ibv infections . iv . 2 . c . construction of a multivalent vaccine based on an ibv vector that protects against chicken anemia virus ( cav ) disease . aim : development of a cav vaccine based on a live attenuated ibv strain that lacks non - essential genes . procedure : ( parts of ) the protection related cav genes v1 , v2 , and v3 were placed into expression cassettes , and subsequently introduced into pfrag3δ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related cav genes v1 , v2 , and v3 can be introduced in and expressed from a live attenuated ibv strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against cav and ibv infections . iv . 2 . d . construction of a multivalent vaccine based on an ibv vector that protects against avian reovirus disease . aim : development of an avian reovirus vaccine based on a live attenuated ibv strain that lacks non - essential genes . procedure : ( parts of ) the protection related avian reovirus genes φ1 , φ2 , and 83 were placed into expression cassettes , and subsequently introduced into pfrag3δ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related avian reovirus genes φ1 , φ2 , and 83 can be introduced in and expressed from a live attenuated ibv strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against avian reovirus and ibv infections . iv . 2 . e . construction of a multivalent vaccine based on an ibv vector that protects against infectious bursal disease . aim : development of an infectious bursal disease virus ( ibdv ) vaccine based on a live attenuated ibv strain that lacks non - essential genes . procedure : ( parts of ) the protection related ibdv gene vp2 were placed into expression cassettes , and subsequently introduced into pfrag3δ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related ibdv gene vp2 can be introduced in and expressed from a live attenuated ibv strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against avian ibdv and ibv infections . iv . 2 . f . construction of a multivalent vaccine based on an ibv vector that protects against marek &# 39 ; s disease . aim : development of a gallid herpes virus 2 ( marek &# 39 ; s disease virus ) vaccine based on a live attenuated ibv strain that lacks non - essential genes . procedure : ( parts of ) the protection related gallid herpes virus 2 gene gb were placed into expression cassettes , and subsequently introduced into pfrag3δ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related gallid herpes virus 2 gene gb can be introduced in and expressed from a live attenuated ibv strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against gallid herpes virus 2 and ibv infections . iv . 2 . g . construction of a multivalent vaccine based on an ibv vector that protects against infectious laryngotracheitis . aim : development of a gallid herpes virus 1 ( infectious laryngotracheitis virus ) vaccine based on a live attenuated ibv strain that lacks non - essential genes . procedure : ( parts of ) the protection related gallid herpes virus 1 genes gb , gc , gd , ge , gh , gi , gk , gl , and gm were placed into expression cassettes , and subsequently introduced into pfrag3δ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related gallid herpes virus 1 genes can be introduced in and expressed from a live attenuated ibv strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against gallid herpes virus 1 and ibv infections . aim : development of an ibv vaccine in which deletion of non - essential genes is combined with a rearranged gene order by moving the n gene upstream of the s gene in the ibv genome . procedure : recombinant viruses were generated as described by casais et al . ( 2001 ). infectious ibv cdna clones were assembled in the vaccinia virus genome by using sequential in vitro ligation of cdna fragments derived from pfrag1 , pfrag2 , and pfrag3 derived plasmids , followed by direct cloning into the genome of vaccinia virus vnoti / tk as described ( 1a ). genes 3a / b and 5a / b were removed from pfrag3 , by pcr mutagenesis leaving the surrounding open reading frames and transcription regulatory sequences intact . in addition , the n gene together with its trs was moved to a position upstream of the s gene resulting in pfrag3δrearranged . recombinant vaccinia viruses were generated by recombination between the fowlpox virus hp1 . 441 and the vnoti / tk - ibv in vitro ligation mixture . recombinant viruses were plaque purified and characterized by pcr and southern blot analysis . finally , infectious ibv lacking genes 3a / b and 5a / b and with a rearranged gene order was generated as follows : chick kidney ( ck ) cells were infected with recombinant fowl pox virus expressing t7 rna polymerase . subsequently cells were transfected with dna isolated from the recombinant vaccinia virus containing the ibv cdna clone lacking genes 3a / b and 5a / b in combination with the rearranged gene order . at 3 days post - infection the culture medium was collected and used to infect ck cells to generate large amounts of recombinant ibv . the recombinant viruses were genetically confirmed by rt - pcr analysis . conclusion : recombinant ibv was generated that lacked the genes 3a / b and 5a / b in combination with a rearranged gene order . these viruses function as a live attenuated vaccine against ibv . when combined with s gene constructs from other ibv serotypes and / or gene constructs from other avian pathogens additional , multivalent vaccines can be generated . approach : recombinant hcov deletion mutant viruses were generated that lack the non - essential genes 4a / b . procedure : recombinant viruses were generated essentially as described by thiel et al . ( 2001 ). infectious hcov cdna clones were assembled in the vaccinia virus genome . vaccinia virus vdhcov - vec - 1 contains a 22 . 5 kbp cdna fragment encoding the 5 ′ end of the hcov genome . this fragment was removed from the vaccinia genome by digestion with bsp 120i , digested with mlui and ligated to a cdna fragment of pmeδ that was obtained by digestion with mlui / eagi . pmeδ contains a cdna fragment that encodes the 3 ′ end of the hcov genome , but lacks gene 4a / b , without disturbing the other genes or transcription regulatory sequences . pmeδ was derived from pme by using conventional pcr mutagenesis methods . the ligation products were ligated to vnoti / tk vaccinia virus dna . recombinant vaccinia viruses were generated as described above . recombinant viruses were plaque purified and characterized by pcr and southern blot analysis . finally , infectious hcov lacking genes 4a / b was generated as follows : chick kidney ( ck ) cells were infected with recombinant fowl pox virus expressing t7 rna polymerase . subsequently cells were transfected with dna isolated from the recombinant vaccinia virus containing the hcov cdna clone lacking genes 4a / b . at 3 days post - infection the culture medium was collected and used to infect human long fibroblast cells ( mrc - 5 ) to generate large amounts of recombinant hcov . the recombinant viruses were genetically confirmed by rt - pcr analysis . conclusion : recombinant hcov was generated that lacked the genes 4a / b . these viruses can function as a live attenuated vaccine against hcov - 229e . v . 2 . generation of a live attenuated vaccine against hcov strain oc43 approach : recombinant hcov deletion mutant viruses were generated that lack the non - essential genes 4a / b , and contain a hybrid s protein gene , which encodes the ectodomain of hcov - oc43 . procedure : recombinant viruses were generated essentially as described by thiel et al . ( 2001 ). infectious hcov cdna clones were assembled in the vaccinia virus genome . vaccinia virus vhcov - vec - 1 contains a 22 . 5 kbp cdna fragment encoding the 5 ′ end of the hcov genome . this fragment was removed from the vaccinia genome by digestion with bsp 120i , digested with mlui and ligated to a cdna fragment of pmeδ - soc43 that was obtained by digestion with mlui / eagi . pmeδ - soc43 contains a cdna fragment that encodes the 3 ′ end of the hcov genome , lacks gene 4a / b , and contains a hybrid s protein gene , without disturbing the other genes or transcription regulatory sequences . the hybrid s protein gene contains the region of the s gene of hcov - oc43 that encodes the s protein ectodomain , while the remainder of the gene is derived from the hcov - 229e gene . pmeδ - soc43 was derived from pmeδ by using conventional ( rt -) pcr mutagenesis methods . the ligation products were ligated to vnoti / tk vaccinia virus dna . recombinant vaccinia viruses were generated as described above . recombinant viruses were plaque purified and characterized by pcr and southern blot analysis . finally , infectious hcov lacking genes 4a / b was generated as follows : chick kidney ( ck ) cells were infected with recombinant fowl pox virus expressing t7 rna polymerase . subsequently cells were transfected with dna isolated from the recombinant vaccinia virus containing the hcov cdna clone lacking genes 4a / b . at 3 days post - infection the culture medium was collected and used to infect human long fibroblast cells ( mrc - 5 ) to generate large amounts of recombinant hcov . the recombinant viruses were genetically confirmed by rt - pcr analysis . conclusion : recombinant hcov was generated that lacked the genes 4a / b . these viruses contain the ectodomain of the s protein of hcov - oc43 and therefore function as a live attenuated vaccine against hcov - oc43 . aim : development of multivalent vaccine based on a live attenuated hcov strain as a vector . approach : expression cassettes of protection related genes of human pathogens were introduced into the hcov genome in combination with attenuating deletion of non - essential genes . the expression cassettes contain the hcov trs ( tctcaact ) in front of ( parts of ) the gene to be expressed . v . 3 . a . construction of a multivalent vaccine based on an hcov vector that protects against respiratory syncytial virus ( rsv ) aim : development of a rsv vaccine based on a live attenuated hcov strain that lacks non - essential genes . procedure : ( parts of ) the protection related rsv genes hn and f were placed into expression cassettes , and subsequently introduced into pmeδ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related rsv genes hn and f can be introduced in and expressed from a live attenuated hcov strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against rsv and hcov infections . v . 3 . b . construction of a multivalent vaccine based on an hcov vector that protects against rotavirus aim : development of a rotavirus vaccine based on a live attenuated hcov strain that lacks non - essential genes . procedure : ( parts of ) the protection related rotavirus genes vp4 , vp6 and vp7 were placed into expression cassettes , and subsequently introduced into pmeδ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related rotavirus genes vp4 , vp6 and vp7 can be introduced in and expressed from a live attenuated hcov strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against rotavirus and hcov infections . v . 3 . c . construction of a multivalent vaccine based on an hcov vector that protects against norwalk - like viruses aim : development of a norwalk - like virus vaccine based on a live attenuated hcov strain that lacks non - essential genes . procedure : ( parts of ) the protection related norwalk - like virus capsid gene was placed into expression cassettes , and subsequently introduced into pmeδ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related norwalk - like virus capsid gene can be introduced in and expressed from a live attenuated hcov strain . this recombinant virus can therefore function as a multivalent vaccine to protect against norwalk - like virus and hcov infections . v . 3 . d . construction of a multivalent vaccine based on an hcov vector that protects against influenza virus aim : development of an influenza virus vaccine based on a live attenuated hcov strain that lacks non - essential genes . procedure : ( parts of ) the protection related influenza virus genes h and n were placed into expression cassettes , and subsequently introduced into pmeδ . after confirmation of all the constructs by restriction and sequence analysis , recombinant viruses were generated as described above . the recombinant viruses were genetically confirmed by rt - pcr analysis . expression of the foreign gene was confirmed by immunoprecipitation analysis . conclusion : ( parts of ) the protection related influenza virus genes h and n can be introduced in and expressed from a live attenuated hcov strain . these recombinant viruses can therefore function as a multivalent vaccine to protect against influenza virus and hcov infections . aim : development of a hcov vaccine in which deletion of non - essential genes is combined with a rearranged gene order by moving the n gene upstream of the s gene in the hcov genome . procedure : recombinant viruses were generated essentially as described by thiel et al . ( 2001 ). infectious hcov cdna clones were assembled in the vaccinia virus genome . vaccinia virus vhcov - vec - 1 contains a 22 . 5 kbp cdna fragment encoding the 5 ′ end of the hcov genome . this fragment was removed from the vaccinia virus genome by digestion with bsp 120i , digested with mlui and ligated to a cdna fragment of pmeδ rearranged that was obtained by digestion with mlui / eagi . pmeδ rearranged contains a cdna fragment that encodes the 3 ′ end of the hcov genome , but lacks gene 4a / b , without disturbing the other genes or transcription regulatory sequences , and in which the n gene , together with its trs , was positioned upstream of the s gene . pmeδ rearranged was derived from pme by using conventional pcr mutagenesis methods . the ligation products were ligated to vnoti / tk vaccinia virus dna . recombinant vaccinia viruses were generated as described above . recombinant viruses were plaque purified and characterized by pcr and southern blot analysis . finally , infectious hcov lacking genes 4a / b was generated as follows : chick kidney ( ck ) cells were infected with recombinant fowlpox virus expressing t7 rna polymerase . subsequently cells were transfected with dna isolated from the recombinant vaccinia virus containing the hcov cdna clone lacking genes 4a / b in combination with the rearranged gene order . at 3 days post - infection the culture medium was collected and used to infect human long fibroblast cells ( mrc - 5 ) to generate large amounts of recombinant hcov . the recombinant viruses were genetically confirmed by rt - pcr analysis . conclusion : recombinant hcov was generated that lacked the genes 4a / b in combination with a rearranged gene order . these viruses can function as a live attenuated vaccine against hcov - 229e . when combined with s gene constructs from hcov - oc43 and / or gene constructs from other human pathogens additional , multivalent vaccines can be generated . 1 . bredenbeek , p . j ., c . j . pachuk , a . f . noten , j . charite , w . luytjes , s . r . weiss , and w . j . spaan . 1990 . the primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus mhv - a59 ; 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