BACKGROUND
the genus flavivirus contains a large number of emerging, vector-transmitted viruses.
of these, the four serotypes of dengue virus  pose the most significant threat to global public health.
the global pandemic of dengue fever has escalated dramatically in recent decades, accompanied by a sharp increase in the more severe manifestations of the disease, dengue hemorrhagic fever and dengue shock syndrome  <cit> .
widespread cessation of vector control, increases in mosquito-breeding sites due to rapid urbanization, and expansion of global travel have all contributed to denv emergence  <cit> .
vector control is a costly and often ineffective response to outbreaks  <cit> .
no antivirals are currently available for any flavivirus  <cit> , and although promising denv vaccine candidates have recently entered clinical trials  <cit> , progress in the development of a denv vaccine has been slow  <cit> .
in response to this exigency, investigators have pursued novel methods to prevent and treat dengue disease.
in particular, there is considerable excitement about the potential to utilize rna interference   to treat flavivirus infection in the host and control flavivirus transmission by the vector  <cit> .
the rnai pathway is composed of two major branches .
the small interfering rna  branch is triggered by perfectly or nearly-perfectly base-paired exogenous dsrna and results in rna degradation, while the cellular microrna branch  is triggered by imperfectly base-paired dsrna and results in translation repression  <cit> .
although sirnas and mirnas are processed via discrete pathways, specific enzymes may participate in both pathways.
for example, recent evidence from drosophila indicates that dicer - <dig> is critical for both rna degradation and translation repression, while dcr- <dig> is required only for rna degradation  <cit> , and that argonaute - <dig> and ago- <dig> proteins overlap in their functions  <cit> .
kumar et al.
<cit>  have demonstrated that introduction of exogenous sirnas can prevent encephalitis caused by west nile virus  and japanese encephalitis virus infections, and genetically-modified mosquitoes expressing sirnas are currently being developed to prevent transmission of denv  <cit> .
however, the impact of rnai triggered by endogenous dsrna produced during virus infection on denv replication, or that of any flavivirus, has received little study.
to date, only two studies have examined whether virus-triggered rnai regulates replication of a flavivirus.
chotkowski et al.
demonstrated that drosophila melanogaster s <dig> cells infected with wnv produced abundant anti-wnv sirnas and that knockdown of ago- <dig>  in these cells increased the rate but not the overall level of wnv replication  <cit> .
moreover, d. melanogaster carrying homozygous null mutations in ago- <dig>  spindle-e  or piwi  supported higher levels of wnv replication than wild type controls, while flies carrying homozygous null mutations in dcr- <dig>  did not  <cit> .
intriguingly, aedes albopictus mosquito c6/ <dig> cells infected with wnv did not produce anti-wnv sirna's, prompting the authors to speculate that the rnai response in this cell line may be weaker than that of drosophila cells  <cit> .
however sanchez-vargas et al.
showed that cells of aedes aegypti mosquitoes, the major vector of denv, produce anti-denv sirna following infection with denv- <dig> in culture and in vivo  <cit> .
moreover in the latter study knockdown of dcr- <dig>  ago- <dig>  or r2d <dig>  all significantly enhanced the rate and level of denv- <dig> replication, with knockdown of dcr- <dig> having the strongest impact.
these findings indicate that components of both the mirna and the sirna branches are involved in modulating viral replication, and that complete functional segregation of the two branches is lacking.
to gain further insight into the ability of rnai to modulate denv infection, in the current study we first investigated whether s <dig> cells are susceptible to denv infection.
s <dig> cells are an attractive substrate for investigation of rnai for three reasons:  the rnai pathway in drosophila is well characterized,  rnai knockdown in s <dig> cells can be accomplished simply be overlaying them with dsrna or sirna  <cit> , and  previously validated sirna's for knockdown of specific rnai enzymes are readily available  <cit> .
after finding that denv replicates in s <dig> cells, we tested whether s <dig> cells respond to denv infection by production of sirna.
finally, we tested the impact of individually knocking down four enzymes of the rnai pathway: dcr- <dig>  dcr- <dig>  ago- <dig> and ago- <dig> on the replication dynamics of denv.
methods
cells
schneider s <dig> cells   <cit>  acquired from the drosophila genomics resource center  were maintained at 28°c in conditioned s <dig> media composed of schneider's drosophila media  supplemented with 10% fetal bovine serum ,  <dig> mm l-glutamine , and 1× penicillin-streptomycin-fungizone® .
media used for dsrna/sirna dilutions  was schneider's drosophila media supplemented with  <dig> mm l-glutamine and 1× psf.
c6/ <dig> cells   <cit>  were maintained at 32°c with 5% co <dig> in minimal essential media  supplemented with 10% fbs,  <dig> mm l-glutamine,  <dig> mm nonessential amino acids  and  <dig>  mg/ml gentamycin .
viruses
to compare the replication of the four serotypes of denv, three isolates of each were selected from a broad array of geographical locations .
each isolate was passaged in c6/ <dig> cells to generate a stock, designated c6/ <dig> p <dig> moi  <dig> , for use in all experiments.
c6/ <dig> cells were infected at moi  <dig> , incubated for two hrs with occasional, gentle rocking under the conditions described above.
five days post infection , supernatant was collected, clarified by centrifugation, stabilized with  <dig>  times volume of 10× spg , and stored at -80°c.
the titer of each c6/ <dig> p <dig> moi  <dig>  stock was determined via serial titration in c6/ <dig> cells as described below.
1cell type for passage followed by total number of passages  in that cell type
<dig> wrceva: provided by dr. robert tesh at the world reference center of emerging viruses and arboviruses at the university of texas at galveston ; niaid: provided by dr. stephen whitehead, niaid, nih; unc: provided by dr. aravinda de silva, department of microbiology and immunology, university of north carolina.
quantification of virus titer
monolayers of c6/ <dig> cells were grown to 80% confluency in 24-well tissue culture treated plates  and infected with serial tenfold dilutions of each stock virus or cell supernatant.
plates were incubated for two hrs with intermittent gentle rocking at 32°c.
inoculated monolayers were overlaid with  <dig> % methylcellulose in optimem  supplemented with 2% fbs,  <dig> mm l-glutamine and  <dig>  mg/ml gentamycin.
focus forming units are referred to as "plaques" hereafter for consistency with previous literature  <cit> ; plaques were detected via immunostaining as previously described  <cit> .
denv- <dig> -  <dig> were detected using denv -  <dig> specific monoclonal antibody 15f <dig>  denv -  <dig> hyperimmune mouse ascites fluid , denv -  <dig> specific hybridoma cell supernatant, and denv-  <dig> hmaf, respectively; all antibodies were the kind gift of dr. stephen s. whitehead, national institute of allergy and infectious disease, national institutes of health, bethesda, md.
infection of s <dig> cells by denv
s <dig> cells were grown to 80% confluency  in six-well tissue culture treated plates .
triplicate wells were infected with each of the  <dig> c6/ <dig> p <dig> moi  <dig>  stocks at a specified moi, based on titer in c6/ <dig> cells  divided by the number of s <dig> cells/well, in a total volume of one ml.
virus was incubated for two hrs at 28°c with occasional, gentle rocking and washed once with one ml of conditioned s <dig> media.
thereafter three ml of conditioned s <dig> media was added to each well.
s <dig> cells were infected at moi  <dig> and incubated for five days at 28°c after which cell supernatants, designated s <dig> p <dig> moi  <dig>  were collected and frozen as described above.
<dig> μl from each s <dig> p <dig> moi  <dig> replicate were then passaged in fresh s <dig> cells as described above.
given the titers on day five for s <dig> p <dig> moi  <dig> ,  <dig> μl of supernatants contained a total of  <dig>  -  <dig>  log10plaque forming units .
cells were incubated for five days and harvested to yield s <dig> p <dig> moi  <dig>  s <dig> cells were infected similarly at moi  <dig>  to yield cell supernatants s <dig> p <dig> moi  <dig> , but these supernatants were not passaged further.
virus titer in all cell supernatants was determined by serial titration in c6/ <dig> cells as described above.
denv replication kinetics in s <dig> cells
triplicate wells of s <dig> cells in six-well plates were infected with the c6/ <dig> p <dig> moi  <dig>  stock of denv- <dig> taiwan at moi  <dig> .
two hrs post infection the inoculum was removed, cells were washed once with conditioned s <dig> media, fresh media was added and  <dig> ml cell supernatant was collected from each well  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> hrs pi and frozen as described above.
fresh media was added to each well for every sampling point so that the total volume of media remained constant.
detection of anti-denv sirnas in s <dig> cells
northern blots were used to detect anti-denv sirnas in infected s <dig> cells.
to assess the production of sirna's in response to infection, one set of s <dig> cells at 80% confluency were infected with denv- <dig> jkt, denv- <dig> tonga, denv- <dig> sleman and denv- <dig> taiwan at moi  <dig>  as described above.
to assess the impact of knocking down components of the rnai pathway on sirna production, a second, concurrent set of s <dig> cells were treated with dsrna to dcr- <dig> or dcr- <dig> and then infected with denv- <dig> jkt, denv- <dig> tonga, denv- <dig> sleman and denv- <dig> taiwan as described below.
three days pi small rnas  were isolated using mirpremier® microrna isolation kit .
rna was quantified, separated on 15% urea polyacrylamide gel using tris borate edta and transferred to hybond™-n+ nylon membrane .
blots were probed with approximately  <dig> nucleotide long digoxigenin  labeled positive-sense probes complementary to nucleotides  <dig> -  <dig> of the 3' untranslated region  of denv- <dig> western pacific,  <dig> -  <dig> of the 3'utr of denv- <dig> tonga,  <dig> -  <dig> of the 3'utr of denv- <dig> sleman and  <dig> -  <dig> of the 3'utr of denv- <dig> taiwan.
the justification for targeting the probe to the 3' utr is based on a recent report that anti-west nile virus sirna's cluster, among other genome locations, in the 3' utr  <cit> .
blots were processed according to protocol defined by the manufacturer for dig probes .
knockdown of enzymes in the rnai pathway
four components of the rnai pathway, ago- <dig>  ago- <dig>  dcr- <dig> and dcr- <dig>  were separately depleted using  <dig> base-pair  dsrna targeting nucleotides  <dig> -  <dig> of dcr- <dig>   <dig> -  <dig> of dcr- <dig>   <dig> -  <dig> of ago- <dig> mrna from d. melanogaster  or a previously validated  <dig> bp sirna against d. melanogaster ago- <dig>  <cit> .
a dsrna targeting nucleotides  <dig> -  <dig> of pgex-2t cloning vector  was used as a control for dsrna knockdown while a renilla luciferase sirna  targeting luciferase was used as control for sirna knockdown.
to generate dsrna, d. melanogaster dna was isolated using the qiagen dneasy blood & tissue kit  and amplified using primers specific to d. melanogaster ago- <dig>  ago- <dig>  dcr- <dig> and dcr- <dig> .
primers contained a t <dig> promoter sequence at the 5' end to allow for transcription using megascript® rnai kit  according to manufacturer's instruction.
transcription of sirna was performed using silencer® sirna construction kit .
<dig>  log <dig> ±  <dig>  log <dig> s <dig> cells were plated on six-well plates and incubated for  <dig> minutes at 28°c.
dsrna/sirna were diluted in one ml of unconditioned s <dig> media to  <dig> nm, applied to the s <dig> cells, and incubated at 28°c for  <dig> hrs.
thereafter three ml of conditioned s <dig> media was added and cells were incubated as described above  <cit> .
cells were re-fed with dsrna/sirna three days following initial treatment.
<dig> 5' to 3' sequence
verification of knockdown
to assess the efficacy of knockdown, seven wells of s <dig> cells were treated with each of the dsrna/sirna's described above.
at two hrs,  <dig> hrs, and daily thereafter through day six post-treatment, cells from one well corresponding to each dsrna/sirna treatment were lysed using ripa buffer  and centrifuged for  <dig> minutes at  <dig>  rpm at 4°c.
supernatants were stored at -80°c in order to analyze all samples concurrently.
total protein in each sample was quantified using bca protein assay kit .
supernatants were separated on a polyacrylamide gel and transferred to immobilon polyvinylidene fluoride transfer membranes .
membranes were blocked with bovine serum albumin and incubated with d. melanogaster specific anti-dcr- <dig> , anti-dcr- <dig> , anti-ago- <dig> , or anti-ago- <dig> antibody   as appropriate.
protein bands were visualized with secondary anti-rabbit or anti-mouse hrp-conjugated igg  using the ecl system .
toxicity assay
to assess whether knockdown of dcr- <dig>  dcr- <dig>  ago- <dig> or ago- <dig> affected the viability of s <dig> cells, a resazurin-based viability assay was performed.
s <dig> cells were propagated to 80% confluency in five  <dig> well tissue culture treated plates .
each treatment was performed in triplicate wells on each plate as follows.
media was removed and designated dsrna/sirna's were added at a concentration of  <dig> nm.
two controls were included in the assay: treatment with  <dig> μl of conditioned s <dig> media was used to measure overall cell viability and treatment with 8% dmso was used to measure the impact of a compound known to be toxic.
plates were incubated for one to five days; on each day  <dig> μl of resazurin from the in vitro toxicology assay kit  was added all the wells of one plate.
the plate was then incubated two hrs and absorbance was read on a plate reader  at  <dig> nm.
the proportion of viable cells was determined by dividing the absorbance of each well on the plate by the average absorbance of the media-treated wells.
denv infection following knockdown of dcr-2
for each of the c6/ <dig> p <dig> moi  <dig>  stocks of  <dig> denv strains , triplicate wells of s <dig> cells in six-well plates were treated with dsrna targeting dcr- <dig> or with control dsrna as described above.
sixteen hrs post treatment wells were infected with the designated virus strain at moi  <dig> and incubated at 28°c.
based on the results of knockdown verification , infected cells were replenished with dsrna  <dig> hrs pi.
cell supernatants were carefully removed and stored in individual tubes at room temperature, leaving one ml residual supernatant per well.
<dig> nm dsrna was added to each well and incubated for  <dig> minutes at 28°c.
each cell supernatant that was removed was added back to its original well containing one ml of residual media.
cell supernatants were harvested  <dig> hrs pi and virus titer was determined as described above.
denv replication kinetics following knockdown of dcr- <dig>  dcr- <dig>  ago- <dig> or ago-2
to monitor the impact of rnai knockdown on denv replication kinetics, sets of six wells of s <dig> cells in six-well plates were treated with one dsrna/sirna targeting dcr- <dig>  dcr- <dig>  ago- <dig>  ago- <dig> or one control dsrna/sirna, as described above.
<dig> hrs post treatment, three wells treated with each enzyme were infected with denv- <dig> taiwan and three with denv- <dig> tonga at moi  <dig>  one ml cell supernatant was collected from each well  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> hrs pi and frozen as described above; one ml of fresh media was then added to each well so that the total volume of media remained constant.
all wells were re-fed dsrna/sirna at  <dig> hrs pi as described above.
statistical analysis
all statistical analyses were carried out using statview .
RESULTS
infection of s <dig> cells by denv
every denv strain achieved a titer >  <dig>  log10pfu/ml in c6/ <dig> cells five days post-infection at moi  <dig>  .
five days after infection of s <dig> cells at moi  <dig>  the  <dig> denv strains reached titers ranging from  <dig>  to  <dig>  log <dig> pfu/ml .
there was a significant positive correlation between titer of the  <dig> denv strains in c6/ <dig>  with the titer of those strains in s <dig>  .
there was no significant difference among the four denv serotypes in titer following this first passage in s <dig> cells , and titer did not change significantly following a second passage in s <dig> cells, s <dig> p <dig> moi  <dig> .
to confirm that the titers observed in s <dig> cells resulted from virus replication rather than carry-over of the inoculum, s <dig> cells were also infected with all  <dig> strains of denv at moi  <dig> ; five days pi all  <dig> strains had achieved titers ranging from  <dig>  to  <dig>  log <dig> pfu/ml .
there was no significant correlation between titers of the  <dig> strains following infection of s <dig> cells at moi  <dig>   and moi  <dig>   or between titers of the  <dig> strains following infection of s <dig> cells at moi  <dig>   and c6/ <dig> cells at moi  <dig>   .
additionally the replication kinetics of one strain, denv- <dig> taiwan, were followed daily for five days ; there was significant difference in virus titer among days post-infection ; specifically, a tukey-kramer post-hoc test revealed that virus titer increased between two hrs and  <dig> hrs  and leveled off thereafter at approximately  <dig>  log10pfu/ml.
detection of anti-denv sirna in s <dig> cells
virus-derived small rnas can range from  <dig> -  <dig> nucleotides depending on secondary structure of the viral genome and processing by rna processing enzymes  <cit> .
virus derived small rnas were detected in s <dig> cells three days after infection with denv- <dig> tvp, denv- <dig> tonga, denv- <dig> sleman and denv- <dig> taiwan by northern blotting  using positive-sense probes designed to detect negative sense sirnas that targeted the positive sense genome of each respective serotype.
no virus-derived sirna's were detected in uninfected control cells.
knockdown of dcr- <dig> or dcr- <dig> resulted in a substantial decrease in the production of virus-derived sirna's in s <dig> cells infected with each of the four isolates above .
the most extreme effect was apparent for dcr- <dig> knockdown followed by infection with denv- <dig> taiwan; in this treatment no virus-derived sirna's were detected at all .
toxicity in s <dig> cells following knockdown of dcr- <dig>  dcr- <dig>  ago- <dig> or ago-2
knockdown of each of the four components of the rnai pathway had no significant effect on cell viability .
a two-factor anova testing the effect of treatment and day post-infection on absorbance revealed a significant effect of treatment  but not day .
however a tukey-kramer post-hoc test revealed that only the dmso-treated cells, which were expected to show reduced viability, differed significantly from control cells , while none of the dsrna/sirna treated cells differed from controls .
denv replication following knockdown of rnai genes
to test whether the rnai response has an effect on denv replication in s <dig> cells, four components of the rnai pathway  were individually depleted via knockdown with an appropriate dsrna or sirna.
the efficacy of depletion of each enzyme was confirmed using western blot analysis .
dcr- <dig> levels were depleted for six days following treatment, but unlike the other three treatments there were no days on which dcr- <dig> expression was undetectable.
dcr- <dig> expression was undetectable until day three post-treatment and showed steady recuperation thereafter.
ago- <dig> expression was undetectable through day five post-treatment.
ago- <dig> expression was undetectable until day three post-treatment and rebounded on day four.
to prevent recovery of expression, all infected cell knockdowns were re-fed dsrna/sirna on day three post initial dsrna/sirna treatment.
as shown in figure  <dig>  all  <dig> denv strains tested achieved significantly higher titers  in cells depleted of dcr- <dig> relative to control cells .
the  <dig> denv strains attained similar titers in cells treated with a control dsrna treatment as compared to untreated cells.
moreover, there was no significant difference among serotypes in the impact of dcr- <dig> knockdown, measured as the difference in titer for a particular replicate virus in knockdown cells versus control cells .
in contrast, variation in the impact of rnai knockdown on the three denv strains within serotypes was detected using factorial anovas for each serotype; when significant differences were detected, a tukey-kramer post-hoc test was used to determine which strains showed significant differences in response to knockdown.
denv- <dig> strains showed significant variation in response to dcr- <dig> knockdown : strain tvp showed a significantly greater increase in titer when dcr- <dig> was knocked down than strains jkt and aush; the latter two did not differ from each other.
denv- <dig> and denv- <dig> strains did not show significant within-serotype variation .
denv- <dig> strains also showed significant variation to dcr- <dig> knockdown : all three strains tested differed significantly from each other.
subsequent analyses focused on two denv strains that had shown the smallest  and an intermediate  response to dcr- <dig> knockdown .
a multistep growth curve revealed that knockdown of dcr- <dig> resulted in enhancement of replication of both strains within  <dig> hrs pi, and by  <dig> hrs pi both strains had achieved a titer  <dig> -  <dig> - fold higher in dcr- <dig> depleted cells than control cells .
a similar pattern was observed following knockdown of dcr- <dig>  ago- <dig> and ago- <dig> ; titers of both denv strains were significantly higher in cells depleted of each enzyme than control cells  <dig> hrs pi .
discussion
the objectives of this study were threefold: first, to monitor the pattern of replication of denv in s <dig> cells in order to assess the utility of s2's for the study of denv, second to investigate the impact of rnai on denv replication; and third to test whether the impact of rnai differs among the four serotypes of denv.
five lines of evidence demonstrate that all four denv serotypes replicated in s <dig> cells.
first, infection of s <dig> cells with denv at an moi  <dig> and moi  <dig>  resulted in titers >  <dig>  and >  <dig>  log10pfu/ml, respectively, even though the input virus inoculum was thoroughly washed away two hours post-infection.
second, titers attained by denv following a second passage in s <dig> cells  were substantially larger than the total amount of virus used to initiate infection .
third, daily monitoring of the titer of denv- <dig> taiwan in s <dig> cells showed that titers increased significantly following one day of infection.
fourth, sirnas were detected in s <dig> cells after infection with each of the four serotypes of denv, indicating that denv infects and replicates in s <dig> cells.
finally, a significant increase in titer was observed for all denv strains when dcr- <dig> was knocked down using dsrnas.
such change in titer following down regulation of an antiviral response is indicative of active replication of denv.
there was no evidence of change in titer of denv between a first and second passage on s <dig> cells.
however future studies to monitor adaptation after extensive serial passage in s <dig> cells are planned.
sessions et al.
<cit>  reported that denv- <dig> ngc attained a peak titer of  <dig>  log10pfu/ml in s <dig> derived d.mel- <dig> cells without prior adaptation.
following serial passages for four months in d.mel- <dig> cells, denv- <dig> ngc titer increased to  <dig>  log10pfu/ml.
consistent with these findings, in the current study peak titers of denv in s <dig> cells infected at moi  <dig>  were approximately  <dig>  log10pfu/ml  <cit> .
however peak titers following infection at moi  <dig> were at least an order of magnitude higher.
like other rna viruses, denv exists as a quasispecies  <cit> , and it is possible that variants that were better able to infect s <dig> cells occurred in the larger virus population used to infect at moi  <dig>  relative to moi  <dig>  .
this hypothesis is supported by the finding that viruses that were taken from the moi  <dig> infection and passaged again onto s <dig> cells achieved a similar titer to the s <dig> p <dig> moi  <dig> infection, even though their founding population was only  <dig>  -  <dig>  log10pfu.
using denv adapted to s <dig> cells, sessions et al.
demonstrated the utility of these cells for investigation of dengue virus host factors   <cit> .
they identified  <dig> dvhf using a genome-wide rnai screen on d.mel- <dig> cells.
findings from the current study indicate that s <dig> cells can also support replication of unadapted denv, thereby offering additional opportunities to leverage the extraordinary depth of knowledge and plethora of tools in drosophila genetics for the study of denv  <cit> .
the titer of each denv strain in s <dig> cells was substantially lower than its titer in c6/ <dig> cells, which are derived from ae.
albopictus, a natural denv vector  <cit> .
at first glance, this result seems to suggest s <dig> cells may not be a useful model to study denv-vector interactions.
however, it has been previously demonstrated that c6/ <dig> cells exhibit a weak, and possibly incomplete, rnai response  <cit> , which may contribute to their ability to support high levels of denv replication.
in contrast, both live mosquitoes  <cit>  and s <dig> cells  <cit>  marshal a vigorous rnai response to infection with flaviviruses and other rna viruses that is capable of limiting viral replication  <cit> .
thus for some areas of study, particularly rnai-virus interactions, s <dig> cells may be preferable to c6/ <dig> cells as an in vitro model.
in this study s <dig> cells infected with denv- <dig>   <dig>   <dig> or  <dig> produced sirnas targeting the denv genome, as has been reported previously for a variety of viruses, including denv, in multiple types of insect cells both in culture and in vivo  <cit> .
in a notable exception to this rule, c6/ <dig> cells failed to produce sirnas when infected with wnv  <cit> .
the production of anti-denv sirna provides confirmation that denv is targeted by an active rnai response in s <dig> cells.
further, a decrease in virus derived small rnas was observed when dcr- <dig> or dcr- <dig> was knocked down prior to infection.
this finding supports the conclusion that enhancement of denv replication following knockdown of components of rnai  resulted from a relaxation of rnai control.
although the current study was designed to detect only sirnas complementary to the positive sense 3' utr, it would be very useful in the future to characterize the entire suite of sirnas produced in response to denv infection.
in drosophila, virus derived small rnas can be generated by dcr- <dig> or dcr- <dig>  <cit>  and subsequently processed by ago- <dig> or ago-2-risc   <cit>  .
knockdown of dcr- <dig> enhanced the replication of each of  <dig> strains of denv, and knockdown of ago <dig>  ago- <dig> or dcr- <dig> enhanced replication of the two denv strains tested.
none of the four knockdowns affected cell viability, supporting the conclusion that the observed augmentation of denv replication was due to knockdown of the targeted enzymes rather than off-target effects.
there was no difference in the impact of the four enzymes on denv replication dynamics, and there was no difference among serotypes in their average response to the knockdown of dcr- <dig>  intriguingly, strains within denv- <dig> and denv- <dig> serotypes showed significant variation in their response to dcr- <dig> knockdown.
these data suggest that denv strains may vary in their sensitivity to rnai, potentially contributing to differences in viral replication in the vector with downstream effects on transmission.
although the current study was not designed to draw inferences about response of specific denv genotypes to rnai or to contrast isolates associated with different grades of disease severity, the s <dig> system could be used to address these questions in the future.
the impact of dcr- <dig> and ago- <dig> knockdowns in this study are generally consistent with the results of sanchez-vargas et al.
<cit> , who found that knockdown of either enzyme in ae.
aegypti in vivo enhanced replication of denv- <dig>  although the impact of ago- <dig> knockdown was delayed in time relative to dcr- <dig>  however our results in s <dig> cells differ from the finding of chotkowski et al.
that loss of dcr- <dig> expression in s <dig> cells did not affect wnv replication  <cit> .
this disparity may reflect methodological differences, particularly differences in expression of rnai-pathway proteins between s <dig> cell lines, or differences between wnv and denv in sensitivity to rnai, and/or differences between the two viruses in their tendency to elicit rnai.
other studies have also revealed variation among viruses in their sensitivity to loss of dcr- <dig> function.
drosophila carrying a homozygous null mutation for dcr- <dig> were hypersusceptible to infection by drosophila c virus  and cricket paralysis virus  <cit> , and loss of function of dcr- <dig> in drosophila also resulted in increased infection by flock house virus, dcv and sindbis virus  <cit> .
in contrast, homozygous knockout of dcr- <dig> in drosophila had no impact on susceptibility to drosophila x virus   <cit> .
in the studies that detected no impact of dcr- <dig> function on replication of wnv or dcv, respectively  <cit> , the authors suggested that synthesis of sirna by dcr- <dig> may counteract the effect of loss of dcr- <dig>
in the current study, knockdown of either dcr- <dig> or ago- <dig> enhanced denv replication to a degree similar to each other and to dcr- <dig> and ago- <dig>  these findings indicate that the proteins are functionally linked between the mirna and sirna braches of the rnai pathway and thus impact viral replication.
these findings are consistent with the report that drosophila carrying a homozygous null mutation for aubergine  exhibit increased susceptibility to dxv infection  <cit>  and support the idea that dcr- <dig> and ago- <dig> also regulate virus replication.
such regulation likely stems from the activity of dcr- <dig> and ago- <dig> in the sirna branch of the rnai pathway.
evidence of such activity includes the requirement of dcr- <dig> for mrna degradation  <cit> , the observation of similar transcript profiles in cells depleted of ago- <dig> and ago- <dig>  <cit> , and the weak association of ago- <dig> with sirnas in cells depleted of ago- <dig>  <cit> .
from this perspective, it would be particularly interesting in future studies to assess the impact of concurrent knockdown of dcr- <dig> and dcr- <dig> or ago- <dig> and ago- <dig> on the dynamics of denv replication.
CONCLUSIONS
our results indicate that rna interference regulates denv replication in drosophila s <dig> cells, and that denv strains, but not serotypes, vary in their sensitivity to such regulation.
s <dig> cells offer a useful model for the study of denv-rnai interactions.
abbreviations
rnai: rna interference; denv: dengue virus; denv-1-4: dengue virus serotypes 1-4; wnv: west nile virus; dcv: drosophila c virus; dxv: drosophila x virus; ago: argonaute; dcr: dicer; sirna: short interfering rna; mirna: microrna; spn: spindle; pi: post infection; dig: digoxigenin; utr: untranslated region; bp: base pair; sirisc: rna induced silencing complex associated with sirna; risc: rna induced silencing complex; mirnp: mirna associated ribo nucleoprotein complex; mirisc: mirna associated risc; dsrna: double stranded rna; moi: multiplicity of infection; psf: penicillin-streptomycin-fungizone®; mem: minimum essential media; hr: hour; df: degrees of freedom; p1: passage 1; p2: passage  <dig>
competing interests
the authors declare that they have no competing interests.
authors' contributions
experiments were conceived by sm and kah and performed by sm.
data was analyzed by sm and kah.
the manuscript was written by sm and kah.
all authors have read and approved the final manuscript.

 the genus flavivirus contains a large number of emerging, vector-transmitted viruses. of these, the four serotypes of dengue virus pose the most significant threat to global public health. the global pandemic of dengue fever has escalated dramatically in recent decades, accompanied by a sharp increase in the more severe manifestations of the disease, dengue hemorrhagic fever and dengue shock syndrome. widespread cessation of vector control, increases in mosquito-breeding sites due to rapid urbanization, and expansion of global travel have all contributed to denv emergence. vector control is a costly and often ineffective response to outbreaks. no antivirals are currently available for any flavivirus, and although promising denv vaccine candidates have recently entered clinical trials, progress in the development of a denv vaccine has been slow. in response to this exigency, investigators have pursued novel methods to prevent and treat dengue disease. in particular, there is considerable excitement about the potential to utilize rna interference to treat flavivirus infection in the host and control flavivirus transmission by the vector. the rnai pathway is composed of two major branches. the small interfering rna branch is triggered by perfectly or nearly-perfectly base-paired exogenous dsrna and results in rna degradation, while the cellular microrna branch is triggered by imperfectly base-paired dsrna and results in translation repression. although sirnas and mirnas are processed via discrete pathways, specific enzymes may participate in both pathways. for example, recent evidence from drosophila indicates that dicer - is critical for both rna degradation and translation repression, while dcr- is required only for rna degradation, and that argonaute - and ago- proteins overlap in their functions. kumar et al. have demonstrated that introduction of exogenous sirnas can prevent encephalitis caused by west nile virus and japanese encephalitis virus infections, and genetically-modified mosquitoes expressing sirnas are currently being developed to prevent transmission of denv. however, the impact of rnai triggered by endogenous dsrna produced during virus infection on denv replication, or that of any flavivirus, has received little study. to date, only two studies have examined whether virus-triggered rnai regulates replication of a flavivirus. chotkowski et al. demonstrated that drosophila melanogaster s cells infected with wnv produced abundant anti-wnv sirnas and that knockdown of ago- in these cells increased the rate but not the overall level of wnv replication. moreover, d. melanogaster carrying homozygous null mutations in ago- spindle-e or piwi supported higher levels of wnv replication than wild type controls, while flies carrying homozygous null mutations in dcr- did not. intriguingly, aedes albopictus mosquito c6/ cells infected with wnv did not produce anti-wnv sirna's, prompting the authors to speculate that the rnai response in this cell line may be weaker than that of drosophila cells. however sanchez-vargas et al. showed that cells of aedes aegypti mosquitoes, the major vector of denv, produce anti-denv sirna following infection with denv- in culture and in vivo. moreover in the latter study knockdown of dcr- ago- or r2d all significantly enhanced the rate and level of denv- replication, with knockdown of dcr- having the strongest impact. these findings indicate that components of both the mirna and the sirna branches are involved in modulating viral replication, and that complete functional segregation of the two branches is lacking. to gain further insight into the ability of rnai to modulate denv infection, in the current study we first investigated whether s cells are susceptible to denv infection. s cells are an attractive substrate for investigation of rnai for three reasons: the rnai pathway in drosophila is well characterized, rnai knockdown in s cells can be accomplished simply be overlaying them with dsrna or sirna, and previously validated sirna's for knockdown of specific rnai enzymes are readily available. after finding that denv replicates in s cells, we tested whether s cells respond to denv infection by production of sirna. finally, we tested the impact of individually knocking down four enzymes of the rnai pathway: dcr- dcr- ago- and ago- on the replication dynamics of denv. methods cells schneider s cells acquired from the drosophila genomics resource center were maintained at 28°c in conditioned s media composed of schneider's drosophila media supplemented with 10% fetal bovine serum, mm l-glutamine, and 1× penicillin-streptomycin-fungizone®. media used for dsrna/sirna dilutions was schneider's drosophila media supplemented with mm l-glutamine and 1× psf. c6/ cells were maintained at 32°c with 5% co in minimal essential media supplemented with 10% fbs, mm l-glutamine, mm nonessential amino acids and mg/ml gentamycin. viruses to compare the replication of the four serotypes of denv, three isolates of each were selected from a broad array of geographical locations. each isolate was passaged in c6/ cells to generate a stock, designated c6/ p moi, for use in all experiments. c6/ cells were infected at moi, incubated for two hrs with occasional, gentle rocking under the conditions described above. five days post infection, supernatant was collected, clarified by centrifugation, stabilized with times volume of 10× spg, and stored at -80°c. the titer of each c6/ p moi stock was determined via serial titration in c6/ cells as described below. 1cell type for passage followed by total number of passages in that cell type wrceva: provided by dr. robert tesh at the world reference center of emerging viruses and arboviruses at the university of texas at galveston; niaid: provided by dr. stephen whitehead, niaid, nih; unc: provided by dr. aravinda de silva, department of microbiology and immunology, university of north carolina. quantification of virus titer monolayers of c6/ cells were grown to 80% confluency in 24-well tissue culture treated plates and infected with serial tenfold dilutions of each stock virus or cell supernatant. plates were incubated for two hrs with intermittent gentle rocking at 32°c. inoculated monolayers were overlaid with % methylcellulose in optimem supplemented with 2% fbs, mm l-glutamine and mg/ml gentamycin. focus forming units are referred to as "plaques" hereafter for consistency with previous literature; plaques were detected via immunostaining as previously described. denv- - were detected using denv - specific monoclonal antibody 15f denv - hyperimmune mouse ascites fluid, denv - specific hybridoma cell supernatant, and denv- hmaf, respectively; all antibodies were the kind gift of dr. stephen s. whitehead, national institute of allergy and infectious disease, national institutes of health, bethesda, md. infection of s cells by denv s cells were grown to 80% confluency in six-well tissue culture treated plates. triplicate wells were infected with each of the c6/ p moi stocks at a specified moi, based on titer in c6/ cells divided by the number of s cells/well, in a total volume of one ml. virus was incubated for two hrs at 28°c with occasional, gentle rocking and washed once with one ml of conditioned s media. thereafter three ml of conditioned s media was added to each well. s cells were infected at moi and incubated for five days at 28°c after which cell supernatants, designated s p moi were collected and frozen as described above. μl from each s p moi replicate were then passaged in fresh s cells as described above. given the titers on day five for s p moi, μl of supernatants contained a total of - log10plaque forming units. cells were incubated for five days and harvested to yield s p moi s cells were infected similarly at moi to yield cell supernatants s p moi, but these supernatants were not passaged further. virus titer in all cell supernatants was determined by serial titration in c6/ cells as described above. denv replication kinetics in s cells triplicate wells of s cells in six-well plates were infected with the c6/ p moi stock of denv- taiwan at moi. two hrs post infection the inoculum was removed, cells were washed once with conditioned s media, fresh media was added and ml cell supernatant was collected from each well and hrs pi and frozen as described above. fresh media was added to each well for every sampling point so that the total volume of media remained constant. detection of anti-denv sirnas in s cells northern blots were used to detect anti-denv sirnas in infected s cells. to assess the production of sirna's in response to infection, one set of s cells at 80% confluency were infected with denv- jkt, denv- tonga, denv- sleman and denv- taiwan at moi as described above. to assess the impact of knocking down components of the rnai pathway on sirna production, a second, concurrent set of s cells were treated with dsrna to dcr- or dcr- and then infected with denv- jkt, denv- tonga, denv- sleman and denv- taiwan as described below. three days pi small rnas were isolated using mirpremier® microrna isolation kit. rna was quantified, separated on 15% urea polyacrylamide gel using tris borate edta and transferred to hybond™-n+ nylon membrane. blots were probed with approximately nucleotide long digoxigenin labeled positive-sense probes complementary to nucleotides - of the 3' untranslated region of denv- western pacific, - of the 3'utr of denv- tonga, - of the 3'utr of denv- sleman and - of the 3'utr of denv- taiwan. the justification for targeting the probe to the 3' utr is based on a recent report that anti-west nile virus sirna's cluster, among other genome locations, in the 3' utr. blots were processed according to protocol defined by the manufacturer for dig probes. knockdown of enzymes in the rnai pathway four components of the rnai pathway, ago- ago- dcr- and dcr- were separately depleted using base-pair dsrna targeting nucleotides - of dcr- - of dcr- - of ago- mrna from d. melanogaster or a previously validated bp sirna against d. melanogaster ago-. a dsrna targeting nucleotides - of pgex-2t cloning vector was used as a control for dsrna knockdown while a renilla luciferase sirna targeting luciferase was used as control for sirna knockdown. to generate dsrna, d. melanogaster dna was isolated using the qiagen dneasy blood & tissue kit and amplified using primers specific to d. melanogaster ago- ago- dcr- and dcr-. primers contained a t promoter sequence at the 5' end to allow for transcription using megascript® rnai kit according to manufacturer's instruction. transcription of sirna was performed using silencer® sirna construction kit. log ± log s cells were plated on six-well plates and incubated for minutes at 28°c. dsrna/sirna were diluted in one ml of unconditioned s media to nm, applied to the s cells, and incubated at 28°c for hrs. thereafter three ml of conditioned s media was added and cells were incubated as described above. cells were re-fed with dsrna/sirna three days following initial treatment. 5' to 3' sequence verification of knockdown to assess the efficacy of knockdown, seven wells of s cells were treated with each of the dsrna/sirna's described above. at two hrs, hrs, and daily thereafter through day six post-treatment, cells from one well corresponding to each dsrna/sirna treatment were lysed using ripa buffer and centrifuged for minutes at rpm at 4°c. supernatants were stored at -80°c in order to analyze all samples concurrently. total protein in each sample was quantified using bca protein assay kit. supernatants were separated on a polyacrylamide gel and transferred to immobilon polyvinylidene fluoride transfer membranes. membranes were blocked with bovine serum albumin and incubated with d. melanogaster specific anti-dcr-, anti-dcr-, anti-ago-, or anti-ago- antibody as appropriate. protein bands were visualized with secondary anti-rabbit or anti-mouse hrp-conjugated igg using the ecl system. toxicity assay to assess whether knockdown of dcr- dcr- ago- or ago- affected the viability of s cells, a resazurin-based viability assay was performed. s cells were propagated to 80% confluency in five well tissue culture treated plates. each treatment was performed in triplicate wells on each plate as follows. media was removed and designated dsrna/sirna's were added at a concentration of nm. two controls were included in the assay: treatment with μl of conditioned s media was used to measure overall cell viability and treatment with 8% dmso was used to measure the impact of a compound known to be toxic. plates were incubated for one to five days; on each day μl of resazurin from the in vitro toxicology assay kit was added all the wells of one plate. the plate was then incubated two hrs and absorbance was read on a plate reader at nm. the proportion of viable cells was determined by dividing the absorbance of each well on the plate by the average absorbance of the media-treated wells. denv infection following knockdown of dcr-2 for each of the c6/ p moi stocks of denv strains, triplicate wells of s cells in six-well plates were treated with dsrna targeting dcr- or with control dsrna as described above. sixteen hrs post treatment wells were infected with the designated virus strain at moi and incubated at 28°c. based on the results of knockdown verification, infected cells were replenished with dsrna hrs pi. cell supernatants were carefully removed and stored in individual tubes at room temperature, leaving one ml residual supernatant per well. nm dsrna was added to each well and incubated for minutes at 28°c. each cell supernatant that was removed was added back to its original well containing one ml of residual media. cell supernatants were harvested hrs pi and virus titer was determined as described above. denv replication kinetics following knockdown of dcr- dcr- ago- or ago-2 to monitor the impact of rnai knockdown on denv replication kinetics, sets of six wells of s cells in six-well plates were treated with one dsrna/sirna targeting dcr- dcr- ago- ago- or one control dsrna/sirna, as described above. hrs post treatment, three wells treated with each enzyme were infected with denv- taiwan and three with denv- tonga at moi one ml cell supernatant was collected from each well and hrs pi and frozen as described above; one ml of fresh media was then added to each well so that the total volume of media remained constant. all wells were re-fed dsrna/sirna at hrs pi as described above. statistical analysis all statistical analyses were carried out using statview.  infection of s cells by denv every denv strain achieved a titer > log10pfu/ml in c6/ cells five days post-infection at moi. five days after infection of s cells at moi the denv strains reached titers ranging from to log pfu/ml. there was a significant positive correlation between titer of the denv strains in c6/ with the titer of those strains in s. there was no significant difference among the four denv serotypes in titer following this first passage in s cells, and titer did not change significantly following a second passage in s cells, s p moi. to confirm that the titers observed in s cells resulted from virus replication rather than carry-over of the inoculum, s cells were also infected with all strains of denv at moi; five days pi all strains had achieved titers ranging from to log pfu/ml. there was no significant correlation between titers of the strains following infection of s cells at moi and moi or between titers of the strains following infection of s cells at moi and c6/ cells at moi. additionally the replication kinetics of one strain, denv- taiwan, were followed daily for five days; there was significant difference in virus titer among days post-infection; specifically, a tukey-kramer post-hoc test revealed that virus titer increased between two hrs and hrs and leveled off thereafter at approximately log10pfu/ml. detection of anti-denv sirna in s cells virus-derived small rnas can range from - nucleotides depending on secondary structure of the viral genome and processing by rna processing enzymes. virus derived small rnas were detected in s cells three days after infection with denv- tvp, denv- tonga, denv- sleman and denv- taiwan by northern blotting using positive-sense probes designed to detect negative sense sirnas that targeted the positive sense genome of each respective serotype. no virus-derived sirna's were detected in uninfected control cells. knockdown of dcr- or dcr- resulted in a substantial decrease in the production of virus-derived sirna's in s cells infected with each of the four isolates above. the most extreme effect was apparent for dcr- knockdown followed by infection with denv- taiwan; in this treatment no virus-derived sirna's were detected at all. toxicity in s cells following knockdown of dcr- dcr- ago- or ago-2 knockdown of each of the four components of the rnai pathway had no significant effect on cell viability. a two-factor anova testing the effect of treatment and day post-infection on absorbance revealed a significant effect of treatment but not day. however a tukey-kramer post-hoc test revealed that only the dmso-treated cells, which were expected to show reduced viability, differed significantly from control cells, while none of the dsrna/sirna treated cells differed from controls. denv replication following knockdown of rnai genes to test whether the rnai response has an effect on denv replication in s cells, four components of the rnai pathway were individually depleted via knockdown with an appropriate dsrna or sirna. the efficacy of depletion of each enzyme was confirmed using western blot analysis. dcr- levels were depleted for six days following treatment, but unlike the other three treatments there were no days on which dcr- expression was undetectable. dcr- expression was undetectable until day three post-treatment and showed steady recuperation thereafter. ago- expression was undetectable through day five post-treatment. ago- expression was undetectable until day three post-treatment and rebounded on day four. to prevent recovery of expression, all infected cell knockdowns were re-fed dsrna/sirna on day three post initial dsrna/sirna treatment. as shown in figure all denv strains tested achieved significantly higher titers in cells depleted of dcr- relative to control cells. the denv strains attained similar titers in cells treated with a control dsrna treatment as compared to untreated cells. moreover, there was no significant difference among serotypes in the impact of dcr- knockdown, measured as the difference in titer for a particular replicate virus in knockdown cells versus control cells. in contrast, variation in the impact of rnai knockdown on the three denv strains within serotypes was detected using factorial anovas for each serotype; when significant differences were detected, a tukey-kramer post-hoc test was used to determine which strains showed significant differences in response to knockdown. denv- strains showed significant variation in response to dcr- knockdown: strain tvp showed a significantly greater increase in titer when dcr- was knocked down than strains jkt and aush; the latter two did not differ from each other. denv- and denv- strains did not show significant within-serotype variation. denv- strains also showed significant variation to dcr- knockdown: all three strains tested differed significantly from each other. subsequent analyses focused on two denv strains that had shown the smallest and an intermediate response to dcr- knockdown. a multistep growth curve revealed that knockdown of dcr- resulted in enhancement of replication of both strains within hrs pi, and by hrs pi both strains had achieved a titer - - fold higher in dcr- depleted cells than control cells. a similar pattern was observed following knockdown of dcr- ago- and ago-; titers of both denv strains were significantly higher in cells depleted of each enzyme than control cells hrs pi. discussion the objectives of this study were threefold: first, to monitor the pattern of replication of denv in s cells in order to assess the utility of s2's for the study of denv, second to investigate the impact of rnai on denv replication; and third to test whether the impact of rnai differs among the four serotypes of denv. five lines of evidence demonstrate that all four denv serotypes replicated in s cells. first, infection of s cells with denv at an moi and moi resulted in titers > and > log10pfu/ml, respectively, even though the input virus inoculum was thoroughly washed away two hours post-infection. second, titers attained by denv following a second passage in s cells were substantially larger than the total amount of virus used to initiate infection. third, daily monitoring of the titer of denv- taiwan in s cells showed that titers increased significantly following one day of infection. fourth, sirnas were detected in s cells after infection with each of the four serotypes of denv, indicating that denv infects and replicates in s cells. finally, a significant increase in titer was observed for all denv strains when dcr- was knocked down using dsrnas. such change in titer following down regulation of an antiviral response is indicative of active replication of denv. there was no evidence of change in titer of denv between a first and second passage on s cells. however future studies to monitor adaptation after extensive serial passage in s cells are planned. sessions et al. reported that denv- ngc attained a peak titer of log10pfu/ml in s derived d.mel- cells without prior adaptation. following serial passages for four months in d.mel- cells, denv- ngc titer increased to log10pfu/ml. consistent with these findings, in the current study peak titers of denv in s cells infected at moi were approximately log10pfu/ml. however peak titers following infection at moi were at least an order of magnitude higher. like other rna viruses, denv exists as a quasispecies, and it is possible that variants that were better able to infect s cells occurred in the larger virus population used to infect at moi relative to moi. this hypothesis is supported by the finding that viruses that were taken from the moi infection and passaged again onto s cells achieved a similar titer to the s p moi infection, even though their founding population was only - log10pfu. using denv adapted to s cells, sessions et al. demonstrated the utility of these cells for investigation of dengue virus host factors. they identified dvhf using a genome-wide rnai screen on d.mel- cells. findings from the current study indicate that s cells can also support replication of unadapted denv, thereby offering additional opportunities to leverage the extraordinary depth of knowledge and plethora of tools in drosophila genetics for the study of denv. the titer of each denv strain in s cells was substantially lower than its titer in c6/ cells, which are derived from ae. albopictus, a natural denv vector. at first glance, this result seems to suggest s cells may not be a useful model to study denv-vector interactions. however, it has been previously demonstrated that c6/ cells exhibit a weak, and possibly incomplete, rnai response, which may contribute to their ability to support high levels of denv replication. in contrast, both live mosquitoes and s cells marshal a vigorous rnai response to infection with flaviviruses and other rna viruses that is capable of limiting viral replication. thus for some areas of study, particularly rnai-virus interactions, s cells may be preferable to c6/ cells as an in vitro model. in this study s cells infected with denv- or produced sirnas targeting the denv genome, as has been reported previously for a variety of viruses, including denv, in multiple types of insect cells both in culture and in vivo. in a notable exception to this rule, c6/ cells failed to produce sirnas when infected with wnv. the production of anti-denv sirna provides confirmation that denv is targeted by an active rnai response in s cells. further, a decrease in virus derived small rnas was observed when dcr- or dcr- was knocked down prior to infection. this finding supports the conclusion that enhancement of denv replication following knockdown of components of rnai resulted from a relaxation of rnai control. although the current study was designed to detect only sirnas complementary to the positive sense 3' utr, it would be very useful in the future to characterize the entire suite of sirnas produced in response to denv infection. in drosophila, virus derived small rnas can be generated by dcr- or dcr- and subsequently processed by ago- or ago-2-risc. knockdown of dcr- enhanced the replication of each of strains of denv, and knockdown of ago ago- or dcr- enhanced replication of the two denv strains tested. none of the four knockdowns affected cell viability, supporting the conclusion that the observed augmentation of denv replication was due to knockdown of the targeted enzymes rather than off-target effects. there was no difference in the impact of the four enzymes on denv replication dynamics, and there was no difference among serotypes in their average response to the knockdown of dcr- intriguingly, strains within denv- and denv- serotypes showed significant variation in their response to dcr- knockdown. these data suggest that denv strains may vary in their sensitivity to rnai, potentially contributing to differences in viral replication in the vector with downstream effects on transmission. although the current study was not designed to draw inferences about response of specific denv genotypes to rnai or to contrast isolates associated with different grades of disease severity, the s system could be used to address these questions in the future. the impact of dcr- and ago- knockdowns in this study are generally consistent with the results of sanchez-vargas et al., who found that knockdown of either enzyme in ae. aegypti in vivo enhanced replication of denv- although the impact of ago- knockdown was delayed in time relative to dcr- however our results in s cells differ from the finding of chotkowski et al. that loss of dcr- expression in s cells did not affect wnv replication. this disparity may reflect methodological differences, particularly differences in expression of rnai-pathway proteins between s cell lines, or differences between wnv and denv in sensitivity to rnai, and/or differences between the two viruses in their tendency to elicit rnai. other studies have also revealed variation among viruses in their sensitivity to loss of dcr- function. drosophila carrying a homozygous null mutation for dcr- were hypersusceptible to infection by drosophila c virus and cricket paralysis virus, and loss of function of dcr- in drosophila also resulted in increased infection by flock house virus, dcv and sindbis virus. in contrast, homozygous knockout of dcr- in drosophila had no impact on susceptibility to drosophila x virus. in the studies that detected no impact of dcr- function on replication of wnv or dcv, respectively, the authors suggested that synthesis of sirna by dcr- may counteract the effect of loss of dcr- in the current study, knockdown of either dcr- or ago- enhanced denv replication to a degree similar to each other and to dcr- and ago- these findings indicate that the proteins are functionally linked between the mirna and sirna braches of the rnai pathway and thus impact viral replication. these findings are consistent with the report that drosophila carrying a homozygous null mutation for aubergine exhibit increased susceptibility to dxv infection and support the idea that dcr- and ago- also regulate virus replication. such regulation likely stems from the activity of dcr- and ago- in the sirna branch of the rnai pathway. evidence of such activity includes the requirement of dcr- for mrna degradation, the observation of similar transcript profiles in cells depleted of ago- and ago-, and the weak association of ago- with sirnas in cells depleted of ago-. from this perspective, it would be particularly interesting in future studies to assess the impact of concurrent knockdown of dcr- and dcr- or ago- and ago- on the dynamics of denv replication.  our results indicate that rna interference regulates denv replication in drosophila s cells, and that denv strains, but not serotypes, vary in their sensitivity to such regulation. s cells offer a useful model for the study of denv-rnai interactions. abbreviations rnai: rna interference; denv: dengue virus; denv-1-4: dengue virus serotypes 1-4; wnv: west nile virus; dcv: drosophila c virus; dxv: drosophila x virus; ago: argonaute; dcr: dicer; sirna: short interfering rna; mirna: microrna; spn: spindle; pi: post infection; dig: digoxigenin; utr: untranslated region; bp: base pair; sirisc: rna induced silencing complex associated with sirna; risc: rna induced silencing complex; mirnp: mirna associated ribo nucleoprotein complex; mirisc: mirna associated risc; dsrna: double stranded rna; moi: multiplicity of infection; psf: penicillin-streptomycin-fungizone®; mem: minimum essential media; hr: hour; df: degrees of freedom; p1: passage 1; p2: passage competing interests the authors declare that they have no competing interests. authors' contributions experiments were conceived by sm and kah and performed by sm. data was analyzed by sm and kah. the manuscript was written by sm and kah. all authors have read and approved the final manuscript. 
while serotypes did not differ in their average response to dcr- knockdown, strains within serotypes showed significant differences in their sensitivity to dcr- knockdown. three strains from each of the four denv serotypes showed replication in s cells following infection at multiplicity of infection and moi 10; each strain achieved titers > log10pfu/ml five days after infection at moi the four serotypes did not differ in mean titer. mosquito-borne dengue virus has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. the objectives of the current study were threefold: first, to assess the utility of drosophila melanogaster s cells for the study of denv, second to investigate the impact of multiple enzymes in the rnai pathway on denv replication; and third to test for variation in the response of the four serotypes of denv to modulation of rnai. these findings indicate that denv can replicate in drosophila s cells and that the rnai pathway plays a role in modulating denv replication in these cells. s cells infected with denv- or produced sirnas, indicating that infection triggered an rnai response. s cells offer a useful cell culture model for evaluation of the interaction between denv and the rnai response. moreover, knockdown of three additional components of the rnai pathway, argonaute, dcr- and ago- also resulted in a significant increase in replication of the two denv strains tested, and the magnitude of this increase was similar to that resulting from dcr- knockdown. rna interference induced by exogenous small interfering rnas has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. knockdown of one of the major enzymes in the rnai pathway, dicer-, resulted in a to 100-fold enhancement of replication of all twelve strains of denv in s cells.
 mosquito-borne dengue virus has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. rna interference induced by exogenous small interfering rnas has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. however, the impact of rnai triggered by authentic virus infection on replication of denv, or any flavivirus, has received little study. the objectives of the current study were threefold: first, to assess the utility of drosophila melanogaster s cells for the study of denv, second to investigate the impact of multiple enzymes in the rnai pathway on denv replication; and third to test for variation in the response of the four serotypes of denv to modulation of rnai.  three strains from each of the four denv serotypes showed replication in s cells following infection at multiplicity of infection and moi 10; each strain achieved titers > log10pfu/ml five days after infection at moi the four serotypes did not differ in mean titer. s cells infected with denv- or produced sirnas, indicating that infection triggered an rnai response. knockdown of one of the major enzymes in the rnai pathway, dicer-, resulted in a to 100-fold enhancement of replication of all twelve strains of denv in s cells. while serotypes did not differ in their average response to dcr- knockdown, strains within serotypes showed significant differences in their sensitivity to dcr- knockdown. moreover, knockdown of three additional components of the rnai pathway, argonaute, dcr- and ago- also resulted in a significant increase in replication of the two denv strains tested, and the magnitude of this increase was similar to that resulting from dcr- knockdown.  these findings indicate that denv can replicate in drosophila s cells and that the rnai pathway plays a role in modulating denv replication in these cells. s cells offer a useful cell culture model for evaluation of the interaction between denv and the rnai response. 