Isolation and genetic characteristics of human genotype 1 Japanese encephalitis virus, China, 2009

Abstract

Background: Several studies have shown that the predominant genotype of Chinese Japanese encephalitis virus (JEV) is evolving from genotype 3 to genotype 1. However, in recent years, almost all genotype 1 isolates were from mosquitoes, and genotype 1 has been less associated with human disease than genotype 3. This study reports the isolation of human genotype 1 JEV and its genetic characteristics to provide additional insights into human JE pathogens that are currently circulating in China.

Methods and results: In 2009, 31 cerebrospinal fluid samples were collected from patients living in Yunnan and Shanxi provinces and were used to inoculate Aedes albopictus C6/36 cells for virus isolation. The JEV strains were identified using immunofluorescent assays and the reverse transcription-polymerase chain reaction. Phylogenetic analyses based on the partial capsid/pre-membrane and full envelope (E) sequences were performed using Clustalx 1.8 software. Three JEV isolates were obtained from a 4-year-old girl and a 2-year-old boy living in Yunnan and an 82-year-old woman in Shanxi. The boy had been immunized with one dose of JE live attenuated vaccine. New isolates were grouped into genotype 1. Amino acid sequence for the viral E protein indicated 95% to 100% identity with each other and with other JEV strains. When compared with a consensus sequence of E protein, two amino acid substitutions were found: Ser(E-123)-Asn in the two Yunnan isolates and Lys(E-166)-Arg in the Shanxi isolate.

Conclusions: Our findings indicate that the genotype 1 of JEV is causing human infections in China. Our observation of a previously vaccinated boy developing JE from genotype 1 virus infection also calls for more detailed studies, both in vitro and in vivo neutralization tests as well as active surveillance, to examine the possibility of a lack of complete protection conferred by the live attenuated JE vaccine against genotype 1 virus.

Figure 1. Phylogenetic tree based on the C/prM gene of the representative JEV strains.

The details of the strains, except SX06CSF-3, SX06CSF-11 (China, Shanxi, human CSF, 2006), and SX06M-5, SX06M-18 (China, Shanxi, mosquito, 2006), are presented in Figure 2. WNV NY99 (NC_009942) was used as an out-group. JEV Genotypes 1–4 are expressed as G1–G4, respectively. An asterisk is used to denote the three isolates used in this study.

Figure 2. Phylogenetic tree based on the full E gene of the representative JEV strains.

WNV NY99 (NC_009942) was used as an out-group. JEV Genotypes 1–4 are expressed as G1–G4, respectively. An asterisk is used to denote the three isolates used in this study.