Patent Application: US-201313904506-A

Abstract:
the present invention belongs to the field of animal health and relates to a nucleic acid sequence which comprises the complete genome of an infectious schmallenberg virus useful for studying viremia and diseases caused by sbv in ruminants , and in the development of vaccines , therapeutics and diagnostics for the prophylaxis , treatment and diagnosis of viremia and diseases caused by sbv .

Description:
the solution to the above technical problem is achieved by the description and the embodiments characterized in the claims . thus , the invention in its different aspects is implemented according to the claims . in one aspect , the invention provides a nucleic acid molecule , in particular a cdna molecule , comprising the genomic sequence of a schmallenberg virus ( sbv ) genome segment , in particular comprising the complete genomic sequence of a genome segment of an infectious schmallenberg virus ( sbv ), wherein said molecule comprises a nucleic acid sequence selected from the group consisting of : a nucleic acid sequence having at least 97 . 8 % sequence identity with the nucleic acid sequence of seq id no : 1 or seq id no : 7 , a nucleic acid sequence having at least 82 . 2 % sequence identity with the nucleic acid sequence of seq id no : 2 , and a nucleic acid sequence having at least 93 % sequence identity with the nucleic acid sequence of seq id no : 3 . preferably , the nucleic acid molecule of the invention comprises the genomic sequence of the s segment of schmallenberg virus , wherein said molecule comprises a nucleic acid sequence having at least 97 . 8 %, preferably at least 98 %, more preferably at least 99 %, still more preferably at least 99 . 5 %, and in particular preferably 100 % sequence identity with the nucleic acid sequence of seq id no : 1 or seq id no : 7 , and wherein this nucleic acid molecule is also termed “ nucleic acid molecule ( s )” or “ dna molecule ( s )” hereinafter . in another aspect , the nucleic acid molecule of the invention comprises the genomic sequence of the m segment of schmallenberg virus , wherein said molecule comprises a nucleic acid sequence having at least 82 . 2 %, in particular at least 85 %, more particular at least 90 % or at least 95 %, preferably at least 98 %, more preferably at least 99 %, still more preferably at least 99 . 5 %, and in particular preferably 100 % sequence identity with the nucleic acid sequence of seq id no : 2 , and wherein this nucleic acid molecule is also termed “ nucleic acid molecule ( m )” or “ dna molecule ( m )” hereinafter . in a further aspect , the nucleic acid molecule of the invention comprises the genomic sequence of the l segment of schmallenberg virus , wherein said molecule comprises a nucleic acid sequence having at least 93 %, in particular at least 95 %, more particular at least 97 %, preferably at least 98 %, more preferably at least 99 %, still more preferably at least 99 . 5 % or at least 99 . 8 %, and in particular preferably 100 % sequence identity with the nucleic acid sequence of seq id no : 3 , and wherein this nucleic acid molecule is also termed “ nucleic acid molecule ( l )” or “ dna molecule ( l )” hereinafter . sequence identity in the context of the invention is understood as being based on pairwise sequence alignments . for purposes of the present invention , pairwise sequence alignments are done with clustalw as implemented in mega5 ( k . tamura et . al ., mega5 : molecular evolutionary genetics analysis using maximum likelihood , evolutionary distance , and maximum parsimony methods . mol . biol . evol . 28 , 2731 - 2739 ( 2011 )), using the default settings ( gap opening penalty of 15 and gap extension penalty of 6 . 66 ; dna weight matrix : clustalw 1 . 6 ; transition weight of 0 . 5 ). sequence identities of the aligned sequences are preferably calculated using bioedit version 7 . 0 . 9 . 0 . the term “ having 100 % sequence identity ”, as used herein , is understood to be equivalent to the term “ being identical ”. as used herein , it is in particular understood that the term “ sequence identity with the nucleic acid sequence of seq id no : x ” is equivalent to the term “ sequence identity with the nucleic acid sequence of seq id no : x over the length of seq id no : x ” or to the term “ sequence identity with the nucleic acid sequence of seq id no : x over the whole length of seq id no : x ”, respectively . in this context , “ x ” is any integer selected from 1 to 10 so that “ seq id no : x ” represents any of the seq id nos mentioned herein . in another aspect , the invention comprises a combination of at least two , preferably two , nucleic acid molecules selected from the group consisting of : the nucleic acid molecule ( s ), i . e ., as defined herein , a nucleic acid molecule comprising the genomic sequence of the s segment of schmallenberg virus , wherein said molecule comprises a nucleic acid sequence having at least 97 . 8 %, preferably at least 98 %, more preferably at least 99 %, still more preferably at least 99 . 5 %, and in particular preferably 100 % sequence identity with the nucleic acid sequence of seq id no : 1 or seq id no : 7 , the nucleic acid molecule ( m ), i . e ., as defined herein , a nucleic acid molecule comprising the genomic sequence of the m segment of schmallenberg virus , wherein said molecule comprises a nucleic acid sequence having at least 82 . 2 %, in particular at least 85 %, more particular at least 90 % or at least 95 %, preferably at least 98 %, more preferably at least 99 %, still more preferably at least 99 . 5 %, and in particular preferably 100 % sequence identity with the nucleic acid sequence of seq id no : 2 , and the nucleic acid molecule ( l ), i . e ., as defined herein , a nucleic acid molecule comprising the genomic sequence of the l segment of schmallenberg virus , wherein said molecule comprises a nucleic acid sequence having at least 93 %, in particular at least 95 %, more particular at least 97 %, preferably at least 98 %, more preferably at least 99 %, still more preferably at least 99 . 5 % or at least 99 . 8 %, and in particular preferably 100 % sequence identity with the nucleic acid sequence of seq id no : 3 , and wherein in particular the combination of the nucleic acid molecule ( s ) and the nucleic acid molecule ( m ), preferably each having at least 98 % or at least 99 % sequence identity with seq id no : 1 and seq id no : 2 , respectively , is preferred , or wherein in particular the combination of the nucleic acid molecule ( s ) and the nucleic acid molecule ( m ), preferably each having at least 98 % or at least 99 % sequence identity with seq id no : 7 and seq id no : 2 , respectively , is preferred . preferably , the nucleic acid molecules described herein are isolated nucleic acid molecules . according to the invention , the combination of the nucleic acid molecule ( s ), the nucleic acid molecule ( m ), and the nucleic acid molecule ( l ) is most preferred , in particular a combination of the nucleic acid molecule ( s ), the nucleic acid molecule ( m ) and the nucleic acid molecule ( l ), each having at least 98 % or at least 99 % sequence identity with seq id no : 1 , seq id no : 2 , and seq id no : 3 , respectively , or in particular a combination of the nucleic acid molecule ( s ), the nucleic acid molecule ( m ) and the nucleic acid molecule ( l ), each having at least 98 % or at least 99 % sequence identity with seq id no : 7 , seq id no : 2 , and seq id no : 3 , respectively . the term “ combination ”, as used herein , in particular refers to any bringing together or admixture of the nucleic acid molecules , of the dna constructs , preferably the cdna constructs or of the rna transcripts to be combined according to the invention , or preferably refers to a composition containing the nucleic acid molecules , the dna constructs , preferably the cdna constructs or the rna transcripts of the combination . preferably , the combination of the nucleic acid molecule ( s ), the nucleic acid molecule ( m ), and the nucleic acid molecule ( l ), is capable of producing infectious schmallenberg virus when transfected into cells . since schmallenberg virus has a negative stranded rna genome , the presence of an rna polymerase , preferably of t7 rna polymerase or the rna polymerase encoded by the schmallenberg virus , in the transfected cells is required . most preferred is the use of the t7 rna polymerase . the presence of the rna polymerase in the transfected cells can be provided , for instance , by co - transfection of a plasmid coding for and expressing the rna polymerase or by penetrating the cells with rna polymerase protein . according to the invention , in this regard , the use of transgenic cells producing rna polymerase is particularly preferred , such as the transfection of the combination of the nucleic acid molecule ( s ), the nucleic acid molecule ( m ), and the nucleic acid molecule ( l ) into bsr - t7 / 5 cells . alternatively , the cells can also be transfected with the mrna that codes for the rna polymerase and which is translated into the rna polymerase when transfected into the host cells . in two exemplary embodiments , the transfection may be performed with or without the co - transfection of at least one , preferably two or three , helper plasmid ( s ). the term “ infectious schmallenberg virus ” according to the invention is in particular understood as a schmallenberg virus which infects mammals and / or insects and causes viremia in the infected mammal and / or insect . as used herein , the term “ viremia ” is particularly understood as a condition in which schmallenberg virus particles reproduce and circulate in the bloodstream of an animal , in particular of a mammal or of an insect . said infection of a mammal and / or insect by the schmallenberg virus being produced by the nucleic acid molecules of the present invention in particular includes attachment of the virus to a host cell , entry of the virus into the cell , uncoating of the virion in the cytoplasm , replication and transcription of the viral genome , expression of viral proteins and assembly and release of new infectious viral particles . preferably , the mammal as mentioned herein is a ruminant , in particular selected from the group consisting of cattle , sheep , goats , deer , elk , giraffes , bison , moose , yaks , water buffalo , camels , alpacas , llamas , antelope , pronghorn , and nilgai . more preferably , the mammal as mentioned herein is a ruminant selected from the group consisting of cattle , sheep and goats . the insect , as mentioned herein , is preferably selected from the group consisting of midges , in particular culicoides spp ., biting flies and mosquitoes . the term “ helper plasmids ” as mentioned herein , is in particular directed to plasmids that contain one or more sbv coding sequence ( s ), e . g . under the control of a t7 promotor , to express the protein ( s ) of sbv . the present invention further provides a dna construct , preferably a cdna construct , comprising the cdna molecule according to the invention , wherein said dna construct is in particular a cdna vector such as a plasmid . herein , the dna construct . preferably the cdna construct , of the present invention which comprises the cdna molecule ( s ) is also termed “ dna construct ( s )”, the dna construct of the present invention which comprises the dna molecule ( m ) is also termed “ dna construct ( m )”, and the dna construct of the present invention which comprises the dna molecule ( l ) is also termed “ dna construct ( l )”. according to the invention , preferred dna vectors or plasmids into which the nucleotide molecule of the present invention can be inserted are pgem - t easy , puc18 , pcdna , px8δt or pt7ribosm2 . the cdna construct , as described herein , is preferably an isolated cdna construct . exemplary cdna constructs of the invention are provided with the sequences set forth in seq id nos : 4 - 6 , wherein seq id no : 4 shows an example of the sequence of a dna construct ( s ), seq id no : 5 shows an example of the sequence of a dna construct ( m ) and seq id no : 6 shows an example of the sequence of a dna construct ( l ). further exemplary cdna constructs of the invention are provided with the sequences set forth in seq id nos : 8 - 10 , wherein seq id no : 8 shows an example of the sequence of a dna construct ( s ), seq id no : 9 shows an example of the sequence of a dna construct ( m ) and seq id no : 10 shows an example of the sequence of a dna construct ( l ). the invention also provides a combination of at least two , preferably two , different dna constructs selected from the group consisting of : the dna construct ( s ), i . e ., as defined herein , a cdna construct which comprises the dna molecule ( s ), the dna construct ( m ), i . e ., as defined herein , a cdna construct which comprises the dna molecule ( m ), and the dna construct ( l ), i . e ., as defined herein , a cdna construct which comprises the dna molecule ( l ), wherein the at least two different cdna constructs are preferably isolated cdna constructs , and wherein in particular the combination of the dna construct ( s ) and the dna construct ( m ) is preferred , preferably each comprising the nucleic acid molecule ( s ) or the nucleic acid molecule ( m ), respectively , having at least 98 % or at least 99 % sequence identity with seq id no : 1 or seq id no : 2 , respectively , or preferably each comprising the nucleic acid molecule ( s ) or the nucleic acid molecule ( m ), respectively , having at least 98 % or at least 99 % sequence identity with seq id no : 7 or seq id no : 2 , respectively . according to the invention , the combination of the dna construct ( s ), the nucleic acid molecule ( m ), and the nucleic acid molecule ( l ), is most preferred , in particular each comprising the nucleic acid molecule ( s ), the nucleic acid molecule ( m ) or the nucleic acid molecule ( l ), respectively , having at least 98 % or at least 99 % sequence identity with seq id no : 1 , seq id no : 2 , or seq id no : 3 , respectively , or in particular each comprising the nucleic acid molecule ( s ), the nucleic acid molecule ( m ) or the nucleic acid molecule ( l ), respectively , having at least 98 % or at least 99 % sequence identity with seq id no : 7 , seq id no : 2 , or seq id no : 3 , respectively . further , the invention provides a preferably isolated rna transcript of the cdna construct of the invention . in the following , the rna transcript of the dna construct ( s ) of the present invention is also termed “ rna transcript ( s )”, the rna transcript of the dna construct ( m ) of the present invention is also termed “ rna transcript ( m )”, and the rna transcript of the dna construct ( l ) is also termed “ rna transcript ( l )”. the invention also provides a combination of at least two , preferably two , different rna transcripts , preferably isolated rna transcripts , selected from the group consisting of : the rna transcript ( s ), i . e ., as defined herein , the rna transcript of the dna construct ( s ), the rna transcript ( m ), i . e ., as defined herein , the rna transcript of the dna construct ( m ), and the rna transcript ( l ), i . e ., as defined herein , the rna transcript of the dna construct ( l ), wherein in particular the combination of the rna transcript ( s ) and the rna transcript is preferred , preferably transcribed from the dna construct ( s ) and the dna construct ( m ), respectively , each comprising the nucleic acid molecule ( s ) or the nucleic acid molecule ( m ), respectively , having at least 98 % or at least 99 % sequence identity with seq id no : 1 or seq id no : 2 , respectively , or preferably transcribed from the dna construct ( s ) and the dna construct ( m ), respectively , each comprising the nucleic acid molecule ( s ) or the nucleic acid molecule ( m ), respectively , having at least 98 % or at least 99 % sequence identity with seq id no : 7 or seq id no : 2 , respectively . according to the invention , the combination of the rna transcript ( s ), the rna transcript ( l ), and the rna transcript ( m ), is most preferred , in particularly transcribed from the dna construct ( s ), the dna construct ( m ) and the dna construct ( l ), respectively , each comprising the nucleic acid molecule ( s ), the nucleic acid molecule ( m ) or the nucleic acid molecule ( l ), respectively , having at least 98 % or at least 99 % sequence identity with seq id no : 1 , seq id no : 2 or seq id no : 3 , respectively , or in particularly transcribed from the dna construct ( s ), the dna construct ( m ) and the dna construct ( l ), respectively , each comprising the nucleic acid molecule ( s ), the nucleic acid molecule ( m ) or the nucleic acid molecule ( l ), respectively , having at least 98 % or at least 99 % sequence identity with seq id no : 7 , seq id no : 2 or seq id no : 3 , respectively . the present invention also provides a cell transfected with the dna construct described herein or with the combination of dna constructs described herein , wherein said cell is preferably an isolated cell . thus , the present invention also provides schmallenberg virus produced by the aforementioned cell , wherein said schmallenberg virus is preferably an isolated schmallenberg virus . furthermore , the present invention also provides a cell , preferably a cultured host cell which comprises the schmallenberg virus produced by or in the presence of one or more of the nucleic acid constructs provided herein . further , the present invention provides a cell transfected with the rna transcript mentioned herein or with the combination of rna transcripts mentioned herein , wherein said cell is preferably an isolated cell . hence , the present invention also provides schmallenberg virus produced by the aforementioned cell , wherein said schmallenberg virus is preferably an isolated schmallenberg virus . the present invention further provides a schmallenberg virus whose genome comprises the nucleic acid molecule of the present invention or the combination of nucleic acid molecules of the present invention , wherein said schmallenberg virus is preferably an isolated schmallenberg virus in another aspect , the present invention provides a method for producing a schmallenberg virus , said method comprising transfecting a cell with the dna construct or with the combination of dna constructs described herein . moreover , the present invention provides a method for producing a schmallenberg virus , said method comprising transfecting a cell with the rna transcript or with the combination of rna transcripts mentioned herein . since schmallenberg virus has a negative stranded rna genome , preferably the method of producing the schmallenberg virus is done in the presence of an rna polymerase , preferably of t7 rna polymerase or the rna polymerase encoded by the schmallenberg virus . most preferred is the use of the t7 rna polymerase . the presence of the rna polymerase in the transfected cells can be provided , for instance , by co - transfection of a plasmid coding for and expressing the rna polymerase . according to the invention , in this regard , the use of transgenic cells producing rna polymerase is particularly preferred , such as the transfection of the combination of the nucleic acid molecule ( s ), the nucleic acid molecule ( m ), and the nucleic acid molecule ( l ) into bsr - t7 / 5 cells . alternatively , the cells can also be transfected with the mrna that codes for the rna polymerase and which is translated into the rna polymerase when transfected into the host cells . in yet another aspect , the present invention provides a composition , said composition comprising the nucleic acid molecule according to the invention or the combination of nucleic acids according to the invention , suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient . production of the nucleic acid molecules described herein is within the skill in the art and can be carried out according to recombinant techniques described , among other places , in sambrook et al ., 2001 , molecular cloning , a laboratory manual , cold spring harbor laboratory press , cold spring harbor , n . y . ; ausubel , et al ., 2003 , current protocols in molecular biology , greene publishing associates & amp ; wiley interscience , ny ; innis et al . ( eds ), 1995 , pcr strategies , academic press , inc ., san diego ; and erlich ( ed ), 1994 , pcr technology , oxford university press , new york , all of which are incorporated herein by reference . establishment of a reverse genetics system for the generation of recombinant sbv , which allows further investigation on the molecular biology of orthobunyaviruses as well as the generation of save and efficient vaccines . sbv was isolated from infected cattle and passaged on kc cells and bhk - 21 cells . rna was extracted from infected cells and transcribed into cdna . pcr fragments of the three rna segments were amplified by using gene specific primers and were inserted into the plasmid px8δt ( 11 ) by restrictions - free cloning ( 13 ). the resulting plasmids px8δt_sbv_s , px8δt_sbv_m and px8δt_sbv_l contain the full - length antigenome of sbv . transfection experiments are done by using bsr t7 / 5 cells , stably expressing the phage t7 polymerase ( 12 ) and plasmid dna of all of the three constructs px8δt_sbv_s , px8δt_sbv_m and px8δt_sbv_l . supernatants of the cells are harvested after various times following transfection and transferred to susceptible cell lines . the cell monolayers are investigated for expression of sbv proteins by indirect immunofluorescence staining . three cdna clones spanning the complete genomic sequence of the segments s , m and l were generated from viral rna by fusion pcr . rna transcripts were produced by bacteriophage t7 polymerase in bsr t7 / 5 cells . the exact 3 ′ end of the rna is specified by self - cleavage of the rna by the hepatitis delta virus antigenome ribozyme sequence . rescue of infectious sbv , growth characteristics of recombinant viruses and manipulation of the full - length genome like the deletion of relevant domains can be demonstrated . a reverse genetic system for the recovery of sbv , the first european simbu serogroup virus , can be established . the new system can be used for the generation of recombinant sbv , by transfection of cells stably expressing phage t7 rna polymerase with the plasmids px8δt_sbv_s , px8δt_sbv_m px8δt_sbv_l allowing expression of antigenomic sbv rna and the viral proteins . by using sbv reverse genetics , defined mutants can be designed enabling the mechanistic investigation of virus - host interactions as well as the molecular basis of sbv pathogenesis . furthermore , the approach will be useful for the design of next generation vaccines like packaged replicons and defective in second cycle virions , chimera or modified deletion mutants . in the following , the construction of the plasmids px8δt_sbv_s , px8δt_sbv_m and px8δt_sbv_l and the transfection and recovery of recombinant sbv , as mentioned above , is described in closer detail . the construction of the plasmids px8δt_sbv_s , px8δt_sbv_m and px8δt_sbv_l was done by using the plasmid vector x8δt ( 11 ). cdna of the schmallenberg virus ( sbv ) rna segments was inserted into this plasmid by restrictions - free cloning ( fusion pcr ) ( 13 ), respectively . the construction of the cdna clones is shown in fig1 a - 1c . the plasmids contain a bacteriophage t7 promotor ( t7 ) before 5 ′ sbv cdna to enable in vitro transcription of cdna into rna , the hepatitis delta virus ribozyme sequence ( hep for the generation of the exact 3 ′ end by self - cleavage of the nascent rna by the hepatitis delta virus antigenome ribozyme and the t7 transcription termination sequence ( t7 term ) downstream the 3 ′ end of the sbv cdna . location of the used primers and nucleotide positions corresponding to the schmallenberg antigenome are indicated by arrows . rna of schmallenberg virus ( bh80 / 11 - 4 ) infected bhk 21 cells was isolated by using qiamp viral rna mini kit ( qiagen ) and transcribed by using the transcriptor high fidelity cdna synthesis kit ( roche ). plasmids were amplified in escherichia coli dh10b ™ cells ( invitrogen ). for megaprimer - pcr and fusion pcr the phusion high fidelity pcr master mix with hf buffer ( new england biolabs ) and phusion high - fidelity master mix ( finnzymes ) were used . plasmid dna was purified by using qiagen plasmid mini or midi kit ( qiagen ). sequencing was carried out using the big dye ® terminator v1 . 1 cycle sequencing kit ( applied biosystems ). nucleotide sequences were read with an automatic sequencer ( 3130 genetic analyzer , applied biosystems ) and analyzed using the genetics computer group software version 11 . 1 ( accelrys inc ., san diego , usa ). primers were synthesized by biomers . net gmbh and are listed in table 1 . sequences derived from plasmid x8δt are underlined , and three additional g residues are in italics . construction of px8δt_sbv_s ( fig1 a ): in a first step segment s cdna was synthesized with primer p_ph_s1f and used as template for the generation of a megaprimer pcr fragment . as primers p_ph_s1f and p_ph_s1r were utilized . by fusion pcr , sbv segment s sequences were introduced into the plasmid px8δt . construction of px8δt_sbv_m ( fig1 b ): in a multi - step cloning procedure the cdna clone px8δt_sbv_m was constituted from four megaprimer pcr fragments which were assembled into plasmid vector px8δt by fusion pcr . in a first step segment m cdna was synthesized with primer p_ph_m1f and p_m3f and used as template for the generation of the megaprimers 1 , 2 , 3 and 4 , respectively . as primers for the generation of megaprimer 1 primers p_ph_m1f and p_ph_m1r , for the generation of megaprimer 2 the primers p_m2f and p_ph_m2r , for the generation of megaprimer 3 the primers p_m3f and p_ph_m3r and for the generation of megaprimer 4 the primers p_m4f and p_ph_mr were used . by fusion pcr the megaprimers were introduced into the plasmid px8δt , successively . construction of px8δt_sbv_l ( fig1 c ): in a multi - step cloning procedure the cdna clone px8δt_sbv_l was constituted from five megaprimer pcr fragments which were assembled into plasmid vector px8δt by fusion pcr . in a first step segment l cdna was synthesized with primer p_ph_l1f and p_l3f and used as template for the generation of the megaprimers 1 , 2 , 3 , 4 and 5 , respectively . as primers for the generation of megaprimer 1 primers p_ph_l1f and p_ph_l1r , for the generation of megaprimer 2 the primers p_l2f and p_ph_l2r , for the generation of megaprimer 3 the primers p_l3f and p_ph_l3r , for the generation of megaprimer 4 the primers p_l4f and p_ph_l4r and for the generation of megaprimer 5 the primers p_l5f and p_ph_lr were used . by fusion pcr the megaprimers were introduced into the plasmid px8δt , successively . transfection experiments are done using bhk 21 cells , clone bsr t7 / 5 , stably expressing the phage t7 rna polymerase ( 12 ), according to lowen et al . ( 10 ). about 6 × 10 5 cells grown to 80 % confluency are transfected with various amounts of plasmid dna e . g . 0 . 25 μg px8δt_sbv_l , 0 . 1 μg px8δt_sbv_s , 1 μg px8δt_sbv_m using a transfection reagent e . g ., lipofectin ( invitrogen ), lipofectamin ( invitrogen ), superfect ( qiagen ) and dac - 30 ( eurogentec ) according to suppliers protocols . transfected cells are incubated for various times ( e . g . 4 - 5 days ) at 37 ° c . the supernatant fluid is collected , clarified by low speed centrifugation and various volumes ( e . g 200 μl ) are inoculated into highly susceptible cells ( kc , bhk 21 ). detection of infectious sbv can be done by indirect if - staining using sbv - specific monoclonal and polyclonal antibodies . establishment of a reverse genetics system using the plasmid pt / ribosm2 for the generation of recombinant sbv , which allows further investigation on the molecular biology of orthobunyaviruses as well as the generation of save and efficient vaccines . sbv was isolated from infected cattle and passaged on kc cells and bhk - 21 cells . rna was extracted from infected cells and transcribed into cdna . pcr fragments of the three rna segments were amplified by using gene specific primers and were subcloned into the plasmid px8δt ( 11 ) by restrictions - free cloning ( 13 ). the resulting plasmids px8δt_sbv_s , px8δt_sbv_m and px8δt_sbv_l contain the full - length antigenome of sbv . afterwards , by using segment - specific primers and the full - length plasmids as template dna , full - length pcr fragments were amplified and inserted into plasmid pt / ribosm2 ( 14 ) either by restrictions - free cloning or by digestion with appropriate restriction enzymes ( e . g . esp3i , bsmbi ) and ligation . the resulting plasmids pt7ribo_sbv_s , pt7ribo_sbv_m and pt7ribo_sbv_l contain the full - length antigenome of sbv . transfection experiments are done by using bsr t7 / 5 cells , stably expressing the phage t7 polymerase ( 12 ) and plasmid dna of all of the three constructs pt7ribo_sbv_s , pt7ribo_sbv_m and px8δt_sbv_l or pt7ribo_sbv_l . supernatants of the cells are harvested after various times following transfection and transferred to susceptible cell lines . the cell monolayers are investigated for expression of sbv proteins by indirect immunofluorescence staining . three cdna clones spanning the complete genomic sequence of the segments s , m and l were generated from viral rna . rna transcripts were produced by bacteriophage t7 polymerase in bsr t7 / 5 cells . the exact 3 ′ end of the rna is specified by self - cleavage of the rna by the hepatitis delta virus antigenome ribozyme sequence . rescue of infectious sbv , growth characteristics of recombinant viruses and manipulation of the full - length genome like the deletion of relevant domains can be demonstrated . the virus rescue is more efficient , compared to example 1 . a reverse genetic system for the recovery of sbv , the first european simbu serogroup virus , can be established . the new system can be used for the generation of recombinant sbv , by transfection of cells stably expressing phage t7 rna polymerase with the plasmids pt7ribo_sbv_s , pt7ribo_sbv_m and px8δt_sbv_l or pt7 — sbv_l allowing expression of antigenomic sbv rna and the viral proteins . by using sbv reverse genetics , defined mutants can be designed enabling the mechanistic investigation of virus - host interactions as well as the molecular basis of sbv pathogenesis . furthermore , the approach will be useful for the design of next generation vaccines like packaged replicons and defective in second cycle virions , chimera or modified deletion mutants . in the following , the construction of the plasmids pt7ribo_sbv_s , pt7ribo_sbv_m , px8δt_sbv_l and pt7ribo_sbv_l and the transfection and recovery of recombinant sbv , as mentioned above , is described in closer detail . the construction of the plasmids pt7ribo_sbv_s , pt7ribo_sbv_m px8δt_sbv_l and pt7ribo_sbv_l was done by using the plasmid vectors x8δt ( 11 ) and pt7ribosm2 ( 14 ). cdna of the schmallenberg virus ( sbv ) rna segments was inserted into this plasmid by standard cloning methods using restriction enzyme bsmb or by restriction - free cloning ( fusion pcr ) ( 13 ), respectively . the construction of the cdna clones is shown in fig2 a - 2c . the plasmids contain a bacteriophage t7 promotor ( t7 ) before 5 ′ sbv cdna to enable in vitro transcription of cdna into rna , the hepatitis delta virus ribozyme sequence ( hep for the generation of the exact 3 ′ end by self - cleavage of the nascent rna by the hepatitis delta virus antigenome ribozyme and the t7 transcription termination sequence ( t7 term ) downstream the 3 ′ end of the sbv cdna . location of the used primers and nucleotide positions corresponding to the schmallenberg antigenome are indicated by arrows . rna of schmallenberg virus ( bh80 / 11 - 4 ) infected bhk 21 cells was isolated by using qiamp viral rna mini kit ( qiagen ) and transcribed by using the transcriptor high fidelity cdna synthesis kit ( roche ). plasmids were amplified in escherichia coli dh10b ™ cells ( invitrogen ). for megaprimer - pcr and fusion pcr the phusion high fidelity pcr master mix with hf buffer ( new england biolabs ) and phusion high - fidelity master mix ( finnzymes ) were used . digestion of the plasmids and dna fragments was done with the restriction enzymes bsmbi ( new england biolabs ) and esp3i ( fisher scientific ). for ligation of plasmid dna and dna fragments , t4 dna ligase ( promega ) was used . plasmid dna was purified by using qiagen plasmid mini or midi kit ( qiagen ). sequencing was carried out using the big dye ® terminator v1 . 1 cycle sequencing kit ( applied biosystems ). nucleotide sequences were read with an automatic sequencer ( 3130 genetic analyzer , applied biosystems ) and analyzed using the genetics computer group software version 11 . 1 ( accelrys inc ., san diego , usa ). primers were synthesized by biomers . net gmbh and are listed in table 2 . sequences derived from plasmids x8δt and pt7ribosm2 are underlined , mutated nucleotides are in lower case and additional g residues and restriction sites are in italics . construction of pt7 — sbv_s ( fig2 a ): in a first step segment s cdna was synthesized with primer p_ph_s1f and used as template for the generation of a megaprimer pcr fragment . as primers p_ph_s1f and p_ph_s1r were utilized . by fusion pcr , sbv segment s sequences were introduced into the plasmid px8δt . plasmid px8δt_sbv_s was used as template , to amplify a full - length megaprimer pcr fragment by using primers p_ph_s2f and p_ph_s1r . by fusion pcr , sbv segment s sequences were introduced into the plasmid pt7ribo_sm2 , resulting in plasmid pt7ribo_sbv_s . construction of pt7ribo_sbv_m ( fig2 b ): in a multi - step cloning procedure the cdna clone px8δt_sbv_m was constituted from four megaprimer pcr fragments which were assembled into plasmid vector px8δt by fusion pcr . in a first step segment m cdna was synthesized with primer p_ph_m1f and p_m3f and used as template for the generation of the megaprimers 1 , 2 , 3 and 4 , respectively . as primers for the generation of megaprimer 1 primers p_ph_m1f and p_ph_m1r , for the generation of megaprimer 2 the primers p_m2f and p_ph_m2r , for the generation of megaprimer 3 the primers p_m3f and p_ph_m3r and for the generation of megaprimer 4 the primers p_m4f and p_ph_mr were used . by fusion pcr the megaprimers were introduced into the plasmid px8δt , successively . plasmid px8δt_sbv_m was used as template , to amplify a full - length pcr fragment by using primers p_m_bsmbi_f and p_m_bsmbi_r . this pcr fragment was digested with bsmbi and ligated into bsmbi - digested plasmid pt7ribo_sm2 , resulting in plasmid pt7ribo_sbv_m . construction of px8δt_sbv_l ( fig2 c ): in a multi - step cloning procedure the cdna clone px8δt_sbv_l was constituted from five megaprimer pcr fragments which were assembled into plasmid vector px8δt by fusion pcr . in a first step segment l cdna was synthesized with primer p_ph_l1f and p_l3f and used as template for the generation of the megaprimers 1 , 2 , 3 , 4 and 5 , respectively . as primers for the generation of megaprimer 1 primers p_ph_l1f and p_ph_l1r , for the generation of megaprimer 2 the primers p_l2f and p_ph_l2r , for the generation of megaprimer 3 the primers p_l3f and p_ph_l3r , for the generation of megaprimer 4 the primers p_l4f and p_ph_l4rand for the generation of megaprimer 5 the primers p_l5f and p_ph_lr were used . by fusion pcr the megaprimers were introduced into the plasmid px8δt , successively . in order to generate pt7ribo_sbv_l , the bsmbi - site within px8δt_sbv_l had to be deleted by site - directed mutagenesis . a pcr fragment ( megaprimer ) was amplified by using primers p_mut l_bsmbif , p_mut l_bsmbir and plasmid px8δt_sbv_l as template dna . by fusion pcr the megaprimer was introduced into the plasmid px8δt_sbv_l , resulting in the plasmid px8δt_mut_l_bsmbi . plasmid px8δt_mut_l was used as template , to amplify a full - length pcr fragment by using primers p_l_bsmbi_f and p_l_bsmbi_r . this pcr fragment was digested with bsmbi and ligated into bsmbi - digested plasmid pt7ribo_sm2 , resulting in plasmid pt7ribo_sbv_l . transfection experiments are done using bhk 21 cells , clone bsr t7 / 5 , stably expressing the phage t7 rna polymerase ( 12 ), according to lowen et al . ( 10 ). about 6 × 10 5 cells grown to 80 % confluency are transfected with various amounts of plasmid dna e . g ., 3 μg pt7robo_sbv_s , 3 μg pt7ribo_sbv_m , 3 μg px8δt_sbv_l or 3 μg pt7ribo_sbv_l using a transfection reagent e . g . lipofectin ( invitrogen ), lipofectamin ( invitrogen ) and superfect ( qiagen ) according to suppliers protocols . transfected cells are incubated for various times ( e . g . 4 - 5 days ) at 37 ° c . the supernatant fluid is collected , clarified by low speed centrifugation and various volumes ( e . g 0 . 1 - 1 . 0 ml ) are inoculated into highly susceptible cells ( kc , bhk 21 ). detection of infectious sbv can be done by indirect if - staining using sbv - specific monoclonal and polyclonal antibodies . seq id no : 1 corresponds to the complete genome sequence of a s segment of an infectious schmallenberg virus ( bh80 / 11 - 4 ), seq id no : 2 corresponds to the complete genome sequence of a m segment of an infectious schmallenberg virus ( bh80 / 11 - 4 ), seq id no : 3 corresponds to the complete genome sequence of a l segment of an infectious schmallenberg virus ( bh80 / 11 - 4 ), seq id no : 7 corresponds to seq id no : 1 , wherein the nucleotide at position 9 is “ a ” instead of “ g ”, 1 . b . hoffmann , m . scheuch , d . hoper , r . jungblut , m . holsteg , h . schirrmeier , m . eschbaumer , k . v . goller , k . wernike , m . fischer , a . breithaupt , t . c . mettenleiter , m . beer , novel orthobunyavirus in cattle , europe , 2011 . emerg . infect . dis . 18 , 469 - 472 ( 2012 ). 2 . m .- m . gariglinany et al ., schmallenberg virus in calf born at term with porencephaly , belgium . emerg . infect . dis . 18 ( 2012 ), doi : 10 . 3201 / eid1806 . 120104 . 3 . m . d . bowen et al ., a reassortant bunyavirus isolated from acute hemorrhagic fever cases in kenya and somalia . virology . 291 , 185 - 190 ( 2001 ). 4 . a . m . q . king , m . j . adams , e . b . carstens , e . j . lefkowitz , eds ., virus taxonomy : ninth report of the international committee on taxonomy of viruses . 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