cell lines and denv-3 strain - bhk-21 cells were propagated at 37c in 
 a humidified incubator with 5% co 2 in minimum essential medium supplemented 
 with 10% foetal bovine serum ( fbs ) . mosquito c6/36 cells were cultured at 30c in 
 leibovitz s medium ( l-15 ) with 5% fbs . both growth media were supplemented with 1% 
 antibiotic solution ( containing penicillin and streptomycin ) . 95016/br - pe/02 strain is a 
 denv-3 clinical sample isolated from an 11-year - old female patient during the denv-3 
 epidemics in the state of pernambuco , brazil in 2002 ( cordeiro et al . virus was isolated in mosquito c6/36 cells 
 ( passage 1 ) and virus serotype confirmed by reverse transcription - polymerase chain 
 reaction ( rt - pcr ) . the infectious clone and replicon described herein were derived from passage 3 
 in insect cells . e. coli strain , yeast strains and yeast transformation - 
 electrocompetent electromax dh10b e. coli cells were acquired from 
 invitrogen . saccharomyces cerevisiae rfy206 ( mata trp1::hisg 
 his3200 ura3 - 52 lys2201 leu2 - 3 ) strain ( finley jr & brent 1994 ) was 
 grown in ypd media and made competent by lithium acetate treatment ( sambrook & russell 2001 ) . s. 
 cerevisiae yph252 ( mat ura3 - 52 lys2 - 801 ade2 - 101 trp1-1 his3-200 
 leu2-1 ) strain ( sikorski & hieter 
 1989 ) was purchased from atcc . yph252 transformation was carried out by 
 electroporation as previously described ( meilhoc et al . plasmid constructions - plasmid psvjs01 , a newly designed shuttle 
 vector , was constructed as follows : a 2-kb fragment was amplified by pcr from prs414 
 ( atcc ) . it contains necessary elements to allow replication ( cen6/ars h1 ) and selection 
 ( trp1 ) in yeast . the 5-end primer used on this pcr reaction included a t7 promoter 
 recognition site and a multiple cloning site ( mcs ) . to assemble psvjs01 , the 2-kb 
 fragment was inserted into not i - linearised pbelobac11 vector ( new 
 england biolabs ) by homologous recombination ( polo et 
 al . purified plasmid dna was amplified in dh10b e. coli strain 
 in selective medium and analysed by restriction pattern on agarose gel 
 electrophoresis . denv-3 denv-3 genome was amplified in five overlapping subgenomic fragments 
 ( table ) using the klentaq la polymerase mix 
 ( clonetech ) , a high fidelity dna polymerase enzymes mix . the first fragment ( f1 ) was 
 amplified using primers psvjs01-f and denv-3 - 1860-r . the forward primer used on this pcr 
 reaction included an rsr ii restriction site , a t7 promoter recognition 
 site and an additional g immediately before the first base of denv-3 genome . this reaction used as 
 template a rt product derived from a denv-3 infectious clone recovered by in vitro 
 ligation ( jjs santos & lhvg gil , unpublished observations ) and introduced a unique 
 bam hi site into the denv-3 infectious clone sequence reported on 
 this paper . the third fragment ( f3 ) was amplified with primers denv-3 - 3481-f and 
 denv-3 - 4570-r while primer set denv-3 - 4370-f and denv-3 - 6700-r was used to amplify 
 fragment f4 . the last fragment ( f5 ) was amplified using primers denv-3 - 6521-f and 
 psvjs01-r . tableprimer set for construction of the dengue virus type 3 ( denv-3 ) infectious 
 clone and replicon pcr fragment   primer i d   primer sequence ( 5-3 )   primer annealing   position ( nt ) 
  gene region   f1   psvjs01-f 
 cggtccgtaatacgactcactatagagttgttartctacgtggaccga   1 - 23   5-utr   denv-3 - 1860-r   tctttcttcaacacaaaggtattc 1,837 - 1,860   e   f2   denv-3 - 1141-f   aactgactcaagatgtcctacc 1,141 - 1,162   e   denv-3 - 3521-r   ggattgccaaacacaagacac 3,501 - 3,521   ns2a   f3   denv-3 - 3481-f   ggtggacaacttcacaatgg 3,481 - 3,500   ns2a   denv-3 - 4570-r   ttccagttctgctttctgtgtc 4,549 - 4,570   ns3   f4   denv-3 - 4370-f   acaatgagaataaaagatgatgaga 4,370 - 4,394   ns2b   denv-3 - 6700-r   gactatagccgacgcgatccat 6,679 - 6,700   ns4a   f5   denv-3 - 6521-f   acaatggaaacactcctactct 6,521 - 6,542   ns4a   psvjs01-r 
 ttcaacatttccgtgtcgcgcggccgcagaaccrgttgattcaacagcaccatt 10,681 - 10,707   3-utr   rf1   psvjs01-f 
 cggtccgtaatacgactcactatagagttgttartctacgtggaccga 1 - 23   5-utr   rep - prm- bam 
 hi - e - r 
 gttttttggggatcctcgtgaagtcaagtggaaag   435 - 454   c / prm   rf2   rep - prm- bam 
 hi - e - f  
 gacttcacgaggatccccaaaaaacacatccatgtcat 2,336 - 2,357   e   denv-3521-r   ggattgccaaacacaagacac 3,501 - 3,521   ns2a 
 a : nucleotide ( nt ) numbering refers to denv-3 full - length 
 genome ; ns : nonstructural ; utr : untranslated region . 
 
 a : nucleotide ( nt ) numbering refers to denv-3 full - length 
 genome ; denv-3 replicon was generated by a pcr - mediated deletion , eliminating almost the entire 
 structural coding region [ from nucleotide ( nt ) 454-nt 2336 ) ] , as depicted in fig . 2 , and 
 cloned as a set of five fragments ( table ) , the first fragment ( rf1 ) 
 was amplified using primers psvjs01-f and rep - prm- bam hi - e - r , 
 generating a 0.5-kb pcr product that retained the first six codons of prm . the second 
 fragment ( rf2 ) was amplified with primers rep - prm- bam hi - e - f and 
 denv-3521-r , producing a 1.3-kb pcr product carrying the last 26 codons of e protein . primers rep - prm- bam hi - e - f and rep - prm- bam hi - e - r 
 were engineered to insert a single bam hi site in the denv-3 replicon 
 sequence . both constructs were assembled into not i - linearised psvjs01 
 vector by homologous recombination ( polo et al . 
 1997 , gibson 2009 ) in yph252 yeast 
 strain . recombinant clones were selected in drop - out ynb agar plates , and then screened 
 by pcr and dna sequencing . functional clones were maintained in yeast to prevent any 
 instability of sequences in bacteria . genome sequence was deposited in the genbank under 
 the accession kc425219 . 
 in vitro transcription and transfection - full - length pcr product from 
 denv-3 infectious clone full - length rna transcripts were in vitro synthesised using the 
 megascript t7 kit ( ambion ) with the addition of a 7-methyl - guanosine cap analog 
 ( ambion ) , following manufacturer s instructions . an aliquot from the reaction was 
 analysed by formaldehyde - agarose gel electrophoresis and rna transcripts were used 
 without further processing . full - length rna transcripts were introduced into bhk-21 
 cells by electroporation using the ecm 830 electro cell manipulator ( btx harvard 
 apparatus ) . 2 x 10   bhk-21 cells were trypsinised and washed twice with 
 serum - free medium , resuspended in 100 l cytomix solution ( ansari et al . 2004 ) and mixed with 5 g of transcribed rna . electroporation 
 was performed with two pulses of 100 s at 1,200 v separated by 1 s interval . as 
 negative control , bhk-21 cells were transfected under the same conditions except no rna 
 was added ( mock ) . after electroporation , cells were allowed to recover for 10 min at 
 room temperature and resuspended in complete growth medium . immunofluorescence assay ( ifa ) - to detect denv-3 virus antigen , 
 transfected cells were seeded onto glass coverslips . at different time points , rna - transfected cells in glass coverslips were rinsed with phosphate buffered saline 
 ( pbs ) , fixed with 50% cold acetone ( v / v ) in pbs for 5 min at 4c and air - dried . fixed 
 cells were incubated with a 1:100 dilution of a polyclonal hyperimmune mouse ascitic 
 fluid ( hmaf ) for 1 h at 37c . this hmaf was raised against group b flaviviruses , 
 including yfv and the four denv serotypes . while its reactivity has not been fully 
 tested , this hmaf reacts predominantly to e protein . after incubation with primary 
 antibody , cells were rinsed twice with pbs and incubated for 1 h at 37c with a 1:100 
 dilution of fluorescein isothiocyanate ( fitc)-conjugated anti - mouse igg antibody 
 ( sigma - aldrich ) , developed in goat . then , cells were rinsed twice with pbs and the 
 nuclei stained with a hoechst 33258 solution ( sigma - aldrich ) . prior visualisation by 
 fluorescent microscopy , cell were washed twice with ddh 2 0 , air - dried and 
 mounted . immunofluorescence analyses were carried out on a dmi 4000b inverted microscope 
 ( leica ) . stability of denv-3 infectious clone in yeast - to evaluate genome 
 stability , cloned denv-3 infectious clone was repeatedly propagated in yeast . recombinant clones were grown in drop - out ynb agar plates at 30c for three days . grown colonies were streaked out in fresh selective agar plates and incubated at 30c for 
 other three days . after 10 consecutive passages , plasmid dna was purified and genome 
 integrity was assessed by dna sequencing and virus rescue . denv-3 replicon rna transcription and transfection - the entire denv-3 
 replicon sequence was amplified by a high - fidelity full - length pcr . the pcr product was 
 purified by phenol - chloroform extraction and ethanol precipitation and used as a 
 template for in vitro transcription . replicon transcripts were synthesised using the 
 mega- script t7 kit ( ambion ) with the addition of a 7-methyl - guanosine cap analog 
 ( ambion ) . denv-3 replicon transient replication assessed by rt - pcr - at different 
 time points , total rna was purified from bhk-21 cells transfected with denv-3 replicon 
 using the invisorb spin cell rna mini kit ( invitek ) , according to manufacturer s 
 instructions . total rna isolation from mock - transfected bhk-21 cells was used as 
 negative control . quantified rna preparations served as templates to rt using 
 superscript iii reverse transcriptase ( invitrogen ) with reverse primer denv-3 - 3521-r 
 ( 5-ggattgccaaacacaagacac-3 ) , following the manufacturer s protocol . standard pcr 
 amplifications were carried out using rt products as templates and specific primers : 
 denv-3 - 1-f ( 5-agttgttagtctacgtggaccg-3 ) and denv-3 - 3521-r ( table ) . virus titration and growth curves - virus titration was determined by 
 focus -forming essay in mosquito c6/36 cells , as described 
 previously ( das et al . briefly , 2 x 10   cells per well were seed in 24-well cell 
 culture dishes 48 h prior to the assay was conducted . cell monolayers were incubated at 
 30c with 0.2 ml of serial dilutions of each virus . after removing the inocula , cells 
 were covered with 1 ml of l-15 overlay medium , containing 1% carboxymethyl cellulose , 2% 
 fbs and 1% antibiotic solution and incubated at 30c . on day 5 , cells were fixed with 30% cold acetone ( v / v ) in pbs for 13 min at room 
 temperature and washed once with pbs . fixed cells were incubated for 1 h at 37c with a 
 hmaf diluted 1:100 in ligation buffer . cells were rinsed with wash buffer and incubated 
 with a 1:500 dilution in ligation buffer of a horseradish peroxidase - conjugated 
 recombinant protein g ( invitrogen ) for 1 h at 37c . cells were rinsed with wash buffer 
 and foci developed by adding 3-amino-9-ethylcarbazole substrate buffer . foci were counted and virus titres , expressed in 
 focus -forming unit per ml , determined by standard methods . for 
 growth curves , 5 x 10   mosquito c6/36 cells per well in a 24-well cell 
 culture dishes were infected in duplicate with wild - type denv-3 ( wt - denv-3 ) and 
 transcript - derived denv-3 at a multiplicity of infection ( moi ) of 0.1 . cells were 
 incubated at 30c for 1 h. after inocula removal , cells were washed twice with pbs and 1 
 ml of growth medium was added to each well . aliquots of the supernatant of infected 
 cells were removed daily and stored at -70c . denv-3 titres in each sample were 
 determined by titration in mosquito c6/36 cells . statistical analysis - statistical analyses were performed using the 
 graphpad prism 4 for macintosh ( graphpad software , inc ) . growth kinetics differences 
 were analysed by a two - way anova with a mixed linear model for repeated measures , 
 followed by a bonferonni s corrected post hoc test for pairwise 
 comparisons at each time point . construction of a newly designed shuttle vector psvjs01 - plasmid 
 vectors and cloning hosts are critical to successful development of flavivirus 
 infectious clone ( ruggli & rice 1999 ) . many 
 strategies to stabilise flavivirus genome aim to reduce plasmid copy number in 
 e. coli by using low - copy plasmid or bac vectors . we sought a 
 similar approach to lower plasmid copy number in yeast by using a chromosome - derived 
 origin of replication . the psvjs01 backbone is based on pbelobac11 , a bac vector . to 
 create the new shuttle vector , a 2-kb fragment was amplified by pcr from prs414 
 containing cen6/ars h1 and trp1 . a t7 promoter recognition site and a mcs were also 
 engineered into this fragment . plasmid psvjs01 was assembled by inserting 2-kb fragment 
 into not i - linearised pbelobac11 vector by homologous recombination . purified plasmid dna was amplified in dh10b e. coli strain and correct 
 cloning of inserts was confirmed by restriction pattern on agarose gel electrophoresis 
 ( data not shown ) . denv-3 genome was amplified from independent isolated cdna 
 clones in five overlapping subgenomic fragments . all five fragments required to produce 
 denv-3 infectious clone along with not i - linearised psvjs01 vector were 
 introduced into yph252 yeast strain by electroporation and a full - length infectious 
 clone ( psvjs01-ic - denv-3 ) was assembly by homologous recombination . sequence analysis 
 confirmed the successful construction of denv-3 infectious clone and identified one 
 silent mutation at position 9490 ( a9490 g ) in denv-3 coding region . denv-3 genome 
 was amplified in five overlapping subgenomic fragments : f1 , f2 , f3 , f4 and f5 . full - length denv-3 infectious clone was assembled into psvjs01 vector by 
 homologous recombination , generating psvjs01-ic - denv-3 . a single 
 bam hi site was engineered and incorporated into denv-3 
 infectious clone sequence . virus rescue and in vitro characterisation of transcript - derived denv-3 
 - full - length pcr amplicons derived from psvjs01-ic - denv-3 # 3 were in vitro synthesised 
 and resulting rna transcripts were electroporated into bhk-21 cells . cells expressing 
 denv-3 proteins were easily detected four days after electroporation with denv-3 
 full - length transcripts , as demonstrated by ifa ( fig . were observed over time 
 clearly showing rna transcripts derived from psvjs01-ic - denv-3 # 3 are infectious in 
 bhk-21 cells . on day 8 , pronounced signs of cytopathic effect were visible in bhk-21 
 cell monolayers . in vitro characterisation by focus -forming assay of 
 the rescued virus showed no significant difference between wt - denv-3 ( fig . transcript - derived denv-3 grew 
 slightly slowly than wt - denv-3 , but this difference was not statistically significant ( p 
 > 0.05 at each time point ) . stability of the denv-3 infectious clone in yeast - in order to further 
 characterise denv-3 infectious clone , the genome stability of functional cdna clones was 
 examined in yeast . recombinant clones were grown in drop - out ynb agar plates for 10 
 subsequent passages separated by three days interval each . no alteration in growth 
 characteristics , colony morphology or coloration was observed during these passages . rna 
 transcripts derived from denv-3 infectious clone after repeated passages of the plasmid 
 in yeast remained infectious upon transfection into bhk-21 cells , as shown in fig . 3b . more importantly , sequence analysis 
 identified no additional mutation in the cloned genome , indicating denv-3 infectious 
 clone could be stably propagated in yeast for at least 10 passages . construction and characterisation of a denv-3 replicon - denv-3 
 replicon was generated by an in - frame deletion removing almost the entire prm - e conding 
 region ( from nt 454-nt 2336 ) , as illustrated in fig . 2 . only the first six codons of prm and the last 26 codons of e were retained 
 in the replicon sequence , as previously described ( pang 
 et al . denv-3 replicon was cloned into not 
 i - linearised psvjs01 as a set of five fragments by homologous recombination technique in 
 yph252 yeast strain , generating plasmid psvjs01-rdenv-3prme . as part of the cloning 
 strategy , a bam hi site was engineered into psvjs01-rdenv-3prme 
 sequence to serve as a genetic marker . pcr amplification and digestion by restriction 
 enzyme confirmed the successful deletion of the target structural protein coding region 
 ( data not shown ) . full - length pcr product derived from psvjs01-rdenv-3prme was in vitro 
 transcribed and resulting denv-3 replicon transcript was electroporated in bhk-21 cells . total rna was purified and replication of denv-3 replicon monitored by rt - pcr over time . 
 on day 3 6b , lane 2 ) , a band corresponding 
 to 1,646-bp in length was amplified by rt - pcr . band size is coherent with the in - frame 
 deletion in denv-3 replicon ( fig . 6b , lane 3 ) , 
 but the intensity of the band was much higher than observed on day 3 , showing denv-3 
 replicon is functional and capable of autonomous replication in bhk-21 cells . this peak 
 in replication of denv-3 replicon on day 4 is consistent with published results in the 
 literature ( alcaraz - estrada et al . however , replication of denv-3 replicon is transient and no pcr amplification was 
 observed on day 10 ( fig 6b , lane 4 ) . digestion 
 of rt - pcr product from day 4 with bam hi ( lane 5 ) generated fragments 
 of 1,192-bp and 454-bp , confirming the genetic marker was retained in denv-3 
 replicon . 2schematic diagram for the construction of a dengue virus type 3 ( denv-3 ) 
 replicon with the yeast shuttle vector psvjs01 . denv-3 replicon was generated 
 by a polymerase chain reaction - mediated deletion into the structural proteins 
 coding region [ from nucleotide ( nt ) 454-nt 2336 ] . denv-3 replicon was assembled 
 into psvjs01 vector by homologous recombination , creating psvjs01-rdenv-3prme . a unique bam hi site was inserted into denv-3 replicon 
 sequence as a genetic marker . 3transcript - derived ic - dengue virus type 3 ( denv-3 ) # 3 protein expression in 
 bhk-21 cells analysed by immunofluorescence assay . bhk-21 cells were 
 electroporated with in vitro transcribed rna from ic - denv-3 # 3 infectious 
 clone . on day 4 after electroporation , cells were fixed and incubated with a 
 polyclonal hyperimmune mouse ascitic fluid raised against group b flaviviruses , 
 followed by an anti - mouse igg antibody conjugated to fluorescein 
 isothiocyanate . images 
 were captured in a dmi 4000b inverted microscope ( leica ) with a 200x 
 magnification factor . a : bhk-21 cells electroporated with transcript - derived 
 ic - denv-3 # 3 ; b : transcript - derived ic - denv-3 # 3 remains infectious after 10 
 repeated passages of the plasmid in yeast . mosquito c6/36 cells were infected with either wild - type dengue virus type 3 
 ( wt - denv-3 ) or transcript - derived ic - denv-3 # 3 . after removing the inocula , cells were 
 fixed and incubated with a polyclonal hyperimmune mouse ascitic fluid raised 
 against group b flaviviruses , followed by a horseradish peroxidase - conjugated 
 recombinant protein g. foci were developed by adding 
 3-amino-9-ethylcarbazole substrate . a : wt - denv-3 foci in 
 mosquito c6/36 cells ; b : transcript - derived ic - denv-3 # 3 foci 
 in mosquito c6/36 cells . 
 
 fig . 5transcript - derived ic - dengue virus type 3 ( denv-3 ) # 3 and wild - type 
 ( wt)-denv-3 growth kinetics in mosquito c6/36 cells . mosquito c6/36 cells were 
 infected in duplicate at multiplicity of infection ( moi ) of 0.1 with wt - denv-3 
 or transcript - derived denv-3 . a sample of the supernatant of infected cells was 
 collected daily and virus titres were determined by titration in mosquito c6/36 
 cells . no statistically significant difference in virus titre ( p > 0.05 ) was 
 found between the viruses at different time points . a : wt - denv-3 growth curve 
 in mosquito c6/36 cells ; b : transcript - derived ic - denv-3 # 3 growth curve in 
 mosquito c6/36 cells ; ffu : focus -forming unit . 6kinetics of dengue virus type 3 ( denv-3 ) replicon rna synthesis in 
 transfected bhk-21 cell . a unique bam hi site was inserted 
 into denv-3 replicon sequence during molecular cloning and used as a genetic 
 marker . a : schematic diagram of rdenv-3prme in - frame deletion and genetic 
 marker analysis ; b : at different time points , total rna was purified from 
 bhk-21 cells transfected with rdenv-3prme replicon . an 1,646-bp fragment 
 spanning the bam hi site was amplified by reverse 
 transcription - polymerase chain reaction ( rt - pcr ) on day 3 ( lane 2 ) and 4 ( lane 
 3 ) and analysed on agarose gel electrophoresis . the rt - pcr product on day 4 was 
 digested with bam hi ( lane 5 ) generating fragments of 1,192-bp 
 and 454-bp . no amplification was observed on day 10 ( lane 4 ) . an 1 kb plus dna 
 ladder ( invitrogen ) was loaded on lane 1 ; mw : molecular weight ; ns : 
 nonstructural ; utr : untranslated region . flaviviruses research has experienced a remarkable progress by the advent of reverse 
 genetics systems in the past decades . however , obtaining flavivirus infectious clones 
 has not been an easy task . the single most reported hurdle in the establishment of these 
 systems is an inherent instability of flavivirus genome in e. coli ( 
 pu et al . 2011 ) . in this paper , the 
 successful construction and characterisation of a complete reverse genetics system for 
 denv-3 has been described . infectious cdna clone methodology has been developed for all four denv 
 serotypes and other major flaviviruses , but most systems utilised established strains or 
 laboratory - adapted clinical isolates as parental virus . even though the value of these 
 studies is unquestionable , it is important to point out the need to expand this 
 methodology for low passage clinical isolates to gain a complete picture in flavivirus 
 biology , particularly in virus pathogenesis , virulence determinants and vector 
 competence ( ubol et al . 2008 , tuiskunen et al . 2011 , cox et al . 2012 ) . our strategy employed the use of homologous recombination in yeast cells to assemble 
 denv-3 infectious clone and replicon . yeast strains are versatile and convenient cloning 
 hosts , frequently used in the construction of reverse genetics systems for denv ( polo et al . homologous recombination in yeast is also a 
 simple and robust method with higher cloning efficiency when compared to standard 
 cloning procedures in e. coli ( gibson 
 2009 , shanks et al . we have 
 also demonstrated the utility of psvjs01 vector for stable propagation of denv-3 genome 
 in yeast . several reverse genetics systems were established using low - copy plasmid or 
 bac vectors in an effort to lower plasmid copy number in e. coli and , 
 as consequence , achieve stability of cloned genomes . in a previous attempt , we were only 
 able to recovery a denv-3 infectious clone by an in vitro ligation strategy using the 
 pbsc shuttle vector ( jjs santos & lhvg gil , unpublished observations ) . despite this 2006 ) , pbsc vector carries a 2 m high copy number origin of 
 replication . we hypothesised that reducing plasmid copy number in yeast using a 
 chromosome - derived origin of replication would allow the successful assembly of a 
 full - length denv-3 infectious clone . ( 2011 ) , no silent 
 mutations were necessary to be introduced to circumvent sequence instability and 
 recovery a functional infectious clone . rna transcripts derived from denv-3 infectious 
 clone were infectious upon transfection into bhk-21 cells . transcript - derived denv-3 
 exhibited growth kinetics , focus formation and size comparable to 
 wt - denv-3 in mosquito c6/36 cell culture . of paramount importance , rna transcripts 
 remained infectious even after repeated passages of the plasmid in yeast . the long - term 
 stability of many infectious clones previously described remains largely undetermined 
 after serial passages of the cloned genome in the harbouring host . only few reports have 
 more closely investigated this stability of continuing propagated flavivirus infectious 
 clones ( hurrelbrink et al . most of their assessment relies on virus rescue 
 from or restriction pattern of passaged plasmid . our work went one step further showing 
 not only the ability of rescuing virus , but also that the nucleotide sequence of denv-3 
 viral genome remain unchanged after successive passages of the plasmid in yeast . we 
 used an approach previously described for a denv-2 replicon by selectively removing 
 almost the entire prm - e coding region ( pang et al . denv-3 replicon was also easily assembled into psvjs01 shuttle vector . 
 in vitro characterisation of denv-3 replicon , we describe an alternative and effective strategy to bypass flavivirus 
 genome instability commonly observed in e. coli by using homologous 
 recombination in yeast and a newly designed psvjs01 vector . this reverse genetics system 
 will be a valuable tool for studying the molecular mechanisms involved in viral genome 
 replication , virion assembly , virus pathogenesis and vaccine development .