airborne microorganisms are typically present in the air we breathe and are potential causes of infectious diseases . influenza viruses are among the respiratory pathogens that can be transmitted by airborne routes [ 14 ] . breathing , coughing , sneezing , and talking during the course of influenza generate a cloud of airborne particles containing influenza virus . the airborne particles have diameters that range from a few millimeters ( large droplets formed during coughing and sneezing ) to submicron ( < 1  m ) ( formed during breathing ) . small particles ( 5  m ) including droplet nuclei from evaporated larger particles can remain airborne for hours ( these concepts and their relevance to influenza viruses are discussed in [ 1 , 314 ] ) . presently , the us centers for disease control and prevention ( cdc ) and the world health organization ( who ) state that influenza virus transmission occurs mostly by large - particle respiratory droplets that travel only a short distance from the source ( 1.829  m ( 6 feet ) according to the cdc and 1  m ( 3.3 feet ) according to the who ) [ 1517 ] . noteworthy , deposition of influenza virus into the lungs ( as small particles ) versus the upper respiratory tract ( as large droplets ) may increase infection risk and illness severity [ 3 , 9 , 1820 ] .   assessments of microbiological air quality are useful for studies of the airborne transmission of pathogens and of other biological particles in the air that may cause noninfectious diseases ( such as allergy to pollen ) . the two principle methods of assessing microbiological air quality are passive monitoring and active sampling . passive monitoring is typically performed using settle plates , which are opened and exposed to the air for a given time , then incubated and resulting bacterial and fungal colonies are analyzed . active sampling is performed with a microbiological air sampler that mechanically draws a known volume of air over , or through , a particle collection device . air samplers are also used to collect airborne viruses , but it is difficult to do so primarily due to their small size and relatively low concentration in ambient air . also , most of the available bioaerosol samplers are not suitable for the collection of viruses for reasons that include ( a ) they typically run for short periods of time ( min ) , making it difficult to capture large volumes , ( b ) most do not separate particles by size , and ( c ) , they are inefficient at collecting submicron particles [ 2325 ] .    the pcis   separates airborne particles in a cascading fashion and simultaneously collects the size - fractionated particles by impaction on polytetrafluoroethylene ( ptfe ) filters . it has collection filters on four impaction stages ( a  d ) , and optionally , an after - filter can be added onto a 5th stage . the pcis is designed to separate and collect airborne particulate matter above the cutpoint of five size ranges : > 2.5  m ( stage a ) , 1.0 to 2.5  m ( stage b ) , 0.50 to 1.0  m ( stage c ) , 0.25 to 0.50  m ( stage d ) , and < 0.25  m ( collected on an after - filter ) ( figure 1 ) . ptfe filters , better known as teflon filters , can collect particles at high efficiency above the cutpoints without the need for coatings . lastly , for the efficient collection of airborne influenza virus , the filters are not prewetted with methanol prior to use . made out of glass , the skc biosampler is purpose - designed to collect particles of a size range that would go through human nasal passages . the skc biosampler has three 0.630  mm tangential sonic nozzles ; air passing through the nozzles creates a swirling flow of collection medium during sampling . compared to other impingers , particle skip and damage to the collected microbial agent are potentially minimized by the collection swirling liquid . whereas the skc biosampler has worked well for the collection of airborne bacteria and fungi in the field , it is nevertheless inefficient at recovering submicrometer and ultrafine virus aerosols , with collection efficiencies of < 10% for particles in the 30 to 100  nm size range . nevertheless , mostly because significantly better alternative devices are not readily available , the skc biosampler has been used for the collection of airborne influenza virus [ 23 , 2931 ] . an adult and a child living in an apartment in gainesville , florida , were diagnosed with influenza caused by type a influenza virus . as both occupants had been vaccinated against influenza a and b viruses in november of 2012 , there was a possibility that they were infected with a new influenza virus strain significantly different than from those in the vaccines . we ( opportunistically ) evaluated airborne material from the apartment that had been collected on pcis filters and in skc biosampler liquid collection media for the presence of viable influenza a virus and/or viral genomic rna . the dwelling that was evaluated in this report is a single - family four - bedroom apartment in gainesville , florida , usa , that has its own heating , ventilation , and air conditioning ( hvac ) system . passive air exchange with the outside was minimal , as windows and an entry door were kept closed due to cold weather . relative humidity ( rh ) and temperature ( t ) were measured using an electronic nist traceable rh and t meter ( thermofisher , catalog number 11 - 661 - 19 ) . virology work was performed in a biosafety level 2-enhanced laboratory at the university of florida . the skc ( skc , inc . , eighty four , pa , usa ) biosampler ( skc catalog number 225 - 9595 ) is a widely - used device for microbiological air quality assessments . skc biosamplers were operated with 115 volt vac - u - go sampling pumps ( skc , inc . , catalog number 228 - 9605 ) . the pumps were warmed up by running them for 5  min prior to use . to protect the pumps against moisture , in - line vapor traps ( skc inc . , catalog number 225 - 22 - 01 ) skc biosamplers were sterilized by autoclaving prior to use then filled with 15  ml of sterile phosphate buffered saline with calcium and magnesium ( pbs ) , 0.5% w / v purified bsa fraction v ( life technologies , grand island , ny , usa ) , and 0.25% ( v / v ) molecular - grade antifoam agent b ( sigma - aldrich , inc . , st . the operating pressure was measured using the pump 's built - in manometer ( pressure gauge ) ; the sampling flow rate was 12.5  l / min , determined using a flow meter attached to the inlet of the skc biosampler prior to use . this suggested that the skc biosampler was operating normally , because at normal atmospheric conditions , the flow rate should always be around 12.5  l / min when there is a pressure drop of at least 0.5  atm . for each unit , a collection time of 30 min was used to sample approximately 375 liters ( 0.375  m ) of air . , catalog number 225 - 370 ) units were used with leland legacy pumps ( skc , inc . , cat number 100 - 3002 ) and operated at a flow rate of 9  l / min . polytetrafluoroethylene ( ptfe ) filters ( 25  mm , 0.5  m pore , skc , inc . ptfe after - filters ( 37  mm , 2.0  m pore , skc inc . the pump 's operating flow rate was calibrated by measuring volume displacement using a defender primary standard calibrator ( skc , inc . , a collection time of 41  min 36  sec was used to sample approximately 0.375  m of air . pcis and skc biosampler units were tandemly situated beneath ceiling vents so that air samplings would be made in areas with active air - flow ( figure 2 ) . the intake openings of the air samplers were at a height of 1  m and perpendicular to the ground , facing the patients . for comparisons of indoor to outdoor counts , outside air was evaluated using a pcis and an skc biosampler in a courtyard outside the apartment . the patients , an adult female ( age 50 ) and young female ( age 10 ) , were diagnosed with influenza at a local clinic in early february 2012 . nasal discharges from both were positive for influenza a virus using a binax now flu a and flu b test ( binax inc . both individuals exhibited classic signs of influenza , starting with a sudden onset fever ( 40c ) , quickly followed by body ache , chills , dry cough , headache , malaise , muscle ache , pain on motion of the eyes , photophobia , red ( and warm ) flushed skin , sneezing , sore throat , watery eyes , and a feeling of severe weakness . less characteristically , the adult but not the child suffered from diarrhea during the first day of illness . the adult had been vaccinated against influenza by injection , and the child by inhaled vaccine , the previous november ( 2012 ) . neither individuals had underlying health conditions and were otherwise healthy and physically fit . within 30 min of collection , virus collection media in the skc biosamplers was concentrated to a volume of about 400  l by ultrafiltration using amicon ultra 15 filter units with a molecular - weight cut off of 100  kd ( millipore , bedford , ma , usa ) . the concentrated skc biosampler fluids were adjusted to a volume of 500  l by addition of pbs with 0.5%  w / v bsa fraction v , and were stored at 80c . the pcis filters for virus tests were individually scraped with flocked swabs wetted with pbs with 0.5%  w / v bsa fraction v then each swab eluted into separate 10  ml aliquots of pbs with 0.5%  w / v bsa fraction v. the eluates were individually concentrated to a volume of about 400  l by ultrafiltration using amicon ultra 15 filter units , the volume adjusted to 500  l by the addition of sterile pbs with 0.5%  w / v bsa fraction v and the concentrated pcis fluids stored at 80c . mdck ( ccl-34 ) and mv1 lu ( ccl-64 ) were obtained from the american type culture collection ( manassas , va , usa ) and were propagated as monolayers at 37c and 5% co2 in advanced dulbecco 's modified eagle 's medium ( admem ) ( invitrogen corp . , carlsbad , ca , usa ) supplemented with 2  mm l - alanyl - l - glutamine ( glutamax , invitrogen corp . ) , antibiotics ( psn ; 50  g / ml penicillin , 50  g / ml streptomycin , 100  g / ml neomycin ( invitrogen corp . ) ) , and 10% ( v / v ) low igg , heat - inactivated gamma - irradiated fetal bovine serum ( hyclone , logan , utah ) . mdck - siat2,6-uf and mv1 lu - siat2,6-uf are cell lines prepared in this laboratory from the atcc mdck and mv1 lu cells and were propagated as described for atcc cells . mdck - siat2,6-uf and mv1 lu - siat2,6-uf overexpress influenza virus 2,6-linked sialic acid receptors . prior to use , all cell lines were treated for 3 weeks with plasmocin and verified free of mycoplasma dna by pcr . aliquots ( 50  l ) of concentrated fluids from the skc biosampler and pcis filters were inoculated onto mdck , mv1 lu , mdck - siat2,6-uf , and mv1 lu - siat2,6uf in serum - free dmem otherwise supplemented as previously described plus l-1-tosylamido-2-phenylethyl chloromethyl ketone ( tpck)-treated mycoplasma- and extraneous virus - free trypsin ( worthington biochemical company , lakewood , nj ) in 5% co2 at 33c . the tpck - trypsin was used at a final concentration 2  g / ml for mdck and mdck - siat2,6-uf cells and at 0.2  g / ml for mv1 lu - siat2,6-uf cells . a commercial solid phase elisa test ( quickvue influenza a and b kit , quidel corp . , san diego , ca , usa ) was used to quickly detect influenza virus in cell cultures . viral rna was extracted from mv1 lu cells using a qiaamp viral rna mini kit , and rt - pcr was performed using primers described in a who manual for influenza virus diagnostics . briefly , vrna was denatured for 5  min at 67c in the presence of superase - in rnase inhibitor ( invitrogen corp . ) , cooled rapidly , and in separate reactions , first strand synthesis was performed with omniscript reverse transcriptase ( rt ) ( qiagen , inc . ) for 1  hr at 37c with primers h1f1 ( 5-agcaaaagcaggggaaaataaaagc-3 ) for influenza a(h1n1 ) 2009 virus , thaf2 ( 5-gcaggggaaaataaaaacaacc-3 ) for former seasonal influenza a(h1n1 ) , and h3a1f6 ( 5-aagcaggggataattctattaacc-3 ) for influenza a(h3n2 ) . pcr was performed using one taq dna polymerase ( new england biolabs , ipswich , ma . primers h1f1 and h1r1264 ( 5-cctactgctgtgaactgtgtattc-3 ) were used for influenza a(h1n1 ) 2009 virus , with an expected product size of 1,264  bp , thaf2 , and sphar11 ( 5-tattttgggcactctcctattg-3 ) for former seasonal influenza a(h1n1 ) , with an expected product size of 990  bp , and h3a1f6 and h3a1r1 ( 5-gtctatcattccctcccaaccat-3 ) for influenza a(h3n2 ) , with an expected product size of 1,127  bp . pcr was performed as initial denaturation step ; 94c ( 30  sec ) ; 30 cycles of 94c ( 30  sec ) , 46c ( 60  sec ) , and 68c ( 1  min 25  sec ) ; terminal extension step at 68c ( 5  min ) ; 4c . gel electrophoresis ( 1.5% ethidium bromide - stained agarose gel ) was used to visualize the pcr product . rna was extracted from aliquots of concentrated skc biosampler and pcis fluids using a qiaamp viral rna mini kit then subjected to rt - pcr analyses . reverse transcription was performed with omniscript rt using primer h3a1f6 , and pcr was performed as described above with primers h3a1f6 and h3a1r1 to generate a 1127  bp amplification product . the consensus sequences of the hemagglutinin and neuraminidase genes of the virus isolated in this work ( designated a / gainesville/07/2013 ( h3n2 ) ) were determined from pcr amplicons . the vrna was reverse transcribed using accuscript high fidelity reverse transcriptase ( agilent technologies , inc . , santa clara , ca ) in the presence of superase - in rnase inhibitor . pcr was performed using phusion polymerase ( new england biolabs ) with denaturation steps performed at 98c . a combination of primers mentioned in the who manual for influenza virus diagnostics   were used ( information to be provided upon request ) . amplicon sequences were analyzed using an applied biosystem 3130 dna analyzer by using bigdye terminator ( v. 3.1 ) chemistry and the same used for amplifications . the hemagglutinin and neuraminidase gene sequences of influenza virus a / gainesville/07/2013 ( h3n2 ) were deposited in genbank ( accession numbers kf061021 and kf061022 ) . plaque assays were performed as in , though mdck ( and not mv1 lu ) cells were used so that our results would be easier to compare and contrast with those of others ( since most laboratories use mdck cells for influenza virus plaque assays ) . the rh of indoor air at the residence ranged from 44 to 45% , and indoor t 's ranged from 21 to 23c during the testing period of this work . viable virus was successfully isolated from samplers located up to 3.7  m away from one of the sick occupants ( table 1 ) . only samplers located at sites 1 , 2 , and 3 , which were close to and in a direct path with sick individuals ( figure 2 ) , collected rt - pcr detectable or viable virus . the pcis collected a range of particle sizes containing influenza virus when it was 1.2  m away from the ill person resting on a couch ( at site 1 ) but presumably only fine to ultrafine particles ( 2.5  m ) at distances of 2.1 and 3.7  m away from the sick persons ( table 3 ) .   for each sample set , influenza virus - specific cpe were apparent earlier in mdck - siat2,6-uf and mv1 lu - siat2,6-uf cells than in conventional mdck and mv1 lu cells . an example is shown in figure 3 for cpe ( granulation of the cytoplasm , enlargement of nuclei , followed by rounding of the cells and detachment from the growing surface ) three days postinfection of mdck and mdck - siat2,6-uf cells that had been inoculated with eluate from the after - filter of a pcis positioned at site 3 . however , for each sample wherein virus was isolated , cpe developed in all the cell types that were used by 6 days postinfection . a quidel quickvue influenza a and b kit was used to quickly confirm the presence of virus in the cells . as observed by others [ 13 , 14 ] , virus was detected by rt - pcr in some samples wherein viable virus was not isolated ( table 3 ) . rt - pcr analyses indicated that influenza a(h3n2 ) had been isolated in cell culture , as primers for the detection of pandemic h1n1 and seasonal h1n1 virus did not amplify a specific product , whereas primers for h3n2 amplified the expected 1,127  bp amplicon corresponding to the anterior half of the hemagglutinin gene sequence ( figure 4 ) . the virus that had been isolated was different from strains present in the laboratory , ruling out laboratory contamination . a simple blast homology search against sequences in genbank as of 27 may 2013 indicated that the ha gene of influenza virus a / gainesville/07/2013 ( h3n2 ) was most similar to that of influenza virus ( a / washington/3262/2012(h3n2 ) ) . the rh within the apartment ( 44 - 45% ) was within the optimal comfort level for humans ( 4060% ) , and the interior t was also comfortable . various studies indicate that aerosol transmission of influenza virus is decreased at an rh greater than about 50% , and that transmission of airborne influenza virus is best under cold and dry conditions ( and references therein ) . since the rh of the apartment was 44 - 45% , and the t was between 22 and 23c , we predicted that viable airborne virus would be detected , and this turned out to be the case . viable  live  virus was present in the liquid collection media of the skc biosampler , and some but not all of the filters of the pcis ( table 3 ) . prior to drawing conclusions , modeling is needed with in vitro generated influenza virus aerosols to determine if the results of table 3 truly reflect a difference in collection efficiency between the skc biosampler and the pcis . it is possible that a large proportion of the collected airborne virus was damaged during impaction with the ptfe filters , yet the actual airborne virus collection efficiency of the pcis was higher than that of the skc biosampler . alternatively , it is also possible that under a constantly flowing airstream during active sampling with a pcis , that influenza virus collected on the ptfe filters gets desiccated ( and thus inactivated ) on the filters .   whereas it is already acknowledged that influenza patients expel large droplets as they cough and sneeze , our findings suggest that influenza patients also produce small aerosol particles ( 5  m ) that contain viable virus . viable virus was detected in areas close to the two patients but not in adjoining rooms separated by curved paths . because fluid dynamics modeling was not used to study air - circulation in this work , it was not clear whether positioning of the samplers in a direct path to sick individuals was important ; perhaps the overriding factor was closeness to the persons . the hemagglutinin gene of influenza virus a / gainesville/07/2013(h3n2 ) is essentially identical to that of a / washington/3262/2012(h3n2 ) ( genbank accession number cy141277 ) . it has less sequence homology to the corresponding gene of a / gainesville/01/2012(h3n2 ) ( genbank accession number kf142471 ) , which circulated in gainesville in 2012 . since florida and washington are situated on opposite coasts of the usa , it is plausible that a / gainesville/07/2013(h3n2 ) stemmed from someone who had traveled to florida from the west coast . sequence analyses of the other genes of a / gainesville/07/2013(h3n2 ) indicated that the virus was similar to other nonvariant h3n2 strains in circulation in the usa , suggesting influenza vaccines were not protective for the two patients of this report . these findings of this work are consistent with recent reports of influenza viruses in fine particle aerosols in the exhaled breath of influenza patients [ 13 , 14 ] , and collectively , these reports challenge the notion that influenza virus is only transmitted through mucous contact with large droplets .
the air we breathe contains microorganisms that can cause infectious respiratory diseases . after two occupants of an apartment 
 were diagnosed with influenza in february of 2013 , efforts were made to detect and isolate airborne influenza virus using two different types of active air samplers : a sioutas personal cascade impactor sampler ( pcis ) and an skc biosampler . 
 the pcis collects size - fractionated particles by impaction on polytetrafluoroethylene filters , whereas the skc biosampler collects airborne particles in liquid media . 
 influenza h3n2 virus was collected by both types of air samplers . 
 the pcis collected a range of particle sizes containing influenza virus near one of the sick individuals but only ultrafine particles when the samplers were positioned farther away . 
 viable virus was present in the liquid collection media of the skc biosampler and some pcis filters . 
 these findings suggest that influenza patients produce ultrafine aerosol particles that contain viable virus .