purification of total rna , isolation , cloning of the small rna fraction , sequencing and analysis on the illumina platform , and northern blots were performed as previously described . northern blot probes : mir-192 : 5-ggctgtcaattcataggtcag-3 , mir-193b : 5-agcgggactttgtgggccagtt-3 , mir-138 : 5-cggcctgattcacaacaccagct-3 and u6 : 5-gccatgctaatcttctctgtatc-3. quantitative pcr for the detection of mir-192 was performed on 10 ng of rna using the taqman ( applied biosystems ) reverse transcription kit according to the manufacturer s instruction . cdna was then analyzed using taqman probes specific for mature mir-192 and mir-20a on a realplex thermocycler ( eppendorf ) . mir-20a was used for normalization as levels are similar between human a549 and ferret lung samples . calu3 ( human bronchial epithelia ) cells were purchased from atcc ( ht - b55 ) and were confirmed to be mycoplasma free . mdck cells expressing full - length primary mir-192 ( mdck192 ) were generated similar to mdck cells expressing mir-142 . 181 or 154 nucleotides of the 3 end of h3/wyoming and h5/vietnam halo mrna were duplicated respectively , and inserted immediately upstream of their cognate stop codons by site directed mutagenesis . this recreated the endogenous open reading frame for ha with a non - overlapping packing sequence separated by a unique cla1 site . the cla1 site , located immediately 3 to the ha mrna stop codon , was used for insertion of mir-192 ( 5-ggctgtcaattcataggtcag-3 ) or scrambled ( 5-atcgataggaatttaccaaat-3 ) targeting sequences . viruses were rescued by standard reverse genetics , plaque purified , sequence confirmed , and quantified by plaque assay . multicycle growth curves were performed in mdck , mdck192 and a549 cells in the presence of tpck trypsin . supernatant was harvested at the indicated time - points and titers assessed by plaque assay . single cycle infections were performed at a multiplicity of infection of one and whole protein was extracted and separated on a 7.5%-10% sds / page gel and transferred to nitrocellulose ( bio - rad ) . membrane was blocked for 1 h with 5% skim milk at 25 c , and then incubated with the indicated primary antibody overnight at 4 c . antibodies specific for actin ( pa5 - 17568 thermoscientific ) , h5 halo ( dpjy02 ( 3b2 ) nr-19870 bei resources , works in hi , wb , ifa , elisa ) , and np ( nr-4544 bei resources ) were added at a concentration of 1 g / ml in 5% skim milk . secondary mouse antibodies ( na931v ge healthcare ) were used at a 1:5000 dilution for 1 h at 25 c . protein was detected using immobilon western chemiluminescent hrp substrate ( millipore ) as directed by the manufacturer . all animal experiments were performed in accordance with mount sinai school of medicine institution of animal care and use committee . 8-week old female mice were purchased from taconic . for these experiments mice were not randomized and investigators were not blinded . mice were anesthetized with a ketamine , xylazine , h2o mixture then infected intranasally with 100 pfu of wt , ctrl or 192 t h5 halo virus . where indicated mice were infected with 1,000 pfu of 192 t h5 halo virus . lungs were harvested at three and five days post infection and homogenized in pbs either for direct titering or protein analysis . briefly , transmission studies were performed in an absl2 + facility at the department of veterinary medicine , university of maryland . animal studies described were carried out according to the guidelines of the animal care and use committee of the university of maryland ( protocol ro-09 - 93 ) . prior to inoculation , 23-week - old female ferrets ( sayre , pa ) were housed in holding bsl2 for 1.5 weeks to establish a baseline for temperature , weight , and general health . serum from each ferret was assayed by np - specific elisa to rule out prior iav exposure . ferrets were inoculated with 10 tissue culture infectious dose 50 ( tcid50 ) , the amount of virus required to infect 50% of infected cell cultures , diluted in pbs ( 0.5ml total volume , 0.25ml / nostril was administered intranasally ) . directly infected ferrets were inoculated one day prior to the introduction of the nave direct contact and respiratory contact ferrets . ferrets were nasal washed ( 0.25ml pbs / nostril ) every day following the administration of anesthesia and assayed by fludetect ( synbiotics corp . ) . nasal washes continued until animals tested negative for two consecutive days . to prevent cross contamination , ferrets were handled in the following order : rc , dc , di . all viruses , including nasal wash samples and virus stocks , were titrated in mdck cells by tcid50 . purification of total rna , isolation , cloning of the small rna fraction , sequencing and analysis on the illumina platform , and northern blots were performed as previously described . northern blot probes : mir-192 : 5-ggctgtcaattcataggtcag-3 , mir-193b : 5-agcgggactttgtgggccagtt-3 , mir-138 : 5-cggcctgattcacaacaccagct-3 and u6 : 5-gccatgctaatcttctctgtatc-3. quantitative pcr for the detection of mir-192 was performed on 10 ng of rna using the taqman ( applied biosystems ) reverse transcription kit according to the manufacturer s instruction . cdna was then analyzed using taqman probes specific for mature mir-192 and mir-20a on a realplex thermocycler ( eppendorf ) . mir-20a was used for normalization as levels are similar between human a549 and ferret lung samples . calu3 ( human bronchial epithelia ) cells were purchased from atcc ( ht - b55 ) and were confirmed to be mycoplasma free . mdck cells expressing full - length primary mir-192 ( mdck192 ) were generated similar to mdck cells expressing mir-142 . 181 or 154 nucleotides of the 3 end of h3/wyoming and h5/vietnam halo mrna were duplicated respectively , and inserted immediately upstream of their cognate stop codons by site directed mutagenesis . this recreated the endogenous open reading frame for ha with a non - overlapping packing sequence separated by a unique cla1 site . the cla1 site , located immediately 3 to the ha mrna stop codon , was used for insertion of mir-192 ( 5-ggctgtcaattcataggtcag-3 ) or scrambled ( 5-atcgataggaatttaccaaat-3 ) targeting sequences . viruses were rescued by standard reverse genetics , plaque purified , sequence confirmed , and quantified by plaque assay . multicycle growth curves were performed in mdck , mdck192 and a549 cells in the presence of tpck trypsin . supernatant was harvested at the indicated time - points and titers assessed by plaque assay . single cycle infections were performed at a multiplicity of infection of one and whole protein was extracted and separated on a 7.5%-10% sds / page gel and transferred to nitrocellulose ( bio - rad ) . membrane was blocked for 1 h with 5% skim milk at 25 c , and then incubated with the indicated primary antibody overnight at 4 c . antibodies specific for actin ( pa5 - 17568 thermoscientific ) , h5 halo ( dpjy02 ( 3b2 ) nr-19870 bei resources , works in hi , wb , ifa , elisa ) , and np ( nr-4544 bei resources ) were added at a concentration of 1 g / ml in 5% skim milk . secondary mouse antibodies ( na931v ge healthcare ) were used at a 1:5000 dilution for 1 h at 25 c . protein was detected using immobilon western chemiluminescent hrp substrate ( millipore ) as directed by the manufacturer . all animal experiments were performed in accordance with mount sinai school of medicine institution of animal care and use committee . 8-week old female mice were purchased from taconic . for these experiments mice were not randomized and investigators were not blinded . mice were anesthetized with a ketamine , xylazine , h2o mixture then infected intranasally with 100 pfu of wt , ctrl or 192 t h5 halo virus . where indicated mice were infected with 1,000 pfu of 192 t h5 halo virus . lungs were harvested at three and five days post infection and homogenized in pbs either for direct titering or protein analysis . animal studies were performed as described previously with minor modifications . briefly , transmission studies were performed in an absl2 + facility at the department of veterinary medicine , university of maryland . animal studies described were carried out according to the guidelines of the animal care and use committee of the university of maryland ( protocol ro-09 - 93 ) . prior to inoculation , 23-week - old female ferrets ( sayre , pa ) were housed in holding bsl2 for 1.5 weeks to establish a baseline for temperature , weight , and general health . serum from each ferret was assayed by np - specific elisa to rule out prior iav exposure . ferrets were inoculated with 10 tissue culture infectious dose 50 ( tcid50 ) , the amount of virus required to infect 50% of infected cell cultures , diluted in pbs ( 0.5ml total volume , 0.25ml / nostril was administered intranasally ) . directly infected ferrets were inoculated one day prior to the introduction of the nave direct contact and respiratory contact ferrets . ferrets were nasal washed ( 0.25ml pbs / nostril ) every day following the administration of anesthesia and assayed by fludetect ( synbiotics corp . ) . nasal washes continued until animals tested negative for two consecutive days . to prevent cross contamination , once an rc ferret tested positive , they became the last to be nasal washed . all viruses , including nasal wash samples and virus stocks , were titrated in mdck cells by tcid50 .
recent gain - of - function studies in influenza a virus h5n1 strains revealed that as few as three amino - acid changes in the hemagglutinin protein confer the capacity for viral transmission between ferrets1 , 2 . 
 as transmission between ferrets is considered a surrogate indicator of transmissibility between humans , these studies raised concerns about the risks of gain - of - function influenza a virus research . here 
 we present an approach to strengthen the biosafety of gain - of - function influenza experiments . 
 we exploit species - specific endogenous small rnas to restrict influenza a virus tropism . in particular 
 , we found that the microrna mir-192 was expressed in primary human respiratory tract epithelial cells as well as mouse lungs but absent from the ferret respiratory tract . 
 incorporation of mir-192 target sites into influenza a virus did not prevent influenza replication and transmissibility in ferrets , but did attenuate influenza pathogenicity in mice . 
 this molecular biocontainment approach should be applicable beyond influenza a virus to minimize the risk of experiments involving other pathogenic viruses .