Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang, an Eastern Province of China

Abstract

Background: Sporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains.

Methodology/principal findings: A total of 570 serum specimens were sampled from community population aged from 0 to 92 years old in Xianju county of Zhejiang Province in 2013-2014. Microseroneutralization test results were analyzed to estimate the population immunity and to observe antibody dynamics in vaccinated children. E genes of 28 JEV strains isolated in Zhejiang Province were sequenced for phylogenetic tree construction and molecular characteristics analysis with other selected strains. Positive JE neutralizing antibody rates were higher in residents ≥35 years old (81%~98%) and lower in residents <35 years old (0~57%). 7 or 8 years after the 2nd live attenuated vaccine dose, the antibodies against for 4 different strains with microseroneutralization test were decreased by 55%~73% on seropositive rates and by 25%~38% on GMTs respectively. JEV strains isolated in recent years were all grouped into GI, while those isolated in the 1980s belonged to GIII. On important amino acid sites related to antigenicity, there was no divergence between the Zhejiang JE virus strains and the vaccine strain (SA14-14-2).

Conclusion/significances: JE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.

Fig 1. Morbidity of Japanese encephalitis in Zhejiang Province, China, 1952–2014.

Data from the National Notifiable Diseases Reporting System, a hospital-based passive reporting system. Laboratory testing method was established to examine reported cases since 2004 in Zhejiang Province.

Fig 2. The counties of sample collection site in Zhejiang Province, China.

Maps were created by an online map service system (http://www.dituhui.com/).

Fig 3. Age-specific seropositive rates of Japanese encephalitis neutralizing antibody and average incidence rates in Zhejiang Province, 2010–2014.

Fig 4. Geometric mean titers (A) and seropositive rates (B) of Japanese encephalitis neutralizing antibody on different year(s) post-booster dose.

Fig 5. Phylogenetic tree on 1500-nt envelope gene of Japanese encephalitis virus strains.

The sequences of Zhejiang strains isolated in 1982–1983 and 2007–2014 in this study are marked in black rhombus and circle, respectively. Phylogenetic analysis was performed by the neighbor-joining method using the MEGA 6.2 software package (www.megasoftware.net). Bootstrap probabilities of each node were calculated using 1000 replicates. Murray Valley encephalitis virus strain MVEV-51 was used as an outgroup. Scale bars indicate the number of nucleotide substitutions per site.