JE is mostly prevalent in China and there are many human JE cases reported recently [7, 9, 17]. However, there is very limited information on JEV strains originated from pigs. Pigs play a critical role to JEV transmission between mosquitos and humans, it is very important to surveillance the JE in the swine population in China.
In 2009, lots of piglets developed viral encephalitis in one pig farm in Yunchen, Shanxi province, where human JE cases broke out in 2006 . In this study, we isolated a new JEV stain by serial passages on BHK-21 cells in Yunchen (Figure 1). The newly isolated JEV, designated SX09S-01, belongs to genotype I by C/prM sequences analysis. Tang  and Nerome  analyzed the molecular characterizations of JEV isolates from swine in Japan. To our knowledge, it's reported that only HEN0701 and SXBJ07 strains have been isolated from swine and belong to the GI JEV in China [20, 21]. In this study, we clearly confirmed that GI JEV strains still circulate in Yuncheng. It suggests that we need to pay more attentions on the prevention and treatment of swine JE in order to control human JE effectively in this region.
To fully characterize the JEV SX09S-01 strain, its complete nucleotide and deduced amino acid sequences were determined. Compared with other strains isolated from different geographic regions at different time periods, a 13-nucleotide region that immediately followed the ORF stop codon was deleted in the genome of SX09S-01 (Figure 2). Similar deletions in this region were also observed in FU , K94P05 , Ishikawa strains (GenBank accession number AB051292), and Ling strain . RNA secondary structure doesn't exist in this region by computational analyses [24, 25]. Ta and Vrati  reported that a 60-nucleotide variable region immediately downstream of the ORF stop codon in JEV genome is not required for viral replication. However, it has recently been suggested that this variable region in the 3'-NTR may play an important role on the rate of viral RNA replication [22, 27, 28], but it still needs further clarification.
JEV E protein forms the viral spikes on the surface and has important biological functions related to virulence and viral host tropism. In this study, we compared the eight critical amino acids in E protein that are closely related with the neurovirulence of JEV with other virulent and attenuated vaccine SA14-14-2 strains and found that SX09S-01 strain has these typical characters of high virulent strain (Table 2) and displays high neurovirulence and low neuroinvasiveness in mice (Additional file 3). These results indicated that neuroinvasiveness of JEV may not have closely relations with the eight amino acids in the E protein and some other gene(s) contributed in part of the level of neuroinvasiveness. Nerome  reported that Sw/Mie/40/2004 is a high neurovirulent and neuroinvasive JEV and there were no amino acids differences in the E protein between Sw/Mie/40/2004 and other low neuroinasiveness strains. In addition, we also found that there was a censuses motif Arg-Gly-Asp (RGD) in the C-terminal of E protein. It has confirmed that protein contained RGD motif can interact with cellular integrins, such as VP1 protein of FMDV . It is not known if JEV E protein interacts with intergrins for virus entry.
JEV can be divided into four genotypes based on a 240-nucleotide highly variable sequence of prM gene [9, 30–32]. However, it results in unreliable information when short sequences (< 300 nt) were used in phylogenetic analyses of flaviviruses . In recent years, full-length genome and E gene have also been used to establish phylogenetic trees for JEV [8, 34–39] and JEV can be divided into five genotypes [7, 12, 40]. JEV E protein plays important roles in both induction of protective immune responses and in the biology of the virus [41, 42]. In order to analyze the evolution of JEV in China, sequence information of JEV strains, originated from mainland China, were collected and phylogenetic trees were constructed on basis of the 24 full-length genomes or 62 E genes. Phylogenetic trees based on the full-length genomes or E genes provided similar topology (Figure 3, 4). JEV strains revealed five distinct phylogenetic groupings, reflecting broad geographical and temporal relationships. In China, JEV isolates are divided into three genotypes, GI, GIII and GV recently reported by Li MH et al . GI strain was firstly isolated in 1979, whilst GIII strains have been isolated since the 1940s. Before the 1980s, GIII JEV was predominant in China. After 2000, there were more and more GIII JEV strains isolated and there is tendency that major genotype of JEV isolates changes from GIII to GI . In Japan, major genotype of JEV isolates had shifted from GIII to GI  and this phenomena were also found in Korea and in Vietnam. In mainland China, current vaccines used in human and pigs are inactivated or attenuated vaccines that both made up of the GIII JEV and it needs to investigate whether the same genotype shift will occur under the selected pressure of JEV vaccines.
In summary, here we have isolated GI JEV SX09S-01 strain from swine in Yuncheng in 2009. Its completed genome was sequenced and molecular characterization was analysized on the level of nucleotide and amino acid. Phyogenetic trees based on the full-length genome and E gene indicate that SX09S-01 strain is most closely related to the XJ69 strain. Future study should be aimed to investigate the efficacy of current vaccines against the SX09S-01 strain and other Chinese GI JEV strains and control the JE of pigs in order to prevent human JE.