during may  october 2013 , 11,300 female mosquitoes belonging to 13 species were trapped at 4 sites in lake neusiedl - seewinkel national park in burgenland state , austria .
unguiculata mosquitoes were collected in austria ( 12 pools , 112 mosquitoes / pool ) . the relative abundance of ur .
39,000 mosquitoes were trapped at 2 fish ponds ( nesyt and novy ) in sedlec , czech republic , near the border with northeastern austria .
unguiculata mosquitoes were grouped into 4 pools ( 2 with 1 mosquito each , 1 with 4 mosquitoes , and 1 with 41 mosquitoes ) . the relative abundance of ur .
the mosquito pools were homogenized in rnase - free water , and rna was extracted by using the qiaamp viral rna mini kit ( qiagen , valencia , ca , usa ) .
the samples were screened for the presence of flavivirus nucleic acid by reverse transcription pcr , using universal flavivirus primers mamd ( 6 ) and cfd2 ( 6,7 ) for amplification of a partial nonstructural protein ( ns ) 5 sequence .
one pooled sample from austria was positive ; the pool contained 9 mosquitoes that had been captured in late august in illmitz , a village east of lake neusiedl ( 47.769997n , 16.752887e ) .
we obtained the complete polyprotein coding sequence and partial 5 and 3 noncoding ends of this novel wnv strain ( genbank accession no .
kj831223 ) , which was designated west nile virus - uranotaenia unguiculata - lake neusiedl - austria-2013 ( wnv - uu - ln - at-2013 ) . primer sequences and amplification protocols are available upon request . the complete polyprotein gene sequence of wnv - uu - ln - at-2013 shares a maximum identity of 83% with lineage 4 wnv strains isolated from ur .
unguiculata mosquitoes and dermacentor marginatus ticks in russia ( 3 ) . at the amino acid level , the entire polyproteins of wnv - uu - ln - at-2013 and the lineage 4 strains from russia share 96% identity ( table 2 ) . compared with the russian lineage 4 strains , a 1,813-nt fragment of the ns5-coding sequence for the putative lineage 6 wnv , isolated from culex pipiens mosquitoes in spain ( 4 ) , shares slightly higher nucleotide and amino acid identities with wnv - uu - ln - at-2013 ( table 2 ) .
wnv , west nile virus ; wnv - uu - ln - at-2013 , west nile virus strain uranotaenia unguiculata - lake neusiedl - austria-2013 ;  , comparison between lineages 6 and 8 was not possible because the available partial sequences do not cover the same nucleotide regions . 
are as follows for the polyprotein genes / polyproteins : wnv - uu - ln - at-2013 ( kj831223 ) , lineage 1a ( af404756/aam81752 ) , lineage 1b ( d00246/baa00176 ) , lineage 1c ( dq256376/abc40712 ) , lineage 2 ( dq116961/aaz91684 ) , lineage 3 ( ay765264/aaw81711 ) , lineage 4 ( fj159129/ach99530 ) , lineage 6 ( spain ) ( gu047875/add69956 ) , lineage 7 ( koutango virus ) ( eu082200/abw76844 ) , lineage 8 ( kj131502/ahv83443 ) , usutu virus ( ay453411/aas59402 ) . 
amino acid sequences ( above the diagonal ) and nucleotide sequences ( below the diagonal ) are based on complete polyprotein genes , with the exception of lineage 6 and 8 strains , for which only partial sequences were available . 
phylogenetic neighbor - joining trees were generated with mega5 software , using clustalw alignments , 1,000 replicates for bootstrap testing , and evolutionary distances computation with the p - distance model ( 8) .
one phylogenetic tree was constructed on the basis of the complete polyprotein - encoding nucleotide sequences of 32 wnv strains representing all previously described lineages for which complete polyprotein - encoding sequences are available .
this tree also showed a close relationship between wnv - uu - ln - at-2013 and the lineage 4 wnv strains from russia ; however , the newly identified strain forms a distinct branch ( figure , panel a ) .
a second phylogenetic analysis that included the proposed lineage 6 virus from spain and that was based on 1,813-nt fragments of ns5 showed a close grouping of wnv - uu - ln - at-2013 virus from austria , the virus from spain , and the lineage 4 viruses from russia ; similarity was slightly higher between the viruses from austria and spain ( figure , panel b ) .
phylogenetic positioning of wnv - uu - ln - at-2013 , a west nile virus ( wnv ) strain newly identified in austria , within the species west nile virus .
a ) phylogenetic position as determined on the basis of the full - length polyprotein - coding nucleotide sequences .
b ) phylogenetic position as determined on the basis of 1,813-nt fragments of ns5 , which enabled inclusion of the proposed lineage 6 virus .
the evolutionary history was inferred by using the neighbor - joining method of mega5 ( 8) with 1,000-fold bootstrap analysis , rooted against the respective sequence of usutu flavivirus .
the envelope protein carries 1 putative n - linked glycosylation site at asparagine residue n-154 , which has been associated with increased wnv pathogenicity and neuroinvasiveness ( 9 ) .
the 3 highly conserved n - linked glycosylation sites at ns1 positions n-130 , n-175 , and n-207 in wnv strains were also calculated for wnv - uu - ln - at-2013 by using netnglyc 1.0 software ( http://www.cbs.dtu.dk/services/netnglyc/ ) .
glycosylation of ns1 at these 3 positions has been implicated in neuroinvasiveness ( 10 ) , as has proline at ns1 aa position 250 ( 11 ) , which is also present in wnv - uu - ln - at-2013 .
the ns2a - encoding nucleotide region contains a foo motif , which can mediate production of ns1 , a variant of ns1 that plays a role in neuroinvasiveness ( 12 ) .
a fifo motif , which has been described for the nonpathogenic mosquito - specific flaviviruses ( 13 ) , could not be determined for wnv - uu - ln - at-2013 .
wnv lineages 14 and putative lineage 6 have been detected in europe , but only wnv lineage 1a has spread across the american continents .
circulation of such a genetically diverse group of wnv strains in europe may partly explain the epidemiologic differences observed between wnv disease in europe and the americas . in europe , the presence of less pathogenic wnv strains may inhibit the spread of more pathogenic strains .
we propose that the wnv - uu - ln - at-2013 strain from austria either constitutes a new lineage ( lineage 9 ) or can be grouped into lineage 4 as sublineage 4c , with the strains from russia and spain as sublineages 4a and 4b , respectively .
however , the short sequence available for the strain from spain does not allow a clear - cut conclusion to be drawn with regard to lineage 4 . we suggest that future designation of new wnv lineages should be restricted to viruses for which at least the complete polyprotein gene sequences have been determined . in addition , rules for defining virus lineages should be established by the international committee on taxonomy of viruses .
these mosquitoes are mainly distributed in the southern half of europe ( 5 ) ; in eastern europe , they have spread from southern ukraine and the volga delta through middle and southwestern asia to iran and pakistan ( 5 ) . in the lake neusiedl area of austria , ur .
unguiculata mosquitoes seem to be an indigenous species , which was first reported in 1970 ( 14 ) . in the czech republic , ur .
unguiculata mosquitoes have been detected only in moravia , in the southern part of the country ( 15 ) .
unguiculata mosquitoes feeding on mammals , including humans , they feed mainly on amphibians and reptiles ( 5 ) .
the pathogenicity of strain wnv - uu - ln - at-2013 in humans and animals has not been elucidated .
genetic data show that the strain carries typical wnv pathogenicity markers and suggest that wnv - uu - ln - at-2013 is not restricted to mosquitoes .
additional monitoring studies involving cell culture and animal isolation experiments are necessary to evaluate the pathogenic potential of this virus for humans and animals .
 west nile virus ( wnv ) is becoming more widespread and markedly effecting public health . 
 we sequenced the complete polyprotein gene of a divergent wnv strain newly detected in a pool of uranotaenia unguiculata mosquitoes in austria . 
 phylogenetic analyses suggest that the new strain constitutes a ninth wnv lineage or a sublineage of wnv lineage 4 . 
The Study
Conclusions