influenza viruses ( iv ) widely spread among diverse hosts and lead to seasonal outbreaks annually , which cost millions of human lives and significant economic losses . in 1997 , highly pathogenic avian influenza ( hpai ) h5n1 was firstly reported to transmit from poultry to human in hong kong . ever since then , this subtype continuously circulated in domestic and wild birds and sporadically caused human infections . up to now , 844 cases from 16 countries have been documented in the world health organization with the fatality rate of ~53% ( 20032015 ) , among which 52 cases were confirmed in china ( http://www.who.int/influenza/human_animal_interface/hai_risk_assessment/en/ ) . however , several episodes of potential human - to - human transmission raised more concerns about h5n1 pandemic . strain - matched vaccines are viewed as the best preventative strategy for ivs , whereas the development of h5n1 vaccines encounters extra obstacles than seasonal flu ones . on one hand , h5n1 virus is lethal to chicken embryos , thus the yield of egg - dependent inactivated vaccines is largely restricted . on the other hand , , novel egg - free avian iv vaccines that could take immediate effect could be an appropriate supplementary . rna interference ( rnai ) is a conserved sequence - specific silencing mechanism in eukaryotes mediated by short double - stranded rnas . these small rnas , sirna and mirna included , suppress target gene expression posttranscriptionally by mrna cleavage or translational inhibition . especially , artificial micrornas ( amirna ) with the backbone of known cellular pre - mirnas reduce the dependence on host factors and enhance safety . this third - generation gene silencing technology has been widely utilized in treatment of cancers and infectious diseases . here , we described the application of amirna in the control of h5n1 as a complement of traditional vaccines . in this study , we exploited the adenovirus delivered , hemagglutinin - specific artificial microrna as a novel antiviral agent . we tested its prophylaxis efficacy in icr mice against four clades of h5n1 , including clade 2.3.2 , clade 2.3.4 , clade 7 , and clade 9 , which were circulating in china from 2004 to 2009 ( ref . we proved that influenza - targeting recombinant adenovirus - delivered artificial mirna ( rad - amirna ) could combat lethal h5n1 infection and ameliorate severity of symptoms in respect of lung viral yields , inflammation , and survival . it has been proved that h5n1 ha was closely related to systemic infection and impaired immune response . four amirnas which scored highest according to online rnai designer ( http://rnaidesigner.invitrogen.com/rnaiexpress/ ) were generated as described in the materials and methods , including ha-300 , ha-903 , ha-1229 , and ha-1405 . the pre - amirna sequences were displayed in table 1 . to evaluate the inhibitory effect of the amirna constructs , we transfected the aforementioned amirna plasmids ( 1 g ) separately into hek 293 t cells 24 hours before influenza challenge . transduced cells were infected with 0.0005 moi h5n1 and lysed with trizol for rna isolation 24 hours later . neg - mirna control plasmid - treated cells were used as mock controls , and its flu gene expression level was set at 100% . as depicted in figure 1 , all amirna treatments significantly lowered h5n1 viral production compared to corresponding controls ( p < 0.01 ) . among four amirnas , ha-1405 exhibited strongest inhibition on clade 2.3.2 ( mean viral load 3.30% ) and clade 2.3.4 h5n1 ( mean viral load 11.10% ) . its mean suppression on clade 9 was 41.17% , only next to ha-903 ( mean viral load 24.53% ) , while the latter one was less efficient against other three h5n1 clades . as to clade 7 , all four amirnas showed similar effect ( ha-300 , 27.63% ; ha-903 , 25.20% ; ha-1229 , 33.00% ; and ha-1405 , 35.57% ) . based on in vitro screen results , we chose ha-1405 to perform further experiments . one difficulty in rnai clinical application is to transduce amirna into target sites with high efficiency and minimal toxicity . our previous data proved that adenovirus vector was one of the ideal candidates considering its tissue tropism and safety . here , we inserted the whole amirna ( ha-1405 ) expression cassette into the e1 region of adc68 and rescued the recombinant adenovirus , termed as rad(ha-1405 ) . to examine whether rad(ha-1405 ) could suppress h5n1 growth in vitro , we performed ha - targeting western blot in vero cells and plaque reduction assay in madin the expression of ha was significantly reduced in rad(ha-1405 ) transduced vero cells when infected with either clade of h5n1 virus . it is noteworthy that ha expression in clade 2.3.2-infecting cells was nearly abolished in presence of 10 virus particle ( vp ) of rad(ha-1405 ) . furthermore , the inhibition in influenza plaque formation was assessed in mdck cells . as shown in figure 2b e , the numbers of plaques were all significantly lowered in the presence of 10 vp of rad(ha-1405 ) against four clades of h5n1 challenge compared to corresponding controls ( clade 2.3.2 , p = 0.0024 ; clade 2.3.4 , p = 0.0031 ; clade 7 , p = 0.0060 ; and clade 9 , p = 0.0023 ) . pretreatment of 10 vp rad(ha-1405 ) slightly decreased the average plaque formation of four h5n1 strains , but no significant difference was detected comparing to corresponding controls ( clade 2.3.2 , p = 0.1928 ; clade 2.3.4 , p = 0.2124 ; clade 7 , p = 0.0502 ; and clade 9 , p = 0.0951 ) . among four clades , rad(ha-1405 ) displayed strongest inhibitory activity against clade 2.3.2 by around fivefold ( mean plaque count , 10 vp of rad(ha-1405 ) = 10.7 , 10 vp of rad - empty = 53.3 ; figure 2b ) , while suppression of other clades ranged from twofold to threefold ( figure 2c  e ) . the inhibition efficiency of adenovirus - expressed amirna was not totally correlated with plasmid screen results ( figure 1 ) . we assumed that difference in cell types , infection doses , and detection sensitivity might lead to this divergence . to assess the effect of replication inhibition on clinical outcome , we inoculated icr mice intranasally with 10 vp of rad(ha-1405 ) or rad - empty ( n = 15 ) 24 hours prior to 5ld50 h5n1 challenge . five mice from each group were euthanized for lung viral rna titration and histological scoring 5 days post infection ( dpi ) . clinical symptoms of rest 10 mice were monitored on a daily basis for 21 days . the mean lung viral copies of rad(ha-1405)-inoculated mice was 35-fold lower in comparison with rad - empty group ( p = 0.0280 ; figure 3a ) . hematoxylin and eosin staining proved that lung sections from control mice displayed severe perivascular and interstitial infiltrates ( mean histological score = 4.6 ) , while no pathology or small - area perivascular infiltrates was observed in rad(ha-1405 ) group ( mean histological score 1.4 ; p < 0.0001 ; figure 3b , c ) . furthermore , consistent with reduced viral titer and less pronounced inflammation responses , rad(ha-1405 ) protected 70% icr mice from lethal infection of clade 2.3.2 ( p = 0.0031 ; figure 3d ) . the survived mice in rad(ha-1405 ) group lost at most 5% of initial weight around day4 to day 8 and gradually recovered ( figure 3e ) . in contrast , control mice suffered constant weight loss and all died or required euthanasia by day 9 . as depicted in figure 4a  c , rad(ha-1405 ) inoculation remarkably decreased ~40 folds of lung viral rna copies ( p = 0.0205 ) and ~1.8-folds of mean histological scores ( p = 0.0063 ) . four out of 10 rad(ha-1405)-treated mice survived clade 2.3.4 infection ( p = 0.0867 ) with maximal 5% weight loss peaking at day 8 ( figure 4d , e ) . all rad - empty - treated mice showed similar weight change pattern at 4 dpi and then continued to lose weight till death . in terms of clade 7 , reduction in lung viral copies was also significant ( p = 0.0341 ) , but the decline was less obvious than above two clades ( ~3-folds in decrease ; figure 5a ) . and average pathologic scores were 2.4 and 4 for rad(ha-1405 ) and rad - empty group ( p = 0.0039 ) , respectively ( figure 5b , c ) . iv - specific amirna rescued 20% icr mice from lethal challenge of clade 7 ( p = 0.4737 ) and alleviated associated symptoms ( figure 5d , e ) . although rad(ha-1405 ) notably decreased lung viral loads ( p = 0.0324 ) and inflammation ( p = 0.0004 ) , all treated mice succumbed to subsequent lethal infection . therefore , we proved that rad(ha-1405 ) could confer substantial protection against clade 2.3.2 h5n1 and ease symptoms caused by other h5n1 clades . it has been proved that h5n1 ha was closely related to systemic infection and impaired immune response . four amirnas which scored highest according to online rnai designer ( http://rnaidesigner.invitrogen.com/rnaiexpress/ ) were generated as described in the materials and methods , including ha-300 , ha-903 , ha-1229 , and ha-1405 . the pre - amirna sequences were displayed in table 1 . to evaluate the inhibitory effect of the amirna constructs , we transfected the aforementioned amirna plasmids ( 1 g ) separately into hek 293 t cells 24 hours before influenza challenge . transduced cells were infected with 0.0005 moi h5n1 and lysed with trizol for rna isolation 24 hours later . neg - mirna control plasmid - treated cells were used as mock controls , and its flu gene expression level was set at 100% . as depicted in figure 1 , all amirna treatments significantly lowered h5n1 viral production compared to corresponding controls ( p < 0.01 ) . among four amirnas , ha-1405 exhibited strongest inhibition on clade 2.3.2 ( mean viral load 3.30% ) and clade 2.3.4 h5n1 ( mean viral load 11.10% ) . its mean suppression on clade 9 was 41.17% , only next to ha-903 ( mean viral load 24.53% ) , while the latter one was less efficient against other three h5n1 clades . as to clade 7 , all four amirnas showed similar effect ( ha-300 , 27.63% ; ha-903 , 25.20% ; ha-1229 , 33.00% ; and ha-1405 , 35.57% ) . based on in vitro screen results , we chose ha-1405 to perform further experiments . one difficulty in rnai clinical application is to transduce amirna into target sites with high efficiency and minimal toxicity . our previous data proved that adenovirus vector was one of the ideal candidates considering its tissue tropism and safety . here , we inserted the whole amirna ( ha-1405 ) expression cassette into the e1 region of adc68 and rescued the recombinant adenovirus , termed as rad(ha-1405 ) . to examine whether rad(ha-1405 ) could suppress h5n1 growth in vitro , we performed ha - targeting western blot in vero cells and plaque reduction assay in madin the expression of ha was significantly reduced in rad(ha-1405 ) transduced vero cells when infected with either clade of h5n1 virus . it is noteworthy that ha expression in clade 2.3.2-infecting cells was nearly abolished in presence of 10 virus particle ( vp ) of rad(ha-1405 ) . furthermore , the inhibition in influenza plaque formation was assessed in mdck cells . as shown in figure 2b  e , the numbers of plaques were all significantly lowered in the presence of 10 vp of rad(ha-1405 ) against four clades of h5n1 challenge compared to corresponding controls ( clade 2.3.2 , p = 0.0024 ; clade 2.3.4 , p = 0.0031 ; clade 7 , p = 0.0060 ; and clade 9 , p = 0.0023 ) . pretreatment of 10 vp rad(ha-1405 ) slightly decreased the average plaque formation of four h5n1 strains , but no significant difference was detected comparing to corresponding controls ( clade 2.3.2 , p = 0.1928 ; clade 2.3.4 , p = 0.2124 ; clade 7 , p = 0.0502 ; and clade 9 , p = 0.0951 ) . among four clades , rad(ha-1405 ) displayed strongest inhibitory activity against clade 2.3.2 by around fivefold ( mean plaque count , 10 vp of rad(ha-1405 ) = 10.7 , 10 vp of rad - empty = 53.3 ; figure 2b ) , while suppression of other clades ranged from twofold to threefold ( figure 2c  e ) . the inhibition efficiency of adenovirus - expressed amirna was not totally correlated with plasmid screen results ( figure 1 ) . we assumed that difference in cell types , infection doses , and detection sensitivity might lead to this divergence . to assess the effect of replication inhibition on clinical outcome , we inoculated icr mice intranasally with 10 vp of rad(ha-1405 ) or rad - empty ( n = 15 ) 24 hours prior to 5ld50 h5n1 challenge . five mice from each group were euthanized for lung viral rna titration and histological scoring 5 days post infection ( dpi ) . clinical symptoms of rest 10 mice were monitored on a daily basis for 21 days . the mean lung viral copies of rad(ha-1405)-inoculated mice was 35-fold lower in comparison with rad - empty group ( p = 0.0280 ; figure 3a ) . hematoxylin and eosin staining proved that lung sections from control mice displayed severe perivascular and interstitial infiltrates ( mean histological score = 4.6 ) , while no pathology or small - area perivascular infiltrates was observed in rad(ha-1405 ) group ( mean histological score 1.4 ; p < 0.0001 ; figure 3b , c ) . furthermore , consistent with reduced viral titer and less pronounced inflammation responses , rad(ha-1405 ) protected 70% icr mice from lethal infection of clade 2.3.2 ( p = 0.0031 ; figure 3d ) . the survived mice in rad(ha-1405 ) group lost at most 5% of initial weight around day4 to day 8 and gradually recovered ( figure 3e ) . in contrast , control mice suffered constant weight loss and all died or required euthanasia by day 9 . icr mice were vaccinated and challenged as above . as depicted in figure 4a  c , rad(ha-1405 ) inoculation remarkably decreased ~40 folds of lung viral rna copies ( p = 0.0205 ) and ~1.8-folds of mean histological scores ( p = 0.0063 ) . four out of 10 rad(ha-1405)-treated mice survived clade 2.3.4 infection ( p = 0.0867 ) with maximal 5% weight loss peaking at day 8 ( figure 4d , e ) . all rad - empty - treated mice showed similar weight change pattern at 4 dpi and then continued to lose weight till death . in terms of clade 7 , reduction in lung viral copies was also significant ( p = 0.0341 ) , but the decline was less obvious than above two clades ( ~3-folds in decrease ; figure 5a ) . and average pathologic scores were 2.4 and 4 for rad(ha-1405 ) and rad - empty group ( p = 0.0039 ) , respectively ( figure 5b , c ) . iv - specific amirna rescued 20% icr mice from lethal challenge of clade 7 ( p = 0.4737 ) and alleviated associated symptoms ( figure 5d , e ) . figure 6 showed the protection efficiency toward clade 9 h5n1 . although rad(ha-1405 ) notably decreased lung viral loads ( p = 0.0324 ) and inflammation ( p = 0.0004 ) , all treated mice succumbed to subsequent lethal infection . therefore , we proved that rad(ha-1405 ) could confer substantial protection against clade 2.3.2 h5n1 and ease symptoms caused by other h5n1 clades . control of hpai h5n1 human infection has been a severe challenge since the 21st century . this virus has evolved into phylogenetically distinct clades and subclades , among which , clades 0 , 1 , 2.1 , 2.2 , 2.3 , and 7 have reported to cause human disease . in china , clades 2.3.2 , 2.3.4 , and 7 co - circulated in poultry and threat public health . although vaccination is the most cost - effective iv intervention strategy , production of h5n1 vaccines faces more technical obstacles than season flu vaccines . moreover , reappearance of drug - resistant h5n1 strains limits the use of currently available antiviral drugs and underscores the urgent demand for novel flu - specific agents . to this end , we developed the sequence - based h5n1 antiviral compound with the help of rnai . small rnas targeting the genome of ebola virus , sars - coronavirus , or hepatitis c virus have all been proved to be effective in preclinical studies . recently , an antisense oligonucleotides , fomivirsen , has been approved by food and drug administration to treat cytomegalovirus . however , to find the way into clinical application , the small rna compounds have to expand their half - life , lower toxicity , and reach target sites precisely . in the previous study , we demonstrated that chimpanzee adenovirus serotype 68 ( adc68 ) was a suitable delivery vector for artificial micrornas in influenza treatment . adenovirus - delivered amirnas targeting conserved internal gene , nucleoprotein , or matrix , protected icr mice completely against homologous a / pr8 , whereas only two rad - amirnas conferred 60 to 70% protection from one clade of h5n1 virus . therefore , we sought to enhance the efficiency toward h5n1 by silencing other flu gene . in this study , we chose the hemagglutinin gene of h5n1 as the target site for the following reasons : ( i ) ha was a main flu pathogenicity determinant and the multi - basic cleavage site of highly pathogenic h5n1 ha was closely related with its systemic infection . ( ii ) h5n1 ha , different from h1n1 and h3n2 , could reduce perforin expression in cd8 + t cells , which might contribute to impaired virus clearance activity and subsequent increase in proinflammatory cytokines . hence , we designed four amirnas based on ha gene from distinct h5n1 clades . through in vitro screen , one amirna , ha-1405 , was incorporated into adc68 genome and extended to in vivo protection experiments . as shown in figure 3 , rad(ha-1405 ) remarkably enhanced mice survival by 70% in face of clade 2.3.2 h5n1 challenge . its efficiency against clade 2.3.4 , clade 7 , and clade 9 h5n1 viruses was 40 , 20 , and 0% , respectively ( figure 46 ) . although the survival rates were not significantly different from controls in the latter three clades of infection , rad(ha-1405 ) indeed prolonged mean survival time , delayed weight loss , and decreased lung viral yields . the effectiveness of rad(ha-1405 ) was comparable but not further enhanced compared to previously reported np- or m - specific amirnas . in conclusion , our data proved the feasibility of rad(ha-1405 ) as an potential antiviral reagent for h5n1 . additional studies , including combination with other amirnas or compounds , are required to further optimize prophylactic efficiency . human embryonic kidney 293 t ( hek 293 t ) , vero and mdck cell lines were purchased from shanghai cell bank , chinese academy of sciences , and maintained in dulbecco 's modified eagle 's medium ( hyclone , beijing , china ) supplemented with 10% fetal bovine serum ( gemini biological products , calabasas , ca ) , 100  u / ml penicillin and 100 g / ml streptomycin ( hyclone ) . transfection was performed with x - treme gene hp dna transfection reagent ( roche , indianapolis , in ) in accordance with manufacturer 's guidance . in brief , indicated dose of plasmid was mixed with x - treme reagent at the ratio of 1:2 in opti - mem ( gbico , grand island , ny ) . the transfection complex was incubated at room temperature for 15 minutes and transferred directly into cells . the plate was cultured at 37 c in 5% co2 incubator for 24 hours and processed to further treatment . h5n1 strains a / duck / hunan/3/2007 ( clade 2.3.2 ) , a / environment / hunan/669/2008 ( clade 2.3.4 ) , a / environment / hunan/135/2007 ( clade 7 ) , and a / chicken / henan/12/2004 ( clade 9 ) were propagated by inoculating the allantoic cavity of 9-day - old specific - pathogen - free ( spf ) chicken embryos and incubated for 48 hours at 35 c . experiments involving h5n1 were conducted in the biosafety level 3 containment facilities at fudan university ( shanghai , china ) . hemagglutinin targeting artificial mirnas were designed by invitrogen 's block - it pol ii mir rnai designer ( http://rnaidesigner . four best - suited single - stranded oligonucleotides were synthesized , annealed for double - stranded oligos , and further cloned into the mirna expression vector pcdna 6.2-gw / mir ( invitrogen , carlsbad , ca ) . neg - mirna control plasmid provided by block - it - pol ii mir rnai expression vector kit served as the negative control . recombinant adenoviruses were constructed as previously described . in brief , the complete artificial amirna expression cassettes from the pcdna6.2-gw / mir vectors were amplified by pcr and ligated with the pshuttle vector ( clonetech , mountain view , ca ) . after being digested with i - ceui and pi - scei , the fragment was subcloned into the e1 region of the chimpanzee - origin adenovirus molecular clone ( adc68 ) . rescued ads were further expended in hek 293 cells and purified by cesium chloride density - gradient centrifugation . virus concentration was measured by spectrophotometry at absorbance of 260  nm . rad - empty was constructed by the same strategy , which was applied as the mock control virus in following studies . mice.six- to eight - week - old female icr mice were obtained from shanghai laboratory animal center , china , and housed in biosafety level 3 laboratory ( fudan university ) . animal study was conducted in accordance with guidelines of institutional animal care and use committee of institute pasteur of shanghai . reduced hemagglutinin expression , western blot was conducted in adenovirus ( ad)-treated vero cells which were subsequently infected with different clades of h5n1 . in brief , vero cells ( plated at 5    10 per well in 12-well plate on the previous day ) were inoculated with indicated dose of rad(ha-1405 ) . twenty - four hours later , cells were infected individually with four different clades of h5n1 viruses at a dose of 0.001 multiplicity of infection ( moi ) and harvested after 24 hours . protein expression levels were detected with rabbit polyclonal antibody for ha ( sino biological , beijing , china ) , followed by horseradish peroxidase - conjugated secondary antibody ( sigma - aldrich , st louis , mo ) . the expression of -actin ( sigma - aldrich ) was measured as a normalization control for protein loading . signals were detected using a chemiluminescence detection system ( ge health life sciences , pittsburgh , pa ) . mdck cells were seeded in six - well plate at the density of 1    10 cells per well 24 hours prior to ad incubation . the confluent monolayer was treated with 10-fold serially diluted rad(ha-1405 ) ( from 10 vp to 10 vp ) or rad - empty ( 10 vp ) and cultured at 37 c for 24 hours . forty to 60 plaque - forming units of h5n1 virus diluted in 100 l serum - free dulbecco 's modified eagle 's medium ( 1% bovine serum albumin ) were added to each well and absorbed at 37 c for 1 hour . mdck cells were washed once with phosphate - buffered saline ( pbs , hyclone ) and overlaid with minimum essential media ( mem , gibco ) containing 0.8% agar and 1% bsa . seventy - two hours later , cells were fixed with 10% formaldehyde and stained with crystal violet . groups of icr mice ( 15 mice per group ) were intranasally ( i.n . ) treated with 1    10 vp of rad(ha-1405 ) in a volume of 20 l 1 day prior to iv challenge . 5ld50 of h5n1 was diluted in 30 l pbs and given i.n . to each mouse after anesthesia . five mice from each group were sacrificed 5 dpi , and lung tissues were taken for virology and histology examination . influenza virus rna copies were determined by quantitative real - time pcr as previously reported , which has been proved in good agreement with traditional titration methods with higher sensitivity . briefly , total rna from treated cells was isolated by trizol ( invitrogen ) following manufacturer 's protocols . purified rna was dissolved in 100 l diethylpyrocarbonate - treated water ( takara , dalian , china ) . the concentration and quality was determined by nanodrop spectrophotometrically ( thermo scientific , wilmington , de ) . one - hundred nanogram of total rna was retro - transcribed using m - mlv reverse transcriptase kit ( promega , san luis obispo , ca ) with a universal primer ( uni 12 , 5-agcraaagcagg-3 ) complementary to the conserved end of influenza genome . flu gene expression was subsequently assayed on 7900ht real - time pcr system ( applied biosystems , foster city , ca ) with following primers : matrix forward 5-aagaccaatcctgtcacctctga-3 , matrix reverse 5-caaagcgtctacgctgcagtcc-3 , -actin forward 5-accgagcgcggctacag-3 , -actin reverse 5-cttaatgtcacgcacgatttcc-3. the amplification was achieved in a 20 l reaction , containing 2 l cdna , 10 l 2  sybr premix ex taq ( tlirnaseh plus ) ( takara ) , 0.4 mol / l forward primer , 0.4 mol / l reverse primer and 0.4 l rox reference dye2 ( 50 ) . reaction was started with denaturation at 95 c for 30 seconds , followed by 40 times cycling at 95 c for 5 seconds , and 60 c for 30 seconds . relative flu gene expression was quantified by the comparative ct method and normalized to -actin expression . all data were analyzed with 7900ht system sds version 2.4 ( applied biosystems , foster city , ca ) . lung tissues were harvested from mice 5 dpi and stored at 80 c before rna isolation . tissues were mechanically homogenized with precellys 24 ( bertin technologies , france ) in ice - cold pbs . homogenate was then centrifuged at 12,000  rpm for 10 minutes , and supernatants were applied for viral load titration as above . viral copies were determined by absolute quantification and the m gene of a / pr8 expressed in pmd18-t vector was used to provide a standard curve . lung tissues were dissected from mice 5 dpi and fixed in 4% paraformaldehide for 24 hours at 4 c . histological changes were scored with following criteria : 1 , no observable pathology ; 2 , perivascular infiltrates ; 3 , perivascular and interstitial infiltrates affecting < 20% of the lobe section ; 4 , perivascular and interstitial infiltrates affecting 2050% of the lobe section ; 5 , perivascular and interstitial infiltrates affecting > 50% of the lobe section . statistical analyses . viral yield , pathological score , and plaque count were compared by student 's t - test or one - way analysis of variance .
influenza virus ( iv ) is a continuously evolving virus that widely spreads in humans and contributes to substantial morbidity and mortality . 
 re - emergence of human infection with avian influenza virus h5n1 poses extra challenge to iv control . 
 artificial microrna ( amirna)-mediated rna interference has become a powerful antiviral approach due to its high specificity and rapid effect . here 
 , we designed several amirnas targeting the hemagglutinin gene of h5n1 , a major determinant of pathogenicity . 
 expression and delivery efficiency were enhanced by presenting functional amirna with chimpanzee adenovirus serotype 68 ( adc68 ) . 
 one amirna , ha-1405 , significantly limited h5n1 replication in vitro and inhibited 96.7% of clade 2.3.2 replication . 
 adc68-conjugated ha-1405 treatment remarkably decreased different clades of h5n1 plaque formation in madin 
 darby canine kidney cells . 
 moreover , prophylactic administration with rad(ha-1405 ) markedly alleviated clinical symptoms and reduced ~3- to 40-folds of lung viral rna copies against four clades of h5n1 in institute of cancer research ( icr ) mice . 
 our results further showed that rad(ha-1405 ) conferred 70 and 40% immediate protection against lethal clade 2.3.2 and clade 2.3.4 h5n1 challenge , respectively . in conclusion , these data provided information that ha - targeting amirna delivered by adc68 could be pursued as a potential agent for highly pathogenic avian influenza viruses prevention .