the commingling of pigs and people at agricultural fairs represents a potentially important and inadequately evaluated interface in the evolution and transmission of zoonotic influenza a viruses .
this interface may be a critical point for the movement of influenza a viruses and/or genes between human and swine populations .
recent cases of influenza a ( h3n2 ) variant virus ( h3n2v ) infection in humans have been epidemiologically associated with exposures to swine at agricultural fairs in north america . since 2009
, we have conducted a prospective influenza a virus surveillance study among pigs exhibited at county fairs in ohio . in july 2012 , as part of routine sampling , h3n2pm viruses ( h3n2 influenza a viruses containing the matrix ( m ) gene from the influenza a ( h1n1 ) pdm09 virus ) were recovered from pigs at a county fair associated with concurrent cases of human h3n2v infections among its participants . here
we report that next generation sequencing of the complete h3n2pm virus genomes , isolated from pigs at the fair , exhibited > 99% similarity at the nucleotide level to h3n2v isolates concurrently recovered from the human cases .
as part of our ongoing surveillance project , nasal swabs were collected from 34 randomly selected pigs ( out of a total of 348 pigs ) exhibited at an ohio county fair on the last day of the fair , 28 july 2012 , approximately 7 days after the pigs arrived at the fair .
visual examination of all pigs in the barns immediately prior to sample collection did not reveal any pigs with overt clinical signs consistent with influenza - like illness ( ili ) although swine exhibitors reported pigs at the fair had a variety of maladies ( diarrhea , vomiting and fever ) 35 days prior to the sampling day , which resulted in the early dismissal of a few pigs from the fair .
swabs were placed singly into vials containing viral transport media and stored at 80 c until they were tested .
real - time reverse transcriptase polymerase chain reaction ( rrt - pcr ) was used to screen the original samples for the presence of influenza a virus , to determine hemagglutinin ( ha ) and neuraminidase subtypes , and to characterize the m gene lineage .
representative influenza a virus isolates were sent to the united states department of agriculture national veterinary services laboratories for confirmational testing and complete genome sequencing using integrated semiconductor sequencing ( ion torrent ) .
all eight segments of two isolates were amplified using gene - specific and universal primers for each segment .
the cdna was purified and cdna libraries were prepared for the ion torrent using the ionxpress plus fragment library kit ( life technologies corp .
, grand island , ny , usa ) with ion xpress barcode adapters ( life technologies corp . ) . prepared libraries were quantitated by qpcr using the ion library quantitation kit ( life technologies corp . ) .
quantitated libraries were diluted and pooled for library amplification using the ion one touch and es systems ( life technologies corp . ) . following enrichment ,
dna was loaded onto an ion 314 chip ( life technologies corp . ) and sequenced using the ion pgm 200 sequencing kit ( life technologies corp . ) .
reference based assemblies were performed using h3n2pm reference sequences generated at national veterinary services laboratories ( full open reading frames ) .
the number of bases used for reference - based assembly was equal to the open reading frame length , i.e. 2280  bp for the pb2 gene , 1701  bp for the ha gene , etc .
the output of the assemblies generated a full - length contiguous sequence for each gene segment spanning the complete open reading frame .
full - length sequences from the two swine - origin influenza a virus isolates reported here , a / swine / ohio/12tosu268/2012(h3n2 ) and a / swine / ohio/12tosu293/2012(h3n2 ) , have been deposited in genbank ( accession numbers jx534958jx534973 ) . on 27 july 2012
, the ohio department of health was notified by a county health department of two individuals who presented with ili to local medical facilities and had significant direct swine contact over a number of days at their local county fair .
specimens were submitted for preliminary testing to the ohio department of health laboratory and then forwarded to the centers for disease control and prevention for final confirmation .
, an additional 20 cases of human illness associated with this county fair were confirmed to be caused by h3n2v .
sequences from the influenza a virus isolates recovered from the swine were compared to the human - origin influenza a h3n2v virus sequences ( partial and full length ) of a / ohio/13/2012(h3n2 ) , a / ohio/14/2012(h3n2 ) , a / ohio/15/2012(h3n2 ) , a / ohio/16/2012(h3n2 ) and/or a / ohio/20/2012(h3n2 ) recovered from people with ili following exposure to swine at the fair ( global initiative on sharing all influenza data and the influenza research database ) .
all segments were analyzed separately , and their evolutionary history was inferred using the maximum parsimony or maximum likelihood methods . a bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed .
the matrix protein tree was obtained using the close - neighbor - interchange algorithm with search level 1 in which the initial trees were obtained with the random addition of sequences ( 10 replicates ) .
the trees are drawn to scale , with branch lengths calculated using the average pathway method and are in the units of the number of changes over the whole sequence .
influenza a ( h3n2 ) viruses containing the m gene from the influenza a ( h1n1 ) pdm09 virus ( h3n2pm ) were detected in 31 of the 34 swine nasal swabs using rrt - pcr and h3n2pm viral isolates were recovered from 29 of the 31 rrt - pcr - positive samples .
analysis of the ha gene placed the concurrently recovered h3n2v and h3n2pm isolates tightly within the contemporary cluster iv h3 viruses circulating in the us swine herd ( figure 1 ) .
the clustering of all ha genes of the human- and swine - origin viruses from this fair suggests common source dissemination .
furthermore , the phylogenetic structure of the ha gene suggests that h3 from the new variant viruses are evolving away from the classical cluster iv h3 .
neuraminidase ( figure 2 ) , the polymerase complex ( figure 3 ) , nucleoprotein ( figure 4 ) and non - structural ( figure 5 ) genes all show a similar trend with near 99% nucleotide similarity between the swine - origin h3n2pm and human - origin h3n2v viruses ( table 1 ) .
the m genes of both the h3n2pm and the analyzed h3n2v influenza a viruses clustered into a single clade with h1n1pdm segments from 2009 and 2012 and were separated from m genes from older , as well as more recent classical h3n2 viruses ( figure 6 ) . for some segments , isolates from pigs and people from illinois ( 2011 ) and nebraska clustered with the h3n2v genes .
this study presents clear molecular evidence that pigs and humans were concurrently infected with the same strain of influenza a virus at an ohio county fair in july 2012 .
the most recent ancestors of all the genetic segments of the h3n2v / h3n2pm viruses can be found in various triple reassortant h3n2 , h1n2 and h1n1 influenza a viruses which have been detected in pigs in north america .
the cocirculation of the genetic elements found in the h3n2v / h3n2pm viruses makes reassortment events occurring in swine the most likely path of emergence .
although similar influenza a h3n2pm viruses have been previously reported in pigs and in commercial swine herds , they had not been reported in exhibition pigs temporally and spatially linked to human cases of h3n2v until july 2012 .
twelve pigs at the laporte county fair in northwestern indiana tested positive for h3n2pm in early july 2012 , but to date , there has been no documented comparison of genomic sequences of the viruses recovered from people and pigs .
the temporal and spatial proximity of the human cases and swine reported here , the frequency of virus isolation from the pigs , and the > 99% sequence identity of the swine- and human - origin viruses originating from this fair clearly demonstrates inter- and intra - species transmission of h3n2pm / h3n2v viruses ( figures 16 ) .
however , the route by which the virus was introduced into the swine and human populations at the fair has not been resolved .
pigs are unique in their ability to serve as hosts of avian- , swine- and human - origin influenza a viruses and thus have been identified as a source of novel reassortant strains with zoonotic potential .
the remarkable nucleotide sequence similarity between the swine - origin and human - origin h3n2 viruses reported here provides evidence that there are few or no adaptive genetic changes needed for the virus to replicate in either host .
none of the nucleotide differences between the h3n2pm and h3n2v isolates resulted in a change in the predicted amino acid sequence , although the ambiguous base call at position 221 in the m genes of the h3n2pm isolates makes it unable to predict the resulting amino acid in that position ( table 1 ) .
historically , as an influenza a virus emerges in a new host , there is commonly a series of genotypic changes that occur as the virus adapts to grow and transmit more efficiently in the new host .
the lack of difference between the genotypes of these isolates suggests that there are virtually no innate species barriers preventing bidirectional interspecies transmission of h3n2pm / h3n2v viruses between humans and pigs .
furthermore , this close phylogenetic relationship between the swine and human viruses is consistent with a recent interspecies transmission .
fortunately , the symptoms exhibited among most human cases of h3n2v in 2012 have been relatively mild and sustained human - to - human transmission of these viruses has not been observed .
however , with the growing number of documented human cases during recent months , the continuing evolution h3n2v is a concern .
agricultural fairs are unique settings where pigs and people from multiple sources and immunological status are concentrated at one location , creating an interface where new or existing influenza a viruses and/or genes can move within or between human and swine populations . the repeated swine 
swine and human  swine interactions occurring at agricultural fairs are distinctly different from those occurring in commercial swine production settings .
this statement is supported by the increasing number of human h3n2v cases in the united states in late summer 2012 and previously documented influenza a virus transmission from humans - to - swine and swine - to - humans at agricultural fairs .
appropriate steps to prevent and/or reduce intra- and inter - species transmission of influenza a viruses at fairs should be undertaken to protect animal and human health while considering short- and long - term implications for the cultural value of livestock exhibitions .
potential strategies to mitigate the transmission of influenza a viruses between pigs and people are available from several sources including the ohio state university 's animal influenza ecology and epidemiology research program website ( http://vet.osu.edu/preventive-medicine/aieerp ) , centers for disease control and prevention and united states department of agriculture information publications .
 evidence accumulating in 20112012 indicates that there is significant intra- and inter - species transmission of influenza a viruses at agricultural fairs , which has renewed interest in this unique human / swine interface . 
 six human cases of influenza a ( h3n2 ) variant ( h3n2v ) virus infections were epidemiologically linked to swine exposure at fairs in the united states in 2011 . in 2012 , the number of h3n2v cases in the midwest had exceeded 300 from early july to september , 2012 . 
 prospective influenza a virus surveillance among pigs at ohio fairs resulted in the detection of h3n2pm ( h3n2 influenza a viruses containing the matrix ( m ) gene from the influenza a ( h1n1 ) pdm09 virus ) . 
 these h3n2pm viruses were temporally and spatially linked to several human h3n2v cases . 
 complete genomic analyses of these h3n2pm isolates demonstrated > 99% nucleotide similarity to the h3n2v isolates recovered from human cases . 
 actions to mitigate the bidirectional interspecies transmission of influenza a virus between people and animals at agricultural fairs may be warranted . 