Following the emergence of USUV encephalitis in birds in 2001 in Europe, 
the subsequent identification of human encephalitis due to USUV in Italy, in 2009, 
raised the possibility that this may represent the beginning of epidemic human USUV encephalitis in Europe 
. Until very recently, epizootiological and phylogenetic evidence has supported the assumption that USUV was introduced into Austria by infected introduced mosquitoes or birds, migrating to Europe from Africa where the virus was originally assumed to circulate relatively harmlessly amongst birds, infecting humans only rarely and without evidence of encephalitis in humans 
. Recently, the evidence of USUV introduction into Europe at least since 1996, indicated that the Italian territory could be considered the epicenter of virus spread to the neighboring countries 
. It was justifiably believed that following the introduction of USUV into Austria, it subsequently radiated from this epicenter, to neighboring countries 
. In addition to an epidemic of chikungunya fever which was introduced into northern Italy in 2007 by an infected human returning from India 
two other arboviruses, bluetongue virus and Schmallenberg virus, have also emerged to cause epidemics in northern Europe in recent years 
. Consequently, in view of our interest in emerging USUV, we determined the genomic sequence of Bologna/09 virus, the first known isolate of USUV to cause human encephalitis. European isolates derived from mosquitoes and birds were compared with the Bologna/09 strain. Our data revealed a close relationship between Bologna/09 and the European avian USUV strains, with a greater divergence from the African USUV isolate. Comparative sequence analysis of the Bologna/09 human encephalitic strain identified two significant amino acid substitutions, one in the DIII-Ir domain of the E protein (S595G) and the other in the non-structural (NS) NS5 protein (D3425E). Interestingly, a similar amino acid substitution in the RdRp domain of the NS5 region, was observed in flavivirus strains that are associated with meningoencephalitis in humans (JEV, WNV, MVEV and KUNV). Importantly, studies of the closely related flavivirus WNV have shown that substitutions in virtually equivalent positions in these two genes were associated with variation in the capacity of WNV to invade the central nervous system (CNS) of laboratory mice under experimental conditions 
. Domain DIII of the E protein of flaviviruses is the likely receptor-binding domain and the major determinant of virus cellular tropism, since this molecule binds to carbohydrate receptors on target cells . Specific amino acid substitutions within domain DIII have been implicated as mediators/moderators of virus infectivity, virulence, antigenicity and escape from neutralizing antibodies for WNV 
. Intriguingly, similar amino acid substitutions in the E domain were found in DENV-2 isolates in Southern-Asia, that were associated with cases of human encephalitis, whether or not they were also associated with classical clinical manifestations of dengue infection 
The flavivirus NS5 protein encodes the RNA dependent RNA polymerase which, together with other NS proteins and the underlying UTR skeletal backbone, is a critical component of the virus replication complex. Substitutions in the conserved domain of this polymerase, in the case of Bologna/09 (D3425E) are likely to impact, on viral replication efficiency via perturbation of the viral replication complex.
Based on our observations, we propose that the two unique substitutions identified in the Bologna/09 isolate may have played a role in the apparent altered tropism and neuroinvasive capacity of this virus for human neurological cells.
Furthermore, previous studies have drawn attention to the functional relevance of ORF secondary structures resulting from synonymous nucleotide substitutions in ssRNA viruses 
. In particular, the study of Tuplin and co-workers conducted on the HCV genome, revealed that SNS in the ORF may play an important role in the folding of the RNA that impacts on essential viral processes, such as translation, replication and packaging 
. Our analyses revealed SNS throughout the entire ORFs of the seven USUV strains and the pattern of these synonymous substitutions correlated with the geographic distribution of the USUV isolates. In particular, of the 41 SNS in the analyzed genomes of the seven USUV strains, 15 were unique for the Bologna 09 strain, another 15 were common to all Italian strains, and 4 were unique for the Austrian-Hungarian strains. Thus, the accumulation of SNS within the ORF of USUV reflects the phylogeographic separation of these viruses and appears to contribute significantly to the generation of genetic diversity and potentially, to the pathogenicity for different host species.
Extension of these observations further suggests the possibility that the SNS identified in the human Bologna/09 strain could correlate with specific changes in RNA secondary structure that result in thermo-dynamically more stable structures when compared with non-human isolates. Clearly, further biological and functional studies will be necessary to assess the significance of SNS in the determination of viral tropism, replication and pathogenicity.
The 5′ and 3′ un-translated regions (UTRs) of the USUV genome are composed of 96 and 667 bases, respectively 
. Among the different flaviviruses, the 5′ and 3′- UTRs form terminal stem-loop sequences that play an essential role in translation and replication of the viral genome 
. Previous studies with WNV have demonstrated that single nucleotide substitutions in the 3′-UTR region can affect RNA processing and replication, as well as polyprotein synthesis and maturation during the replication cycle 
. Among the different functional regions within the 3′- UTR, region III has been the most studied 
because it forms a long stable hairpin (LSH) that is conserved in all the flaviviruses studied thus far 
. It is important to note that in our study, specific nucleotide substitutions were identified in the LSH region of the 3′ - UTR of the strain Bologna/09 and that these substitutions were not identified amongst the other USUV strains for which complete genome sequence is presently available. The two nucleotide positions where the sequence was detected in the Bologna/09 strain genome (nt 641 and 650) were located in the 3′ region of the 3′ terminal structure. It needs to be emphasized that, with the exception of the Bologna/09 strain, this is a completely conserved sequence within the LSH in the different USUV genomes. Previous studies showed that the specific binding site for eukaryotic translation elongation factor (eEF1A) is located in the LSH domain within the 3′ - UTR and that nucleotide substitutions in this region decreased eEF1A binding in in vitro
. The eEF1A delivers the aminoacylated tRNAs and the GTP to the A site on the ribosome during protein synthesis 
, thus mediating an important function of the viral replication process.
Blackwell and co-workers demonstrated that a single nucleotide substitution within this region was responsible for molecular re-orientation leading to increased interaction between EF1a and the 3′ SLH viral RNA in WNV and increased WNV replication 
Analysis of the RNA folding free energy both on the 5′ and 3′-UTRs observed from different strains suggests divergent evolution of USUV. Furthermore, these data could indicate that the RNA folding free energy of the UTR regions could have arisen following host adaptation of USUV from birds to humans possibly under the selective influence of the different temperatures in humans and birds.
Perhaps the key message of this study is that the genomic sequence of the human Bologna/09 USUV isolate exhibits several specific differences when compared with the genomes of USUV isolates from mosquitoes or birds. We recognize that our results are based on the identification and isolation of a single human case of clinically neuroinvasive disease. However, in summary, we have identified two amino acid substitutions, one in the DIII domain of the E protein and one in the RdRp conserved domain of the NS5 protein, together with unique synonymous substitutions throughout the genome.
In conclusion, whilst more evidence will be required, the concept that a human neurovirulent strain of USUV with unique genetic characteristics has been identified in northern Italy requires serious further consideration in view of the potential public health implications.