Patent Application: US-74404108-A

Abstract:
the invention relates to a method for producing a modified viral strain of a virus which is a member of the reoviridae family and , in particular , relates to vaccinal viral strains of the orbivirus genus .

Description:
the new approach to vaccine design uses a novel reverse genetics method developed by the inventors . it builds on the discovery that bluetongue virus transcripts are infectious by transfection [ 1 ], and allows the replacement of targeted segments with cloned versions of the viral genes . the method uses the novel approach of transfecting virus permissive cells with btv transcripts mixed with bacteriophage t7 in vitro transcripts of btv segments derived from cloned genes . virus containing the replacing genome segment is isolated by screening virus plaques . this new method of reverse genetics differs from the existing reverse genetics technologies used in the reoviridae family : 1 ) the helper virus - dependent method of roner et al . [ 3 ] successfully applied to mammalian orthoreoviruses . viral transcripts and viral dsrna are mixed with t7 in vitro transcripts and rescued using a helper virus infection ; 2 ) the helper virus - dependent method of komoto et al . [ 2 ] used to alter a capsid protein of rotavirus . the t7 transcript is generated in the cell using the vaccinia t7 rna polymerase system , and rescued using a helper virus strain ; and 3 ) the plasmid - based method of kobayashi et al . [ 4 ] used to make mutation in mammalian orthoreovirus genes . all the viral genome segments are generated in the cell using the vaccinia t7 rna polymerase system . the new approach uses reverse - genetics to produce vaccine strains which contain the immunologically relevant bluetongue proteins ( vp2 and vp5 ) from the serotype of interest with a universal background of the other viral proteins . this is coupled with inactivation of one or more essential viral genes through extensive mutation , by reverse genetics , which are provided by a complementing cell line . the virus produced can only be grown in the complementing cell line and is capable of only a single round of replication in other cells such as those of a vaccinated animal . the targeting of one or more btv enzymatic proteins ( polymerase , helicase and capping proteins ) or alternatively btv non - structural viral proteins for inactivation by reverse - genetics will allow the use of a diva strategy for surveillance purposes . the new approach also eliminates the problem of under - attenuation and reduces the time delay from identification of a new serotype to production of a vaccine strain , associated with attenuating new strains . the probability of reversion to virulence is greatly reduced through the use of extensive mutations in the viral genes targeted . the probability of reassortment with wild - type virus producing an infectious virus is also much reduced by the fact that the vaccine strain only undergoes a single replication cycle in the vaccinated animal . the new approach also avoids the need to confirm the inactivation of vaccine batches associated with inactivated vaccines . the present technique has been used to produce a disc ( disabled infectious single cycle ) vaccine for btv , wherein an essential gene ( vp6 ) was manipulated through the reverse genetics system and its function was destroyed through a large deletion . the vp6 deletion mutant ( btv1 delta vp6 ) was recovered using the reverse genetics technique in combination with a complementing cell line which supplied the vp6 protein in trans . characterisation of the growth properties of btv1 delta vp6 showed that it has the necessary characteristics for a btv disc vaccine i . e ., i ) expression of viral proteins in non - complementing mammalian cells ; ii ) no detectible infectious virus generated in non - complementing mammalian or insect cell lines ; and iii ) robust replication in the complementing vp6 cell line . additionally , the ability to create a virus express a foreign protein / peptide has been demonstrated using an ns3 complementing cell line in combination with a btv which has the enhanced green fluorescent protein ( egfp ) inserted in the centre of the ns3 gene . this allows the production of vaccine strains containing an immunological marker which can be detected in vaccinated animals to distinguish them from infected animals , i . e ., the diva concept ( distinguishing infected and vaccinated animals ). cell lines and virus . bsr cells ( a clone of bhk - 21 ) were cultured in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) supplemented with 5 % v / v foetal bovine serum ( fbs ), at 35 ° c . in 5 % co 2 . btv stocks were generated by infecting bsr cells at a multiplicity of infection ( moi ) of 0 . 1 and harvesting the medium at 3 - 4 days post - infection . viral stocks were stored at 4 ° c . purification of bluetongue virus cores . bsr cultures were infected with btv at an moi of 0 . 02 - 0 . 1 . transcriptionally active btv - 1 cores were purified as previously described and stored at 4 ° c . [ 1 ]. synthesis and purification of bluetongue virus mrna in vitro . btv cores were incubated at 40 μg / ml at 30 ° c . for 5 - 6 hours in btv core transcription buffer ( 100 mm tris hcl ph8 . 0 , 4 mm atp , 2 mm gtp , 2 mm ctp , 2 mm utp , 500 μm s - adenosylmethionine , 6 mm dtt , 9 mm mgcl 2 , 0 . 5 u / μl rnasin ® plus [ promega ]). btv core - derived mrnas were purified using the previously described method , and stored at − 80 ° c . [ 1 ]. rt - pcr amplification of btv - 1 genome segments . cdna copies of each btv - 1 genome segment were amplified from viral dsrna in a sequence independent manner using the flac method [ 18 ]. briefly , the hairpin anchor primer was ligated to viral dsrna as described , followed by cdna synthesis from gel purified genome segments with superscript ™ iii ( invitrogen ) at 10u / μl , 55 ° c . for 1 hour . pcr amplification was performed using 5 ′ phosphorylated flac 2 primer ( 5 ′ gagttaattaagcggccgcagtttagaatcctcagaggtc3 ′; seq id no : 1 ) with kod hot start dna polymerase ( novagen ). paci and noti sites are in bold type . t7 plasmid clones used for the synthesis of btv transcripts . cdna plasmid clones were constructed for btv - 10 genome segment 10 ( pns3bsmbi ), segment 5 ( pvp5bsmbi ), and segment 2 ( pvp2bsmbi ), and for all ten segments of the btv - 1 genome . a mutant version of the btv - 10 segment 10 clone , containing an introduced haeii site ( pns3hae ), and a mutant version of the btv - 1 segment 8 clone , containing an introduced bglii site ( pbtv1s8bgl ) were also constructed . the functional cassette in each plasmid clone contained a t7 promoter and a bsmbi , bsai , or bpii site , with the btv genome segment located between these elements . the btv genome segment in each clone was positioned relative to the other two sequence elements such that the t7 transcript derived from plasmid digested with bsmbi , bsai , or bpii was predicted to have exactly the same sequence as the mrna strand of the corresponding btv genome segment ( fig2 ). synthesis of btv transcripts from cdna plasmid clones . t7 plasmid clones were digested with bsmbi , bsai , or bpii then extracted once with phenol / chloroform and once with chloroform . each digested plasmid was precipitated with isopropanol in the presence of 0 . 15m sodium acetate . dna pellets were washed twice in 70 % ( v / v ) ethanol and dissolved at 1 μg / μl in 10 mm tris hcl ph8 . 0 . transcripts with a 5 ′ cap analogue were generated from the digested t7 plasmid clones using the mmessage mmachine ® t7 ultra kit ( ambion ), using a 4 : 1 ratio of anti - reverse cap analogue to rgtp . t7 btv transcripts were extracted once with phenol / chloroform followed by one extraction with chloroform . unincorporated rntps were removed by size fractionation using microspin ™ g - 25 columns ( ge healthcare ) according to the manufacturer &# 39 ; s instructions . the t7 btv transcripts were precipitated with an equal volume of isopropanol in the presence of 0 . 15m sodium acetate . rna pellets were washed twice in 70 % ( v / v ) ethanol and dissolved in sterile diethylpyrocarbonate ( depc ) treated water , and stored at − 80 ° c . denaturing agarose gel electrophoresis . purified btv ssrna was analyzed by electrophoresis on 1 % agarose in mops ( morpholinepropanesulfonic acid ) electrophoresis buffer in the presence of formaldehyde , using standard techniques [ 19 ]. transfection of cultured cells to recover bluetongue virus with one or two cdna - derived genome segments . btv mrnas derived from transcribing cores were mixed with one or more t7 btv transcripts in opti - mem ® i , in the presence of 0 . 1 u / μl rnasin ® plus ( promega ). the rna mixture was incubated at 20 ° c . for 30 minutes before mixing with lipofectamine ™ 2000 reagent ( invitrogen ) [ see below ]. confluent bsr monolayers in 6 well plates were transfected with 1 . 5 μg btv mrna mixed with 0 . 75 μg of each t7 btv transcript using lipofectamine ™ 2000 reagent according to the manufacturer &# 39 ; s instructions . at 4 hours post - transfection the culture medium was replaced with a 6 ml overlay consisting of minimal essential medium ( mem ), 2 % fbs , 1 . 5 % w / v agarose type vii ( sigma ). assays were incubated at 35 ° c ., 5 % co 2 for 72 - 96 hours to allow plaques to appear . transfection of cultured cells to recover bluetongue virus entirely from cdna - derived genome segments . 300 - 400 ng of each t7 btv transcript were mixed , as described above , to produce a complete genome set of t7 btv transcripts . transfection of bsr monolayers was performed as described above . preparation of dsrna from transfection - derived btv plaques . each plaque was picked into 500 μl dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ), 5 % fbs , and 200 μl was used to infect 1 . 5 × 10 6 bsr . infected cells were incubated at 35 ° c . in 5 % co 2 for 72 - 96 hours to allow amplification of the btv . viral dsrna was purified from infected bsr cells as previously described [ 1 ]. screening transfection - derived btv plaques for reassortants containing the introduced genome segments . where the genome segment being introduced migrated at a different rate on polyacrylamide gels , screening was done by electrophoresis of the dsrna on 9 % polyacrylamide gels in tris / glycine buffer ( ph8 . 3 ). gels were post - stained for 30 minutes with ethidium bromide . where screening was not possible on the basis of the migration rate , rt - pcr ( reverse transcription polymerase chain reaction ) followed by restriction endonuclease digestion was used to discriminate between reassortants and wild - type btv . cdna was synthesized from 100 ng heat denatured viral dsrna with superscript ™ iii ( invitrogen ) using forward and reverse primers flanking the target region , at 55 ° c . for 1 hour . the target region was pcr amplified using taq dna polymerase with the same forward and reverse primers and digested with restriction endonucleases . products were resolved by electrophoresis in agarose gels containing ethidium bromide , in tris - borate - edta buffer . sequence analysis of rt - pcr products was done using dye terminators on abi 3730xl sequencing machines using the value read service of mwg biotech [ 20 ]. construction of pns3hae and pbtv1s8bgl . pns3bsmbi was altered to contain an additional haeii site by site - directed mutagenesis using primers s10_mt_hae — 409f and s10_mt_hae — 409r by the method of weiner et al [ 17 ]. similarly the wildtype btv - 1 s8 clone was altered to introduce a bglii site using primers 5 ′ btv1_s8_bglii and 3 ′ btv1_s8_bglii . clones were screened for the presence of the introduced site by haeii or bglii digestion , and the expression cassette sequenced to identify clones containing no adventitious mutations using the value read service of mwg biotech . construction of stable cell lines expressing the btv vp6 or ns3 protein . the coding regions for btv - 10 vp6 and btv - 1 ns3 were amplified by pcr and cloned into the puromycin selectable plasmid pcaggs / mcs - pm1 [ 29 ], to obtain pcaggnp6 and pcagg / ns3 , respectively . bsr cells were transfected with pcaggnp6 or pcagg / ns3 using lipofectamine ™ 2000 reagent ( invitrogen ) and 48 hours post - transfection were trypsinized and selected with puromycin at 7 . 5 μg / ml . isolated resistant colonies were cultured and the expression of the vp6 or ns3 protein was tested by immunoblotting using an appropriate antibody . the vp6 and ns3 expressing lines were termed bsr vp6 and bsr ns3 . the recovery of btv , using the complementing bsr vp6 or bsr ns3 cell line . a btv - 1 segment 9 clone with an out of frame deletion of nucleotides 301 - 743 ( out of 1049 nt ) was constructed , pbtv1 s9delta ( fig1 ). the corresponding mutant virus , btv1 delta vp6 , was recovered , using the bsr vp6 cell line in the place of wildtype bsr cells during transfection . similarly , a btv - 1 segment 10 clone with the enhanced green fluorescent protein ( egfp ) inserted in the centre of the ns3 gene was constructed , pbtv1 s10gfp . the corresponding gfp expressing virus , btv1 s10gfp , was recovered , using the bsr ns3 cell line during transfection . passaging of btv1 delta vp6 and btv1 s10gfp . btv1 delta vp6 was passaged on the bsr vp6 cell line , and the titre determined by plaque assay also using the bsr vp6 cell line . btv1 s10 gfp was passaged on the bsr ns3 cell line , and the titre determined by plaque assay also using the bsr ns3 cell line . multi step growth curves of btv1 delta vp6 . bsr or c6 / 36 cells were infected at an moi of 0 . 5 in twelve well dishes in 250 μl of dmem or l15 medium , respectively . wells were washed three times in 1 ml pbs and incubated under standard growth conditions in 1 ml of growth medium . wells were harvested at time intervals and the total virus determined by plaque assay titration on the bsr vp6 complementing cell line . primers . ecot7_s9_f ( 5 ′ ctaggaattctaatacgactcactatagttaaaaaatcgcatatgtc agctgc3 ′; seq id no : 15 ). ecobsmb_s9_r ( 5 ′ cagtgaattcgtctccgtaagtgtaaaatcgccctacg3 &# 39 ;; seq id no : 16 ) btv1s10t7ecori ( 5 ′ cggaattctaatacgactcactatagttaaaaagtgtcgctgccatg cta3 ′; seq id no : 17 ) ns3bsmbi rev ( 5 ′ gtaagtgtgtagtatcgcgcacc3 ′; seq id no : 18 ) reassortment of genome segments by co - transfection with btv mrna from two serotypes . the recovery of infectious btv from core - derived transcripts through the transfection of permissive cells has been demonstrated [ 1 ]. with the aim of producing a reverse genetics system for btv , the introduction of genome segments from one btv serotype into another was investigated as an intermediate step , prior to the introduction of cdna - derived genome segments . infectious core - derived transcripts were prepared from btv - 1 and btv - 9 as previously described [ 1 ]. the transcripts from the two serotypes were either generated simultaneously in the same transcription reaction or prepared separately and then mixed . confluent bsr monolayers were transfected with the transcript mixtures and virus was amplified from the resulting plaques . the dsrna was purified from each amplified plaque and the origin of genome segments was determined by electrophoresis on non - denaturing page gels , which allow the discrimination of some genome segments from different isolates . when co - synthesized transcripts from btv - 1 and btv - 9 were used , progeny viruses were generated which had genome segments from both parental sources of transcripts [ reassortants ] ( fig1 a ). lane 1 contains a reassortant which has segment 1 and segment 4 of btv - 1 in a genetic background of segments which migrate as btv - 9 . similarly , lane 2 contains a reassortant with the segment 3 of btv - 9 in a btv - 1 genetic background , and lane 3 contains a reassortant with segment 1 of btv - 9 in a btv - 1 genetic background . when btv - 1 and btv - 9 transcripts were prepared separately and mixed prior to transfection , reassortant progeny viruses were also generated , indicating that co - synthesis of transcripts is not necessary for reassortment to occur ( fig1 b ). these data demonstrated that co - transfection with a mixture of viral transcripts is a viable strategy for the introduction of genome segments from a separate source into the btv genome . the introduction of a btv segment derived from a cdna clone into the btv - 1 genome . the targeted replacement of a genome segment with a t7 transcript derived from a cdna clone was subsequently investigated as a model for the introduction of cloned sequences into the btv genome . the introduction of the btv - 10 segment 10 t7 transcript into the genome of btv - 1 was chosen to allow the rapid screening of plaques based on the faster migration rate of segment 10 of btv - 10 compared to btv - 1 , on page gels . the btv - 10 segment 10 t7 transcript was produced from pns3bsmbi which has a t7 promoter to generate the correct 5 ′ end sequence and a bsmbi site to generate the correct 3 ′ end sequence ( fig2 ). btv - 1 transcripts produced from transcribing cores were mixed with the btv - 10 segment 10 t7 transcript and used to transfect confluent bsr monolayers . a 5 : 1 molar ratio of t7 transcript to the corresponding core - derived mrna was found to be best and was used in all experiments . increasing the ratio of t7 transcript to btv1 transcripts reduced the total number of plaques recovered ( data not shown ). typically ˜ 50 plaques were recovered from each well following the transfection of a six well dish with 1 . 5 μg core - derived transcripts plus 0 . 75 μg t7 transcript . virus was amplified from these plaques and the dsrna purified . the origin of genome segment 10 was initially determined by electrophoresis of the dsrna on page gels . dsrna genome profiles containing the faster migrating segment 10 from btv - 10 were obtained with a sufficiently high frequency ( 15 - 80 %) to make screening of plaques a viable option ( fig3 a ). the identity of segment 10 was confirmed using rt - pcr followed by sequencing of a region showing variability between type 1 and type 10 ( fig3 b , c and d ). these data demonstrated the recovery of the plasmid - derived btv - 10 segment 10 into the genome of viable btv - 1 . btv naturally produces reassorted progeny genomes when a cell is infected with two different strains [ 21 ]. to abolish the possibility of natural reassortment between two viruses being the origin of the segment 10 reassortants , a btv - 10 segment 10 clone containing an introduced silent haeii site ( pns3hae ) as a marker was made by the site directed mutagenesis of pns3bsmbi . bsr monolayers were transfected with a mixture of btv - 1 core - derived mrnas and the btv - 10 segment 10 t7 transcript containing the introduced mutation , derived from pns3hae . the recovery of virus containing this mutant btv - 10 segment 10 sequence was initially screened for by its increased migration rate on page gels ( fig4 a ). the introduction of the haeii site into segment 10 of the btv genome was confirmed by rt - pcr of dsrna from plaque purified virus , followed by haeii digestion ( fig4 b ), and by sequencing of the rt - pcr product ( fig4 c and d ). segment 10 was determined to be the same as the segment encoded in pns3hae throughout its length , by sequencing a full - length rt - pcr product ( data not shown ). the simultaneous introduction of two btv - 10 segments derived from cdna clones into the btv - 1 genome . to assess this possibility of simultaneously altering two genome segments the introduction of the outer capsid protein encoding segments ( segments 2 and 5 ) from btv - 10 into a background of btv - 1 genome segments was investigated . replacement of these genome segments with the segments from another serotype would enable the serotype of the virus to be altered . t7 transcripts derived from segments 2 and 5 of btv - 10 were prepared from pvp2bsmbi and pvp5bsmbi respectively and mixed with btv - 1 core - derived mrnas at a 5 : 1 ratio of each t7 transcript to the corresponding core - derived transcript . confluent bsr cell monolayers were transfected with the rna mixture , and dsrna prepared from the recovered plaques . the origin of segments 2 and 5 were initially assessed by their migration rate on page gels ( fig5 a ). both segments 2 and 5 from btv - 10 were recovered together at high frequency ( 20 - 80 %). the identity of the segments was confirmed by rt - pcr followed by restriction digestion ( fig5 b and c ). the complete sequence of segment 2 and segment 5 was determined to be that of btv - 10 by rt - pcr amplification and sequencing ( data not shown ). no progeny ( 0 out of 19 plaques from three independent experiments ) were recovered which contained only segment 2 or only segment 5 from btv - 10 , suggesting that viruses containing segment 2 from one parent and segment 5 from the other parent are either of reduced viability , or are generated at a lower frequency than the double reassortants . this phenomenon was further supported when the introduction of segment 2 or segment 5 from btv - 10 into btv - 1 was attempted singly and no reassortant progeny were recovered ( data not shown ). the recovery of stv entirely from t7 transcripts . while the above method is a viable reverse genetics system which allows the manipulation of btv genome segments the screening of reassortant plaques from wildtype plaques could hinder the recovery of slow growing mutants . the ideal reverse genetics system would permit the assembly of infectious virus entirely from t7 transcripts . to maximise the probability of having a viable clone for every genome segment rt - pcr amplification of each genome segment was performed with dsrna of btv - 1 , using the sequence - independent flac method developed for dsrna templates [ 18 ]. each rt - pcr product was cloned into puc19 [ 22 ] and the complete sequence of each clone was compared with the complete sequence of each rt - pcr product in order to determine whether a representative molecule had been cloned in each case ( data not shown ). alternative clones were sequenced when coding changes or any differences within 200 nt of the ends of the cloned genome segment were present . once a complete set of ten clones was obtained each genome segment was pcr amplified using the high fidelity iod hot start dna polymerase ( novagen ) to introduce a t7 promoter directly upstream of the genome segment and a restriction enzyme site directly downstream ( fig2 ). these functional cassettes were also cloned in puc19 . t7 transcripts synthesized using the restriction digested plasmid clones were determined to be of the expected size when resolved on 1 % denaturing agarose gels ( fig6 a and b ). t7 transcripts made from restriction digested plasmids were mixed in equal ratio by weight , and 3 - 4 μg in total used to transfect confluent bsr monolayers . transfected monolayers were overlaid with agarose and plaques appeared at 3 - 6 days post - transfection ( fig7 a ). dsrna from amplified plaques was compared with btv - 1 stock virus on page gels , and found to be indistinguishable , confirming that btv - 1 had been recovered ( fig7 b ). to substantiate further that btv could be derived from t7 transcripts a mutant of the btv - 1 segment 8 t7 clone was made which contained an introduced silent bglii site , pbtv1s8bgl . plaques were recovered from transfections with a complete set of t7 transcripts where the segment 8 bglii marker transcript replaced the wildtype s8 transcript ( fig8 a ). dsrna from amplified plaques was found to be indistinguishable when compared with btv - 1 stock virus on page gels ( fig8 b ). plaques were amplified by infection of bsr cells and the s8 segment amplified by rt - pcr using primers ns2_bam_r and ns2_bam_t7 — f . digestion of the rt - pcr product demonstrated that a bglii site had been introduced ( fig9 a ). the rt - pcr products were sequenced using the btv1 — s8 — 627r primer confirming the introduction of the marker sequence ( fig9 b ). these data demonstrate that it is possible to recover btv from a complete genomic set of t7 transcripts , and introduce viable mutations using this system . recovery of the btv1 delta vp6 virus . to generate a disc vaccine strain a large out of frame deletion in the essential protein vp6 was made in a wildtype segment 9 clone to generate pbtv1 s9delta ( fig1 ). a complete genomic set of in vitro synthesised t7 transcripts was made as previously described , but containing the deleted segment 9 transcript instead of the wildtype segment 9 transcript . the corresponding mutant virus , btv1 delta vp6 , was recovered , using the bsr vp6 cell line in the place of wildtype bsr cells during transfection . cytopathic effect ( cpe ) indistinguishable from btv cpe was visible in transfected wells by 5 days post - transfection , indicating the recovery of infectious virus ( data not shown ). btv1 delta vp6 replicates in the bsr vp6 cell line . to determine whether the btv1 delta vp6 virus replicates robustly in the bsr vp6 cell line it was compared to wildtype btv - 1 replication in wildtype bsr cells . cpe produced by the infection of the bsr vp6 cell line with btv1 delta vp6 was equivalent to that produced when wildtype btv - 1 was used to infect wildtype bsr cells , indicating that there is no gross defect in the replication of btv1 delta vp6 ( see fig1 ). the plaque - forming potential of btv1 delta vp6 was assessed using the bsr vp6 cell line ( see fig1 ). the bsr vp6 cell line was found to complement the growth of btv1 delta vp6 such that plaques of a normal appearance were produced , enabling the normal titration of btv1 delta vp6 . btv1 delta vp6 virus could be grown to comparable titres to wildtype btv - 1 ( over 10 7 infectious units / ml ) using the bsr vp6 cell line . these data demonstrate that the btv1 delta vp6 virus replicates efficiently in the complementing bsr vp6 cell line . characterisation of genome segment 9 of btv1 delta vp6 . btv1 delta vp6 was propagated on the bsr vp6 cell line , and the viral double - stranded rna extracted and purified as previously described . the btv1 delta vp6 genome lacks the wildtype s9 segment and contains a smaller genome segment , corresponding to the replacement of the wildtype s9 segment with the deleted s9 segment ( fig1 a ). the s9 segment from btv1 delta vp6 was amplified by rt - pcr using primers annealing at the ends of the s9 segment ( ecot7_s9_f and ecobsmb_s9 — r ), generating the expected product of 650 nt ( fig1 b ), and sequenced with the same primers . the observed sequence of the amplified genome segment was identical to that in the pbtv1 s9delta ( data not shown ), demonstrating that the virus recovered was constructed correctly . characterisation of the growth of btv1 delta vp6 on non - complementing cell lines . an important characteristic of a disc vaccine strain is that it should be unable to complete a replication cycle in the host organism . to evaluate whether btv1 delta vp6 is defective , the mammalian line bsr ( a bhk - 21 sub clone ), and the insect cell line c6 / 36 , lines in which btv replicates efficiently , were used as proxies for the mammalian and insect hosts . both cell lines were infected with btv1 delta vp6 , and the total virus produced monitored by plaque assay using the bsr vp6 complementing cell line , over 72 hours . btv1 delta vp6 did not replicate in either cell line , whereas wildtype btv - 1 showed efficient replication in both lines ( fig1 a and b ). this data demonstrates that the disruption of the vp6 gene has rendered the virus incapable of replicating when the vp6 protein is not supplied by the complementing cell line . btv1 delta vp6 expresses viral proteins in non - complementing bsr cells . any disc vaccine must express virus proteins in the host organism , in order to induce an immune response . to evaluate whether btv1 delta vp6 can express viral proteins wildtype bsr cells were used as a proxy for the mammalian host . detection of the non - structural protein , ns2 , was used as a marker for viral protein expression . in a comparison of the protein expression of the btv1 delta vp6 virus with wildtype btv - 1 the ns2 protein was expressed at increasing levels with time in both btv1 delta vp6 infected bsr and btv - 1 infected bsr ( fig1 ). the level of protein expression from the btv1 delta vp6 virus was lower than that for wildtype btv - 1 , as would be expected for a defective virus . this data demonstrates that the btv1 delta vp6 virus expresses virus protein in non - complementing bsr cells . marker antigens can be added to the btv genome . the ability to express a marker antigen / peptide from the btv genome would allow a diva compliant vaccine strain to be created ( diva : differentiating infected from vaccinated animals ). to demonstrate that this is possible using the reverse genetics approach in combination with complementing cell lines , a clone containing an in frame fusion of ns3 with egfp was made , pbtv1 s10gfp . a complete genomic set of in vitro synthesised t7 transcripts was made as previously described , but containing the ns3 - egfp fusion transcript instead of the wildtype segment 10 transcript . the corresponding mutant virus , btv1 s10gfp , was recovered as described , using a bsr ns3 complementing cell line in the place of wildtype bsr cells during transfection . when non - complementing c6 / 36 cells were infected with btv1 s10gfp the expression of egfp was confirmed by its fluorescence under uv light ( data not shown ) characterisation of genome segment 10 of btv1 s10gfp . the genome of btv1 s10gfp lacks the wild type s10 segment and contains a bigger segment , corresponding to the s10 gfp fusion ( fig1 a ). the s10 segment from btv1 s10gfp was amplified by rt - pcr using primers annealing at the ends of the s10 segment ( btv1s10t7 ecori and ns3bsmbi rev ), and the expected product of 1446 nt was amplified ( fig1 b ). the rt - pcr product from btv1 s10gfp was sequenced and the presence of the egfp gene was confirmed ( see fig1 ), demonstrating that the virus recovered was constructed correctly . two changes which increase the efficiency of the bluetongue virus recovery from plasmid - derived t7 transcripts have been made . both of these changes are alterations to the transfection method described above . each improvement results in a ˜ 10 fold increase in the efficiency of recovering virus from plasmid - derived transcripts , and together result in ˜ 100 fold increase in the recovery of virus . the cells are transfected twice ( instead of once ), with a complete set of ten plasmid derived transcripts on each occasion , as described above . the transfections are performed 18 hours apart , and result in an increase in virus recovery of ˜ 10 fold , over using a single transfection . the increase in recovery of virus using the double transfection method was observed when using ssrna made from btv cores ( fig1 ) and when using a complete set of t7 transcripts made from ten cdna clones ( fig1 ). modification # 2 , omission of genome segments 2 , 5 , 7 , and 10 from the first transfection : the cells are transfected twice , as in modification # 1 , but the t7 transcripts encoding genome segments 2 , 5 , 7 , and 10 are omitted from the first transfection . the second transfection uses a complete set of ten transcripts . the transfections are performed 18 hours apart , and result in a further increase in virus recovery of ˜ 10 fold over using the double transfection described above ( fig2 ). the two approaches described represent alternative reverse genetics systems for btv , using either a mixture of authentic viral transcripts and t7 transcripts , or a complete genomic set of t7 transcripts . they extend the discovery that btv transcripts are infectious when used to transfect permissive cells [ 1 ], and demonstrate that in vitro synthesized t7 transcripts with a cap analogue at the 5 ′ end can functionally substitute for transcripts synthesized by core particles . the recovery of progeny virus with genome segments originating from two separate core - derived mrna preparations established the principle of introducing exogenous transcripts into the genome of btv by mixing with authentic viral transcripts ( fig1 b ). the observation that mixing the mrna preparations after transcription was effective in producing reassortants allowed for the possibility of using plasmid - derived transcripts in combination with core - derived mrnas to introduce targeted mutations into the btv genome . the introduction of the btv - 10 segment 10 transcript into the genome of btv - 1 was investigated to determine whether the facile introduction of plasmid - derived transcripts into infectious btv could be achieved . this model system showed that using an excess of the t7 transcript generated reassortant plaques at a frequency which made the screening of individual plaques practical ( 15 - 80 %). the initial screening of plaques by the rate of migration of the segment 10 dsrna on page gels ( fig3 ) was confirmed by sequencing of the rt - pcr product ( fig3 ). the high efficiency of reassortment between the t7 transcript and authentic viral transcripts has meant that a selectable marker approach was not required . the introduction of the haeii site marker mutation into segment 10 of btv confirmed that reassortants were derived from the in vitro synthesized segment 10 t7 transcript ( fig4 ). the haeii - containing segment 10 was recovered with a similar efficiency to wildtype btv - 10 segment 10 . both segment 10 reassortant viruses demonstrated no gross replication deficiency compared to wildtype btv - 1 ( data not shown ). this shows that genome segment 10 from btv - 10 is functionally compatible with a background of btv - 1 genome segments both at the levels of rna packaging and replication , and ns3 / ns3a protein function . the simultaneous reassortment of two t7 transcripts into the btv genome to replace the antigenically important outer capsid proteins of btv - 1 with those from btv - 10 cdna clones was shown to be possible using an excess of both t7 transcripts ( fig5 ). progeny plaques containing the btv - 10 segment 2 and 5 were recovered at a 20 - 80 % frequency , but no reassortants were isolated containing only segment 2 or segment 5 from btv - 10 . this demonstrates that together segments 2 and 5 of btv - 10 can functionally substitute for the corresponding btv - 1 genome segments , and suggests there is incompatibility between segment 2 and segment 5 from these two serotypes at some level . the encoded proteins , vp2 and vp5 , are highly variable due their exposure to immune selective pressure on the surface of the virus particle . our favoured explanation is that the vp2 and vp5 proteins have co - evolved and that the three dimensional structure of vp2 from one serotype is not necessarily compatible with the vp5 from another serotype . this is consistent with the previously reported incompatibility of the vp2 and vp5 proteins from some serotype combinations observed in the generation of btv virus - like particles [ 23 , 24 ]. incompatibility of segment 2 and segment 5 in some serotype combinations at an rna packaging level is another possibility . the simultaneous introduction of both outer capsid proteins from another serotype allows the possibility of producing vaccine strains to different serotypes based on a consistent genetic background . the high amino acid sequence divergence between the vp2 proteins of btv - 1 and btv - 10 ( 40 % amino acid identity ) suggest that the assembly of varied vp2 + vp5 pairs onto the conserved core of the btv virion will be possible . the recovery of btv - 1 from a complete set of t7 transcripts was investigated to determine whether virus with a fully defined genome could be recovered from cdna clones . transfection of bsr monolayers with the ten t7 transcripts was found to lead to the production of plaques ( fig7 ). the recovery of a bglii marker mutation into the s8 segment confirmed that the virus recovered was derived from the t7 transcripts used in the transfections ( fig8 and 9 ). the recovery of infectious btv from t7 transcripts alone demonstrates that t7 transcripts synthesized in the presence of cap analogue are functionally equivalent to authentic viral transcripts at all stages of the replication cycle . the t7 transcripts must be translated , selected during genome packaging , and act as templates for negative strand synthesis if virions are to be generated . furthermore , after negative strand synthesis the resulting dsrna genome segment must be competent for transcription in the next round of infection . the recovery of btv from t7 transcripts leads to the recovery of ˜ 100 fold less plaques than when an equivalent quantity of core - derived viral transcripts are used . the lower efficiency may derive from the fact that only a proportion of t7 transcripts generated in the presence of a cap analogue have the cap analogue incorporated at the 5 ′ end . in addition to being poorly translated the uncapped transcripts may be defective during rna packaging , negative strand synthesis or during transcription in the next round of infection . importantly the uncapped transcripts have a 5 ′ triphosphate moiety which is known to be a pathogen - associated molecular pattern ( pamp ) recognised by rig - i and leading to the induction of antiviral responses [ 25 - 28 ]. alternatively , the technical issues associated with generating ten ssrna molecules with the conserved terminal sequences intact may contribute to the lower recovery observed with t7 transcripts . the recovery of btv entirely from plasmid - derived transcripts allows the generation of btv mutants with a consistent genetic background . this approach will be useful in the recovery mutants which are expected to have a slow replication phenotype , as the screening of plaques for the desired mutant among wildtype plaques is not required . in such cases there would be no background of faster replicating virus which may hamper the recovery of the slower replicating mutants . this approach could also be used to recover primary / low passage isolates of btv , avoiding gradual alteration of these strains to cell culture conditions . the recovery of reassortants containing one plasmid - derived genome segment requires the construction of a single clone or pcr product and is applicable to any genome segment . this single construct approach can be used to investigate individual viral genes without the need to construct a full set of ten clones . as reoviridae members have a common replication strategy both the reassortment and t7 only reverse genetics approaches may be applicable to a wide range of viruses which lack a reverse genetics system . the use of in vitro synthesized t7 transcripts in both approaches obviates the requirement to supply t7 rna polymerase by infecting with a recombinant poxvirus , which may interfere with the replication of the virus being recovered . alternative reverse genetics strategies have been used successfully for other genera in the reoviridae [ 2 - 4 ]. the first reverse genetics system was a helper virus system for the mammalian orthoreoviruses [ 3 ]. this approach combined reovirus infection of permissive cells and transfection with viral dsrna , viral mrna , a t7 transcript , and in vitro translated viral mrna . another helper virus approach has allowed the replacement of a rotavirus outer capsid protein with the corresponding protein from another serotype [ 2 ]. the expression of the introduced genome segment was driven in vivo by the recombinant t7 vaccinia virus system , and selective pressure against the equivalent helper virus protein was provided by the use of antibody selection . most recently mammalian orthoreovirus has been recovered using a plasmid - based system similar to the t7 driven systems first used with negative strand viruses [ 4 ]. in this case expression of all ten genome segments was driven in vivo by the recombinant t7 vaccinia virus system . all the successful reverse genetics strategies have several notable features in common ; 1 ) the genome segments derived from cdna clones are provided as message sense transcripts in the transfected cell . 2 ) the cdna - derived transcripts used have the same 5 ′ end and 3 ′ end sequences as the corresponding viral transcript . the 5 ′ ends are generated through the use of a t7 promoter with the appropriate sequence , and the 3 ′ ends are generated through the use of the hepatitis delta ribozyme in vivo or a restriction enzyme site in vitro . all genome segments in reoviridae members have short conserved sequences at their extreme 5 ′ and 3 ′ ends the functions of which are still being elucidated . 3 ) like the authentic viral transcripts the cdna - derived transcripts are capped , either in vitro with a cap analogue or in vivo through the cross - capping activity associated with the vaccinia t7 rna polymerase recombinant [ 13 ]. to achieve infectious virus recovery gene expression must be sufficient to allow the assembly of progeny core particles , which themselves are transcriptionally active and lead to an amplification of gene expression . a high level of gene expression is needed to assemble these incomplete virions , and without the presence of the cap structure at the 5 ′ end of the cdna - derived transcripts their stability and level of translation would much reduced [ 14 ]. disc viruses lacking a viral gene have been generated using the combination of the btv reverse genetics system and complementing cell lines . the viruses recovered fulfil the following criteria for a btv disc virus vaccine strain : 1 ) the expression of viral proteins in non - complementing mammalian cells ( fig1 ); 2 ) no detectible infectious virus generated in non - complementing mammalian or insect cell lines ( fig1 ); and 3 ) robust replication in the corresponding complementing cell line ( fig1 ). additionally , the ability to express foreign proteins or peptides has been demonstrated using the insertion of the egfp protein into the ns3 open reading frame , allowing the production of vaccine strains containing an immunological marker which may be detected in vaccinated animals to distinguish them from infected animals , the diva concept ( distinguishing infected and vaccinated animals ). the t7 transcripts or viral ssrna have two functions in the replication cycle of members of the reoviridae family ; 1 ) to be translated to generate the viral proteins ; and 2 ) to act as replication intermediates for the synthesis of new double - stranded genome segments . for rescue to be successful in a cell , using the single transfection approach , a proportion of the transcripts must remain available to be packaged and replicated in assembling progeny virus particles . this is expected to be a limiting step in the efficiency of virus recovery , as unlike a normal infection new transcripts are not being continually synthesised from an infecting core particle . to increase the efficiency of recovery a second transfection was performed to introduce additional transcripts for packaging at a time when morphogenesis would be expected to have reached the packaging stage . the predicted increase in the recovery of virus was observed using viral ssrna or t7 transcripts ( fig1 and 19 ), and was found to be ˜ 10 fold . to further increase efficiency of recovery genome segments were omitted from the first transfection so that morphogenesis could not proceed beyond the assembly of the inner layer of the capsid . this approach was adopted to arrest assembly at the stage where packaging is expected to occur . the genome segment coding assignments for the omitted segments are : segment 2 encodes vp2 ( outer capsid ), segment 5 encodes vp5 ( outer capsid ), segment 7 encodes vp7 ( middle layer of capsid ), and segment 10 encodes ns3 ( required for virus egress ). the consequence of omitting segments 2 , 5 , 7 , and 10 is that the middle layer and outer layer of the triple layered capsid are not synthesised , and the egress protein ns3 is not present . it was found that arresting morphogenesis in this way increased the recovery of virus a further ˜ 10 fold when a complete set of ten transcripts was provided in the second transfection . 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