Isolation and molecular characterization of genotype 1 Japanese encephalitis virus, SX09S-01, from pigs in China

Japanese encephalitis (JE) is mostly prevalent in eastern and southern Asia, such as China, India and Japan [1, 2]. JE has extended its geographic regions and it has been recently reported in Australia and USA [2, 3]. There are approximately 30,000-50,000 JE cases and up to 10,000-15,000 deaths occur worldwide each year [2, 4]. In fact, the actual number of cases caused by Japanese encephalitis virus (JEV) is most likely to be much higher, and up to 175,000 each year [5]. Most of cases occurred in the young children and older people. Approximately 25%-30% of cases caused by JEV are fatal, and 50% result in permanent neuropsychiatric sequelae [2, 6].

In China, JE is one of the most important viral encephalitis and has been reported in most provinces of mainland China, except for Xinjiang Uygur Autonomous Region and Qinhai Province [6, 7]. There are still 8,000-10,000 cases reported annually and nearly 80% of globally reported cases occur in China although JE vaccines are widely used [8]. Particularly, in August 2006, an outbreak of JE occurred in Yuncheng, Shanxi Province, China Sixty-six human cases were reported with 19 fatalities [9].

JEV, the etiological agent of JE, belongs to a mosquito-borne Flavivirus within the family Flaviviridae[10]. JEV exists in a zoonotic transmission cycle between animals/water birds and human by Culex mosquitoes, and humans are a dead-end host [11]. The viral genome is a single-stranded positive sense RNA molecule approximately 11 kb in length and contains a single long open reading frame (ORF) that encodes a polyprotein flanked by 5' and 3' nontranslated regions (NTRs). The polyprotein consists of three structural proteins, designated capsid protein (C), membrane (M, a mature form of its precursor protein prM), and envelope protein (E), as well as seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). The prM and E proteins play critical roles in several biological activities, such as hemagglutination, neutralization, viral binding to cellular receptors and membrane fusion. In addition, the E protein has a major role in determining the neuorovirulence or neuroinvasiveness. Phylogenetic analyses mainly focused on partial sequences derived from either the C/prM or E gene and JEV can be divided into five genotypes (GI-GV) based on the nucleotide sequence of E gene [1, 7, 12].

Swine is an important reservoir and overwintering host for JEV and thus plays a critical role in the human encephalitis epidemics. However, the detailed information on JEV from pigs was lacking. Here, we report the isolation of a GI JEV from piglets that developed viral encephalitis in Yuncheng in July 2009 and this virus is designated SX09S-01 strain. To investigate its molecular characters, the completed genome was sequenced and analyzed. Eight critical amino acids in the E protein were found to be closely related to the JEV neurovirulence. Phyogenetic trees were constructed on the basis of the 24 full-length JEV genomes and 62 completed E genes mostly selected from China. Phyogenetic analysis indicated that SX09S-01 strain was most closely related to the XJ69 strain.

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 [9]. 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 [18] and Nerome [19] 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 [1], K94P05 [22], Ishikawa strains (GenBank accession number AB051292), and Ling strain [23]. RNA secondary structure doesn't exist in this region by computational analyses [24, 25]. Ta and Vrati [26] 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 [19] 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 [29]. 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 [33]. 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 [7]. 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 [19] and this phenomena were also found in Korea[39] and in Vietnam[43]. 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.

The authors declare that they have no competing interests.