Patent Application: US-72276905-A

Abstract:
the invention relates to the field of influenza vaccine production . influenza vaccines have been produced in embryonated hens &# 39 ; eggs for over 50 years , but recently there have been considerable efforts to develop cell culture systems for vaccine production . the invention provides a nucleic acid comprising an influenza gene segment and a bacteriophage polymerase promotor or a complementary strand of said nucleic acid , and a cell comprising such a nucleic acid capable of producing desired influenza virus . furthermore , the invention provides a composition comprising a cell or material derived from a cell according to the invention and a virus or material derived from a viral particle according to the invention .

Description:
generation of recombinant influenza a virus using a t7 rna polymerase based reverse genetics system for a long time , the fundamental research of influenza a viruses has been hampered by the lack of availability of efficient reverse genetics systems . although the earliest reverse genetics techniques for negative stranded rna viruses were in fact developed for influenza a virus ( 7 , 18 ), the rescue of this virus exclusively from recombinant dna was achieved only recently ( 9 , 20 ). influenza a virus is a negative strand rna virus . during the virus replication cycle , three types of rna are produced : negative sense genomic viral rna ( vrna ), positive sense rna complementary to the genomic rna ( crna ) and positive sense messenger rna ( mrna ). whereas the vrna and crna contain essentially unmodified ends , the mrna is capped and has a poly ( a ) tail ( 16 ). rna polymerase i ( poli ) is a nucleolar enzyme that transcribes ribosomal rna ( rrna ) and is abundantly expressed in growing cells . like vrna , rrna has no cap and no poly ( a ) tail ( 23 ). hobom and colleagues ( 19 , 21 , 29 ) successfully produced artificial influenza virus vrna - like segments with precise 5 ′ and 3 ′ ends using poli . transcription of cdna cloned in the context of a poli promoter - terminator cassette allowed the generation of vrna - like molecules with correct 5 ′ and 3 ′ ends ( 29 ). subsequent studies which involved helper influenza virus demonstrated that these genomic vrna molecules could be recognized and replicated by the influenza virus polymerase complex and packaged into progeny influenza viruses . this system allowed the generation of influenza viruses containing mutations in one of the viral gene segments or an additional gene segment , thus allowing studies of viral genes and their products . as the result of the use of helper virus , selection of transfectant virus was required , this is rather cumbersome . neumann et al . designed a poli system for the recovery of influenza a viruses entirely from cloned cdna ( 20 ). cdnas encoding full - length vrnas of the influenza a virus were cloned between the human poli promoter and the mouse poli terminator . in principle , transfection of these eight plasmids into eukaryotic cells should result in the synthesis of all eight influenza vrnas . human embryonic kidney cells ( 293t ) were cotransfected with these eight vrna expression plasmids and plasmids expressing the viral nucleoprotein and the polymerase proteins pb2 , pb1 and pa from an rna polymerase ii ( polii ) promoter . the vrnas synthesized by the cellular poli were packaged into rnps and amounts greater than 1 × 10 3 plaque - forming units of infectious virus per ml ( pfu / ml ) of supernatant were recovered . cotransfection with plasmids expressing the remaining viral structural proteins led to a substantial increase in virus production , namely 3 × 10 4 to 5 × 10 7 pfu / ml ( 20 ). fodor et al . reported a similar system for the recovery of influenza a virus ( 9 ). this system depended on eight plasmids encoding all eight vrna cdnas , flanked by a human poli promoter but which contained a hepatitis δ virus ribozyme ( hδvrib ) sequence rather than a poli terminator sequence . these plasmids were cotransfected into vero cells with four plasmids expressing the pb1 , pb2 , pa and np proteins from an adenovirus type 2 major late promoter . using equal amounts of each of the expression plasmids , fodor et al . reported a rescue rate of 1 to 2 infectious viral particles from 10 6 transfected cells ( 9 ). we had designed a similar reverse genetic system to produce recombinant influenza virus a / pr / 8 / 34 . we concluded that virus titers of ˜ 10 4 can be obtained without virus replication in the transfected cell culture which can be boosted to & gt ; 10 7 when the virus is allowed to replicate ( 4 ). since these poli - driven systems required the cotransfection of 12 - 16 plasmids , the use of cell lines that can be transfected with high efficiencies were necessary for efficient production of recombinant virus . subsequently , hoffmann et al . developed a bidirectional poli - polii transcription system for the generation of influenza a virus from only eight plasmids ( 12 ). in this bidirectional system , the vrna cdna was inserted between the human poli promoter and the minimal mouse poli terminator sequences . this entire cassette was inserted between a pol promoter and a polyadenylation site . this allowed the transcription of vrna and mrna from the poli and polii promoters respectively , from a single construct . cotransfection of eight poli - polii plasmids , each encoding one of the influenza a virus gene segments , in 293t cells cocultured with madin darby canine kidney ( mdck ) cells resulted in the recovery of infectious influenza a virus , with yields up to 2 × 10 7 pfu / ml supernatant ( 12 ). the use of one template for the synthesis of both mrna and vrna reduced the number of plasmids required for virus generation . the efficiency of virus generation in this system was reported to be similar to that of the unidirectional ( 12 - 16 plasmid ) poli system . whereas polii promoters are often compatible with the transcription machinery from different species , transcription from poli promoters exhibits stringent , though not absolute , species specificity . this species - specificity is conferred by the interaction of transcription factors with the promoter and , to a lesser extent , in the protein - protein interactions between these factors ( 23 ). the species - specificity of the poli - based reverse genetics systems forms a major disadvantage . the reverse genetics systems described above employed a human poli promoter , limiting the production of recombinant virus to cells of primate origin , such as 293t cells or vero cells . while poli promoters have been characterized for several species including human , mouse , rat , and pig ( 8 , 14 , 17 ), they remain unknown for many others . canine and avian cells are routinely used for influenza a virus research and vaccine production , but the canine and avian poli promoters have not yet been described . to improve the flexibility of influenza virus reverse genetics technology , we sought to develop a universal reverse genetics system . we have chosen to design a system based on the expression of the gene segments of influenza a virus under the control of a bacteriophage t7 rna polymerase promoter ( pt7 ). because the bacteriophage t7 rna polymerase ( t7pol ) can be supplied to cells by transfection or through the use of stably modified cell lines , this system is not restricted to cells from a particular species . t7pol - based reverse genetics systems are used for the rescue of non - segmented negative strand viruses . schnell et al . were the first to rescue a non - segmented negative strand virus solely from cloned cdna ( 27 ). a cdna clone was made encoding the full - length anti - genomic rna of the rabies virus ( rv ). this cdna was flanked by pt7 and a hδvrib sequence next to a t7pol terminator sequence ( tt7 ). following transcription by t7pol , a precise 3 ′ end of the genome is generated by autolytic cleavage of the hδvrib sequence at the 3 ′ end . this plasmid was cotransfected with expression plasmids encoding the viral n protein and the polymerase proteins l and p under the control of pt7 into cells expressing the t7pol . this procedure led to the rescue of recombinant rv , but only from approximately 1 of 2 × 10 7 transfected cells ( 27 ). since then , similar systems have been described for the paramyxoviridae , rhabdoviridae and filoviridae families of nonsegmented nsv ( 10 ). for successful recovery of non - segmented negative strand viruses from cdna , very often positive - sense antigenomic rna ( crna ) is produced rather than the negative - sense vrna . it is thought that the simultaneous presence of naked negative - sense vrna and positive sense mrna encoding the viral proteins will result in hybridization , preventing the assembly of the genome into ribonucleoprotein complexes ( rnps ) ( 27 ). negative strand viruses normally do not encounter this problem since they always keep their genome in the rnp form , which prevents hybridization . recovery of sendai virus ( 15 ), human parainfluenza virus type 3 ( 6 ) and human metapneumovirus ( 11 ) has been reported with cdna encoding negative - sense genomic rna ; however , the efficiencies were significantly lower than results with positive - sense rna . this principle has also been applied for the rescue of recombinant influenza virus . hoffmann et al . ( 13 ) also determined the efficiency of recombinant influenza virus production from antigenomic positive - sense rna . in contrast to non - segmented and segmented negative strand viruses replicating solely in the cytoplasm , influenza a virus could be rescued from both genomic and antigenomic vectors with similar efficiencies . one limiting factor in virus rescue systems using pt7 is that residues at the + 1 to + 3 positions can affect transcription . it was observed that transcription of a cdna can be increased by the introduction of 2 or 3 g residues directly downstream of pt7 ( 22 ). this observation has been applied for the rescue of , e . g . recombinant rv ( 27 ), vesicular stomatitis virus ( 28 ), respiratory syncytial virus ( 3 ), and human metapneumovirus ( 11 ). apparently , for these viruses , the additional g residues at one of the genomic termini did not affect virus replication but had a positive effect on the t7pol - driven transcription . t7pol - based systems have been used extensively for influenza virus reverse genetics studies ( 18 ), but plasmid - based production of recombinant influenza virus has not been described to date . here we describe such a t7pol based reverse genetics system for the production of recombinant influenza virus for the first time . madin - darby canine kidney ( mdck ) cells were cultured in emem ( biowhittaker ) supplemented with 10 % fcs , 100 iu / ml penicillin , 100 μg / ml streptomycin , 2 mm glutamine , 1 . 5 mg / ml sodiumbicarbonate , 10 mm hepes and non - essential amino acids . 293t cells were cultured in dmem ( biowhittaker ) supplemented with 10 % fcs , 100 iu / ml penicillin , 100 μg / ml streptomycin , 2 mm glutamine , 1 mm sodiumpyruvate and non - essential amino acids . bsr - t7 cells , a baby hamster kidney cell line stably expressing t7 rna polymerase ( 2 ). bsr - t7 cells were grown in dmem supplemented with 10 % fcs , 100 iu / ml penicillin , 100 μg / ml streptomycin , 2 mm glutamine , 1 mm sodiumpyruvate and 0 . 5 mg / ml g418 ( life technologies , breda , the netherlands ). influenza virus a / pr / 8 / 34 , being adapted for replication in embryonated chicken eggs and may not replicate optimally in mammalian cell cultures , was passaged seven times at a low multiplicity of infection in mdck cells grown in episerf media ( gibco brl ) supplemented with 10 iu / ml penicillin and 10 μg / ml streptomycin . after the seventh passage , virus titers of 10 8 tcid 50 / ml were obtained routinely . transient calcium phosphate - mediated transfections of 293t cells were performed essentially as described ( 24 ). cells were plated the day before transfection in gelatinized 100 mm diameter culture dishes to obtain 50 percent confluent monolayers . after overnight transfection the transfection medium was replaced with fresh medium supplemented with 2 % fcs for virus production or 10 % fcs for all other transfections . cells were incubated for 30 to 72 hours , after which supernatants were harvested and cells were analyzed for fluorescence if appropriate . plasmid pegfp - n1 ( clontech , bd biosciences , amsterdam , the netherlands ) was transfected in parallel in all experiments and the percentage of fluorescent cells was measured in a facscalibur ( becton dickinson ) flow cytometer , confirming that the transfection efficiency ranged from 95 - 100 percent . virus - containing supernatants were cleared by centrifugation for 10 minutes at 300 × g . virus titers in the supernatant were determined either directly or upon storage at 4 ° c . for less than one week , or at − 80 ° c . for longer than one week . transient transfection of mdck cells was performed essentially as described previously ( 1 ). briefly , 240 μl of optimem i medium ( gibcobrl ) was added to 10 μl of lipofectamin 2000 and incubated at room temperature for 5 minutes . to this mixture , the intended amount of dna , adjusted to a volume of 50 μl using optimem i media was added . this mixture was incubated at room temperature for 20 minutes . after incubation , 200 μl mdck culture medium ( see above ) without penicillin and streptomycin was added and this mixture was added to 1 × 10 6 mdck cells in suspension in a 6 - well plate . after 5 hours incubation , cells were washed twice with pbs and cultured in 2 ml mdck culture medium without penicillin and streptomycin . this medium was replaced with mdck culture medium containing 2 % fcs after overnight incubation . for transient transfection of bsr - t7 cells , 400 . 000 cells were plated in a 6 - wells culture dish a day before transfection to obtain 50 - 70 % confluent monolayers . serum free dmem ( 240 μl ) was added to 10 μl of lipofectamin 2000 and incubated for 4 minutes at room temperature . to this mixture , dna adjusted to 50 μl with serum free dmem was added , and incubated at room temperature for 20 minutes . before transfection , medium was replaced with 2 ml of serum free dmem . after incubation , the transfection mixture was added drop wise to the cells and incubated for 5 hours at 37 ° c . after transfection , cells were washed with pbs once and 2 ml of dmem supplemented with 2 % fcs for virus production or with 10 % fcs for facs analyses was added . eukaryotic expression vectors encoding t7pol ( par3126 and par3132 ) were used . whereas plasmid par3126 encodes a wild type t7pol , plasmid par3132 expresses a t7pol containing a nuclear localization signal ( nls ), that effectively targets the t7pol to the cell nucleus ( 5 ). eukaryotic expression plasmids from which the influenza a virus polymerase proteins are expressed employing a mouse hydroxy - methylglutaryl - coenzyme a reductase promoter , phmg - pb1 , phmg - pb2 , phmg - pa and phmg - np ( 25 ). the hδvrib of ppoli - cat - rt ( 25 ) was amplified by pcr and cloned in the xbai - bamhi sites of psp72 . a tt7 sequence , digested with bamhi - ecorv , was cloned in the bamhi - hpai sites of psp72 - hδvrib , resulting in psp72 - hδvrib - tt7 ( ms24 ). an oligonucleotide encoding the pt7 was ligated in the ndei - xbai sites of psp72 - hδvrib - tt7 in the appropriate context to introduced bbsi sites , resulting in vector psp72 - pt7 - hδvrib - tt7 ( ms25 , fig1 ). a green fluorescent protein ( gfp ) open reading frame flanked by ncrs from segment 5 of influenza virus a / pr / 8 / 34 , was cloned in the bbsi sites of psp72 - pt7 - hδvrib - tt7 , using psp - hu - gfp - mu ( 4 ) as a template . this gfp minigenome was cloned both in the sense and antisense orientations , and contained either 0 / 2 / 3 additional g residues directly downstream of pt7 ( fig1 ). to clone the gene segments of influenza virus a / pr / 8 / 34 in psp72 - pt7 - hδvrib - tt7 , the bidirectional influenza virus a / pr / 8 / 34 constructs described by de wit et al . ( 4 ) were used as a template for pcr ( the fourth 3 ′ nucleotide was in correspondence to the influenza virus a / pr / 8 / 34 sequences reported at the national influenza sequence database ). primers containing an aari restriction site were used for cloning segment 1 , 2 , 3 , 4 , 6 , 7 , 8 and blunt end ligation was used for segment 5 ; the gene segments were cloned in the bbsi sites in an antisense orientation containing 2 additional g residues after pt7 . a bidirectional vector psp72 - pt7 - hδvrib - tt7 - pcmv ( ms65 , fig1 ) was produced by cloning a cmv promoter ( pcmv ) downstream of tt7 to allow production of mrna from the corresponding gene segments . pcmv was amplified by pcr using primers containing asei restriction sites . psp72 - pt7 - hδvrib - tt7 was partially digested with asei and pcmv was ligated downstream from the tt7 in the appropriate direction for production of mrna from the gene segment . again , the influenza virus a / pr / 8 / 34 segments were cloned to obtain each of the bidirectional t7pol - driven influenza virus a / pr / 8 / 34 constructs . we also generated a set of bidirectional vectors from which tt7 was deleted . this was done by digesting psp72 - pt7 - hδvrib - tt7 - pcmv with bamhi - bpeei , treatment with klenow enzyme , and relegation , to generate psp72 - pt7 - hδvrib - pcmv ( ms90 , fig1 ). again , the influenza virus a / pr / 8 / 34 segments were cloned to obtain each of the bidirectional t7pol - driven influenza virus a / pr / 8 / 34 constructs . all plasmids were sequenced using a big dye terminator v3 . 1 cycle sequencing kit ( applied biosystems ) and a 3100 genetic analyser ( applied biosystems ), according to the instructions of the manufacturer . 293 t cells were transfected , as described above , with 5 μg from each of the unidirectional plasmids containing a gene segment of pr / 8 / 34 , 5 μg of the expression plasmids hmg - pb2 , hmg - pb1 , hmg - pa , hmg - np each and 15 μg of par3132 . alternatively , we transfected 5 μg from each of the bidirectional plasmids containing a gene segment of pr / 8 / 34 and 15 μg of par3132 . supernatants were harvested 72 hours after transfection and 1 ml was used to infect a confluent monolayer of mdck cells . prior to inoculation , mdck cells were washed twice with pbs , and 1 ml of 293t supernatant was used to inoculate a confluent monolayer of mdck cells in a 6 - well plate ; 40 μg of trypsin ( 2 . 5 %, bio whittaker ) was added during infection . plates were stored at 37 ° c . for 1 hour and washed twice with pbs , after which 2 ml of emem ( biowhittaker ) supplemented with 4 % bsa , 100 iu / ml penicillin , 100 μg / ml streptomycin , 2 mm glutamine , 1 . 5 mg / ml sodiumbicarbonate , 10 mm hepes , non - essential amino acids and 20 μg / ml trypsin ( infection medium ) was added . at 3 days post infection , the supernatants of the cultures were harvested and tested for ha activity as an indicator for infection of the cells . virus titrations were performed as described previously ( 26 ). briefly , ten - fold serial dilutions of the transfected cell supernatants were prepared in infection medium . prior to inoculation , the cells were washed twice with pbs . 100 μl of the diluted culture supernatants was used to inoculate a confluent monolayer of mdck cells in 96 wells plates . after 1 h at 37 ° c . the cells were washed again with pbs and 200 μl of fresh infection medium was added to each well . at 3 days post infection , the supernatants of the cultures were tested for ha activity as an indicator for infection of the cells in individual wells . the infectious titers were calculated from 10 replicates according to the method of spearman - karber ( 26 ). gfp minigenome assays with a unidirectional t7pol - based reverse genetics system a unidirectional vector containing pt7 , hδvrib and tt7 was constructed . a gfp open reading frame flanked by the non coding regions ( ncrs ) of segment 5 of influenza virus a / pr / 8 / 34 was cloned in psp72 - pt7 - hδvrib - tt7 in the sense ( s ) and antisense ( as ) orientation with 0 , 2 or 3 additional g residues ( fig1 and appendices 2 and 3 ). these constructs were named s - 0g , s - 2g , s - 3g , as - 0g , as - 2g and as - 3g respectively . we tested which of these options resulted in the best performance . we transfected 293t cells with one of the gfp minigenomes ( s - 0g , s - 2g , s - 3g , as - 0g , as - 2g , as - 3g ), a t7pol expression plasmid ( par3132 ), and four plasmids expressing the pb2 , pb1 , pa and np proteins ( phmg - pb2 , phmg - pb1 , phmg - pa , phmg - np ). as controls , we performed the same transfections from which phmg - np was omitted , which should result in the lack of replication of the gfp minigenome . at 30 hours after transfection the cells were analyzed for fluorescence in a facscalibur . the results are depicted in fig2 . from the left panel it can be seen that the highest proportion of gfp positive cells were observed upon transfection of the gfp minigenome in the antisense orientation , with two additional g residues . the other gfp minigenome constructs also yielded a proportion of gfp positive cells , but somewhat smaller . when the mean fluorescence of the gfp positive cells was compared ( fig2 , right panel ), again the gfp minigenome in the antisense orientation with two additional g residues displayed the best performance . in this experiment , the gfp minigenome in the sense orientation with two additional g residues displayed the poorest performance , and other constructs were intermediate . while we observed some variation with respect to the proportion of gfp - expressing cells and levels of gfp expression between the different gfp minigenome plasmids from experiment to experiment ( data not shown ), the gfp minigenome in the antisense orientation with two additional g residues in general performed best , and this construct was thus selected for subsequent experiments . one problem that we potentially needed to solve was the expression of t7pol . for paramyxovirus reverse genetics , a t7pol expressed primarily in the cell cytoplasm is used , which is desirable since paramyxovirus replication also takes place in the cytoplasm . influenza viruses replicate in the cell nucleus , and expression of t7pol in the cytoplasm may thus not be the best option . we thus wished to compare the level of gfp expression when either a cytoplasmic version of t7pol was used ( plasmid ar3126 ), or a t7pol containing a nuclear localization signal ( nls , plasmid par3132 ). the results of this experiment are shown in fig3 . when a wild type t7pol expression plasmid was used , the mean gfp fluorescence in the positive cells was 521 . the level of gfp expression could be enhanced significantly by using a t7pol that contained a nuclear localization signal ( mean fluorescence 1106 ). when both the t7pol constructs with and without the nuclear localization signal were combined ( 1 : 1 ratio , keeping the total amount of transfected plasmid unchanged ), an intermediate level of gfp expression was observed ( mean fluorescence 775 ). in numerous independent experiments , using a wide variety of gfp minigenome plasmids , these results were reproducible ; a 2 to 10 - fold increase in gfp expression was observed when a nuclear version of t7pol was used ( data not shown ). thus , in subsequent experiments , we have made use of the t7pol containing a nuclear localization signal . for several paramyxovirus reverse genetics systems , the t7pol is not supplied by plasmid transfection but through the use of a cell line that allows the stable expression of t7pol . for this purpose , baby hamster kidney cells ( bsr - t7 ) are available . we tested whether bsr - t7 cells could be used for the transcription of the influenza virus gfp minigenome which could subsequently be replicated by the influenza virus polymerase complex , resulting in gfp expression ( fig4 ). as can be seen in fig4 , high gfp fluorescence in 293t cells is strongly dependent of the expression of the t7pol . in bsr - t7 cells , relatively high levels of gfp expression were observed upon cotransfection of the gfp minigenome with the influenza virus polymerase complex , in contrast to transfections from which the phmg - np plasmid was omitted . upon the addition of a plasmid expressing a nuclear version of t7pol , gfp expression was found to be even higher . the relatively high levels of gfp expression in bsr - t7 cells suggest that the stable expression of t7pol is more efficient than transient expression by transfection . however , the experiment in which nuclear t7pol was provided by transfection suggests that for influenza virus reverse genetics , a stable cell line expressing a nuclear t7pol rather than the wild type t7pol would be even more efficient . production of recombinant virus with a unidirectional t7pol - based reverse genetics system next , the gene segments of influenza virus a / pr / 8 / 34 were cloned in vector psp72 - pt7 - hδvrib - tt7 for the generation of recombinant influenza virus a / pr / 8 / 34 . we transfected 293t cells with the eight constructs encoding the gene segments of influenza virus a / pr / 8 / 34 , pt7pol ( par3132 ), phmg - pb1 , phmg - pb2 , phmg - pa and phmg - np . after transfection , trypsin was added to the medium to allow replication of the produced viruses . at 72 h after transfection , supernatants were harvested and used to inoculate mdck cells . at 3 days after inoculation , a ha - test was performed on the supernatant of these mdck cells as an indication of virus replication . the ha - test was positive . subsequently , the virus titer of the 293t and mdck supernatant was determined . the virus titer in the 293t supernatant was shown to be 1 . 6 × 10 1 tcid 50 / ml ; the virus titer in the mdck supernatant was shown to be 2 . 0 × 10 7 tcid 50 / ml . somewhat lower virus titers in 293t cells and mdck cells were obtained when trypsin was not added to 293t cells after transfection ( data not shown ). this thus represents the first plasmid - only recombinant influenza a virus rescue that did not employ a poli promoter . we next wished to develop a bi - directional reverse genetics system under the control of the pt7 . a plasmid vector was produced by cloning pcmv in psp72 - pt7 - hδvrib - tt7 , resulting in vector psp72 - pt7 - hδvrib - tt7 - pcmv ( fig1 ). a gfp open reading frame flanked by the non - coding regions ( ncrs ) of segment 5 of influenza virus a / pr / 8 / 34 was cloned in psp72 - pt7 - hδvrib - tt7 - pcmv in antisense orientation with 2 additional g residues . because we anticipated that this plasmid would give rise to gfp expression without the need for minigenome replication by the influenza virus polymerase complex ( pcmv is in the sense orientation with respect to the minigenome ), we also made a similar construct containing the minigenome ( 0 g residues ) in the sense orientation with respect to pt7 ( hence antisense with respect to pcmv ). the minigenome plasmids were transfected in 293t cells along with plasmids expressing the nuclear t7pol and phmg - pb1 , phmg - pb2 , phmg - pa and phmg - np . cells were analyzed by facs after 30 hours ( fig5 ). transfection of the sense gfp minigenome ( s - 0g ) with an incomplete influenza virus polymerase complex resulted in very few gfp positive cells ( fig5 , left panel ) with very low gfp expression ( fig5 , right panel ). in the presence of the complete influenza virus polymerase complex , ˜ 7 % of cells were gfp positive with a mean fluorescence of ˜ 1200 . using the antisense gfp minigenome plasmid , a relatively large proportion of the cells (˜ 10 %) expressed gfp in the absence of a complete influenza virus polymerase complex , but only at low levels ( mean gfp fluorescence 182 ). upon cotransfection of the complete influenza virus polymerase complex , the proportion of cells expressing gfp did not increase , whereas the level of gfp expression per cell increased significantly ( mean gfp fluorescence 1205 ). thus , from this experiment we could conclude that the bidirectional expression vector was functional ; we observed low levels of gfp expression without the need for the influenza virus polymerase complex , as a result of production of gfp mrna from the pcmv . of note , this was confirmed by transfection of 293t cells with the as - 2g gfp minigenome plasmid alone , resulting in similar levels of gfp expression (˜ 19 % of cells expressing at a mean fluorescence of 128 , data not shown ). in addition , we observed increased levels of gfp expression in the presence of the influenza virus polymerase complex as a result of replication of the minigenome transcribed from pt7 . thus , the bidirectional pt7 - pcmv expression plasmid was functional . production of recombinant virus with a bidirectional t7pol - based reverse genetics system next , the gene segments of influenza virus a / pr / 8 / 34 were cloned in vector psp72 - pt7 - hδvrib - tt7 - pcmv for the generation of recombinant influenza virus a / pr / 8 / 34 . we transfected 293t cells with the eight constructs encoding the gene segments of influenza virus a / pr / 8 / 34 and pt7pol ( par3132 ). after transfection , trypsin was added to the medium to allow replication of the produced viruses . at 72 h after transfection , supernatants were harvested and used to inoculate mdck cells . at 3 days after inoculation , a ha - test was performed on the supernatant of these mdck cells as an indication of virus replication . the ha - test was negative , indicating that no recombinant virus was recovered . from minigenome reporter assays using the bidirectional vectors to express the pb2 , pb1 , pa and np genes , we obtained evidence that protein expression from these plasmids was very low ( data not shown ). we hypothesized that the tt7 sequence was interfering with transcription form pcmv , resulting in low production of the encoded genes . we thus generated a new bidirectional plasmid , from which the tt7 sequence was removed ( psp72 - pt7 - hδvrib - pcmv ). the gene segments of influenza virus a / pr / 8 / 34 were cloned in vector psp72 - pt7 - hδvrib - pcmv for the generation of recombinant influenza virus a / pr / 8 / 34 . in initial attempts , again no recombinant virus was produced . however , upon some optimization of the amount of plasmids used for transfection we successfully produced recombinant virus . the amounts of plasmids used for this experiment were 10 μg each of the constructs encoding pb2 , pb1 , pa and ha , and 5 μg each of the constructs encoding np , na , ma , and ns . while the recombinant virus titers in the 293t cells were undetectable , subsequent inoculation of mdck cells resulted in a virus with an initial titer of 1 . 3 × 10 5 tcid50 / ml . to provide further evidence for the universal nature of the reverse genetics system based on t7pol , we tested the replication of the gfp minigenome in mdck cells rather than 293t cells . although the experiments with bsr - t7 cells already provided evidence that the t7pol reverse genetics system works in cells of non - primate origin ( fig4 ), mdck are more widely used for influenza virus research and vaccine production . as can be seen in fig6 , the t7pol - based reverse genetics system was found to be functional in mdck cells . combined with the results in bsr - t7 cells ( fig4 ), these experiments indicate that the t7pol reverse genetics system indeed represents a “ universal ” system , applicable to wide variety of cell types . from this experiment , it can also be concluded that production of recombinant influenza viruses from non - primate cells is now possible . here we have shown , for the first time , the production of recombinant influenza a virus a / pr / 8 / 34 ( mdck - adapted nibsc strain ) using a t7pol - based system in 293t cells . however , there are no assumptions that limit the use of these methods to influenza a virus a / pr / 8 / 34 ; they can be applied to all influenza viruses of types a , b and c as well as other segmented negative stranded rna viruses . there are also no assumptions that limit the use of these methods to 293t cells , bsr - t7 cells and mdck cells ; the t7pol can be supplied by , for instance , transfection of a wide range of cell lines in which recombinant virus could then be produced . there is also significant flexibility with respect to the plasmid vectors for this t7pol - based reverse genetics technology , and the elements that they contain . here , we used the rna polymerase of bacteriophage t7 to produce vrna or crna - like rna molecules but various other rna polymerases such as the bacteriophage sp6 rna polymerase could be used also . in the experiments shown here , the t7 rna polymerase was modified to contain the nuclear localization signal of the sv40 large t antigen , but rna polymerases may be modified using a variety of other nuclear targeting signals ( e . g . those of the hnrnp k protein ). we here employed the ribozyme sequence of the hepatitis delta virus but other ribozyme sequences have been described that could be used alternatively . finally , the system described here is not dependent on the use of influenza virus polymerase protein expression vectors based on the mouse hydroxy - 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14 . recombinant virus is produced as above , based on a high - throughput virus backbone ( eg derived from the vaccine strain a / pr / 8 / 34 ) with the ha and na genes of a relevant epidemic virus ( eg a / moscow / 10 / 99 ). after the production of recombinant virus by transfection , the virus is amplified in the appropriate cellular substrate ( eg eggs , mdck cells , vero cells ) to sufficiently high amounts . upon propagation in embryonated chicken eggs , the allantoic fluid is cleared by centrifugation for 10 min . at 1000 × g and filtration through a 0 . 45 micrometer filter . the virus is now pelleted by centrifugation for 1 . 5 hours at 150 . 000 × g at 4 ° c . and 25 % sucrose in pbs and centrifuged for 1 . 5 hours at 250 . 000 × g at 4 ° c . the top layer containing ha and na proteins are than dialized against pbs and purity and quantity of the protein preparation are confirmed using 12 . 5 % sds - polyacrylamide gels stained with coomassie brilliant blue . ferrets are immunized with ˜ 10 micrograms ha / na proteins intramuscularly . if desired , vaccinations can be performed using subsequent multiple dosing , or using adjuvants ( mf59 , iscom ). antibody titers against ha and na in serum samples collected before and after vaccination are determined using hemagglutination inhibition assays , neuraminidase inhibition assays , elisa , virus neutralization assays , etc . vaccinated and control animals are challenged 6 weeks after vaccination using 1 × 10e5 50 percent tissue - culture infectious dosis ( tcid - 50 ) of influenza virus a / moscow / 10 / 99 or a heterologous virus isolate . after challenge , nasal or pharyngeal swab samples are collected from the animals on a daily basis for 10 days , and the amount of virus excreted by the infected animals are determined by quantitative pcr analyses or virus titrations . thus , the obtained vaccine - induced immunity can be confirmed by quantifying the rise in antibody titers and the level of protection against infection with the challenge virus .