bovine lung fragments , deep nasal swab specimens , and trans - tracheal aspiration liquids were submitted to the laboratoire dpartemental danalyses de sane - et - loire ( mcon , france ) and tested for classical respiratory pathogens .
the unit mixte de recherche , interactions htes  agents pathognes 1225 ( toulouse , france ) , received and tested 134 samples by using real - time reverse transcription pcr for influenza d virus as previously described ( 3 ) .
we tested 25 archived samples per year for 20102013 and 34 samples collected during january  march 2014 .
six ( 4.5% ) were positive for influenza d virus : 1 each in 2011 and 2012 and 4 in 2014 ; cycle threshold ( ct ) values ranged from 15 to 35 ( table 1 ) .
co - infections were detected with pasteurella multocida , mannheimia haemolytica , histophilus somni , bovine respiratory syncytial virus , and/or bovine herpesvirus 1 in 4 of the influenza d  positive specimens .
two samples ( nos . 5831 and 5920 , collected in 2014 ) were negative for all tested respiratory pathogens , despite reports of clinical signs in the animals ( table 1 ) . * ct , cycle threshold ; bcov , bovine coronavirus ; bohv-1 , bovine herpesvirus i ; bpiv3
, bovine parainfluenzavirus 3 ; brsv , bovine respiratory syncytial virus ; flud , influenza d virus ; i d , identification ; n , negative ; nc , not communicated ; nt , not tested ; p : positive ( ct<35 ) , wp : weakly positive ( 35<ct<40 ) . 
the specimen with the lowest ct value , d / bovine / france/2986/2012 ( ct 15 ) was selected for further molecular characterization , and its full genome was amplified by pcr ( primers in table 2 ) and sequenced on a 3130xl applied biosystems capillary sequencer ( applied biosystems , foster city , ca , usa ) .
the 7 gene segments of d / bovine / france/2986/2012 clearly clustered with us influenza d strains from pigs and cattle ( c / ok , c / bovine / minnesota/628/2013 , c / bovine / minnesota/729/2013 , and c / bovine / oklahoma/660/2013 ) ( figure ) , which suggests a common origin of these new influenza viruses .
we found no evidence of reassortment between influenza c and d ( c / ok - like ) viruses .
in addition , the splicing pattern of the matrix gene segment and the reduced 5-n - acetyl binding pocket in the hemagglutinin - esterase ( he ) protein of d / bovine / france/2986/2012 was similar to that of c / ok and different from that of human influenza c virus , confirming the similarity of d / bovine / france/2956/2012 and the newly described swine and bovine us influenza d virus strains .
the estimated ranges of evolutionary distances ( in number of substitutions per site using the maximum composite likelihood model ) between d / bovine / france/2986/2012 and the 4 us influenza d viruses ranged from 0.8 to 5.7% and were as follows : 1.9%2.1% , 0.8%0.9% , 2.1%2.5% , 2.3%2.7% , 1.8%3.8% , 3.6%4.2% , and 5.1%5.7% for polymerase basic ( pb ) 2 , pb1 , polymerase 3/polymerase acidic , nucleoprotein , matrix , nonstructural protein , and he gene segments , respectively .
phylogenetic trees of the 7 gene segments of d / bovine / france/2986/2012 influenza virus at the nucleotide level .
maximum - likelihood analysis with 500 bootstrap replicates ( bootstrap values > 75 are indicated on the tree nodes ) .
the gene sequences of d / bovine / france/2986/2012 ( in large bold underlined font ) were compared with representatives of all the orthomyxoviridae genera : all the viral strains used in ( 1 ) .
p , polymerase , nucleoprotein , pb , polymerase basic  scale bars indicate nucleotide substitutions per site .
forty unique amino acid substitutions were identified throughout the genome , but the limited available data on influenza d genomes make a functional interpretation of the substitutions difficult to determine . in addition , although the he proteins of human influenza c and c / ok viruses contain 7 and 6 potential glycosylation sites , respectively , d / bovine / france/2986/2012 has just 5 : at positions 28 , 54 , 146 , 346 , and 613 ( atg numbering ) , identical to 5 of the 6 identified for c / ok virus . for influenza a viruses , modifications of n - linked glycosylation sites in the globular head of the hemagglutinin protein
have been linked to changes in virulence , antigenicity , receptor - binding preference , fusion activity , and immune evasion ( 4 ) .
for example , an increase in glycosylation site numbers has been associated with early stages of influenza a(h1n1 ) virus evolution , and changes in the positional conversion of the glycosylation sites have been associated with later evolutionary stages of the virus ( 5 ) .
the missing potential glycosylation site in d / bovine / france/2986/2012 is located at position 513 , probably likely in the f3  =  he2 fusion domain of the protein ( 6 ) and not in the globular head of the protein . thus speculating on the putative time course of virus emergence between c / ok - like and d / bovine / france/2986/2012-like strains
further studies are needed to understand the phenotype(s ) associated with aa substitutions in influenza d viruses .
webster et al . suggested the existence of a common ancestor for influenza a , b , and c viruses and a more recent common ancestor to influenza a and b viruses only considering the different genome organizations between influenza a / b and influenza c viruses ( 6 ) .
recently estimated the time of divergence between influenza c and d virus gene segments at 334 years for pb1 to 1,299 years for he ( 7 ) .
the time of emergence and evolutionary rate of influenza d viruses need to be examined as more data become available .
a puzzling question raised by our current study is the geographic origin of influenza d strains : were cattle in france contaminated by their north american counterparts or vice versa ?
did co - evolution occur ? did the pathogen originate from a distinct location or from a distinct host ?
retrospective studies with archived samples would help date the emergence of influenza d viruses and enable an understanding of their evolution .
the pathogen may have spread to swine and cattle in recent years only ; efforts should be made to find the virus host range and its reservoir species and to evaluate the public health relevance of this new pathogen .
finally , surveillance projects with larger cohorts , as well as experimental infections , need to be conducted before 1 ) the causality between respiratory symptoms and influenza d virus infection in cattle can be established , 2 ) recommendations on samples to collect can be given , and 3 ) prevalence can be compared in different geographic areas . although the causative agent(s ) of some respiratory infections in the field remain(s ) unknown ( g. meyer , pers .
comm . ) and although 2 of the positive specimens in our study originated from young cattle with respiratory symptoms but no identified respiratory pathogen , further studies are also warranted to provide an understanding of the pathobiology of influenza d virus in cattle and its putative role in complex bovine respiratory disease .
 a new influenza virus , genus d , isolated in us pigs and cattle , has also been circulating in cattle in france . 
 it was first identified there in 2011 , and an increase was detected in 2014 . 
 the virus genome in france is 94%99% identical to its us counterpart , which suggests intercontinental spillover . 