Recurrence of Japanese Encephalitis Epidemic in Wuhan, China, 2009-2010

Recurrence of Japanese Encephalitis Epidemic in Wuhan,
China, 2009–2010
Quan Hu1*., Banghua Chen1., Zerong Zhu1, Junhua Tian1, Yu Zhou2, Xiaoyong Zhang3, Xin Zheng4*
1Wuhan Centers for Disease Control and Prevention, Wuhan, China, 2Division of Allergy and Immunology, Joseph Stokes Jr. Research Institute at The Children’s Hospital
of Philadelphia, Department of Pediatrics, Philadelphia, Pennsylvania, United States of America, 3 Institute of Virology, University Hospital of Essen, Essen, Germany,
4Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Abstract
Background: Japanese encephalitis (JE) was once epidemic in most areas of China, including Wuhan, a city located in the
central part of China. The incidence of JE dramatically decreased due to nationwide immunization with the live attenuated
JE virus (JEV) vaccine, and no JE cases were reported during 2005–2008 in Wuhan. In 2009 and 2010, 31 JE cases reoccurred
in this area. In this study, we investigated the causes of JE recurrence.
Methods and Findings: All JE cases were laboratory-confirmed by detecting the JEV-specific IgM antibody with an IgM-
capture enzyme-linked immunosorbent assay (ELISA). All patients were children between 2 months and 9 years of age with
a median age of 2 years. Of the 31 cases, 9 had received one or two doses of the JEV vaccine, 11 had not been immunized
previously with the JEV vaccine, and 11 had an unclear immunization history. Through reverse transcription polymerase
chain reaction (RT-PCR), sequencing, and phylogenetic analysis, two new strains of JEV were isolated from Culex
tritaeniorhynchus and identified as genotype 1 JEV, rather than genotype 3, which circulated in this area previously.
Conclusions: Vaccine failure or missed vaccination may have caused JE recurrence. Local centers for disease control and
prevention need to improve immunization coverage, and the efficacy of the JE vaccine needs to be reevaluated in a
population at risk for disease.
Citation: Hu Q, Chen B, Zhu Z, Tian J, Zhou Y, et al. (2013) Recurrence of Japanese Encephalitis Epidemic in Wuhan, China, 2009–2010. PLoS ONE 8(1): e52687.
doi:10.1371/journal.pone.0052687
Editor: Young-Min Lee, Utah State University, United States of America
Received July 20, 2012; Accepted November 19, 2012; Published January 9, 2013
Copyright:  2013 Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Technology & Science Youth Talent Foundation from Hubei Health Department (No. QJX2008-47). [
edu.cn/gljg/kcy/file/zxjlzl/cgzt/08n(wst)xdqnrcjjxm(48 h).pdf] The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: powerhu21@yahoo.com (QH); Xin11@hotmail.com (XZ)
. These authors contributed equally to this work.
Introduction
Japanese encephalitis (JE) is an acute epidemic disease of the
central nervous system caused by infection with the Japanese
encephalitis virus (JEV), which primarily affects children and
adolescents [1,2]. It was recently estimated by the World Health
Organization (WHO) that the annual case frequency of JE is
67,897 in JE-endemic areas, most of whom are children under 15
years old. The case mortality rate is 20–30%, and neurologic or
psychiatric sequela occurs in 30–50% of survivors [2–5]. JE occurs
throughout most of Asia and parts of the western Pacific [6,7,8].
Extensive JE vaccination programs have been implemented in
JE endemic countries. Asian countries, like Japan and Korea,
which have had major epidemics in the past, have already
controlled JE through extensive JE vaccination programs.
However, JE is still a life-threatening disease to people living in
endemic areas in developing countries, mainly due to the
difficulties of controlling the JE vector and amplifier [9]. In the
1990s, outbreaks were reported in Australia and on the island of
Saipan. In both, mosquito vectors were believed to be involved
[10,11].
JEV is an arthropod-borne virus (arbovirus) that is transmitted
in an enzootic cycle between mosquitoes and amplifying
vertebrate hosts, mainly pigs and wading birds [12,13,14]. JEV
is the most common pathogen leading to viral encephalitis in Asia.
JEV strains have been divided into five genotypes, and genotypes 1
and 3 are distributed widely in Asia, including China, Japan,
Korea, India, Vietnam, and the Philippines [15].
JE cases have been reported in most provinces of China except
Xinjiang Uygur Autonomous, and Qinghai Province [1,16]. Since
an extensive JE vaccination program started for children in the
1970s, the number of JE cases has significantly decreased
nationwide, from 174,932 cases of morbidity in 1971 to 5,097
cases in 2005 [16]. However, outbreaks still occur in some
provinces, especially in the middle and western areas of China
[16,17]. Here, we report that 31 JE cases occurred from 2009 to
2010 in Wuhan, which is located in the central part of China and
is the capital of Hubei Province.
In Wuhan, the incidence rate of JE dramatically decreased in
the early 1990s (Figure 1), when a booster JE vaccination
campaign began to immunize children under 15 years old in
rural areas with live attenuated vaccine (SA14-14-2, manufactured
PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e52687
by Chengdu Institute of Biological Products, China) in April every
year at their own expense [18,19]. Between 2005 and 2008, no JE
cases were reported. In the present study, we collected epidemi-
ological data from JE patients, piglets, and mosquitoes in the areas
of co ... terization of JEV strains to reveal the underlying causes of
the JE epidemic in Wuhan.
Wuhan is situated in the middle of Hubei Province, China (East
Longitude 113u419–115u059, North Latitude 29u589–31u229) and
has a humid subtropical (Koppen Cfa) climate with abundant
rainfall and four distinctive seasons. The annual average
temperature is 15.8–17.5uC, and the average annual rainfall is
1269 mm. The geographical and environmental conditions are
very suitable for mosquitoes to grow and reproduce. JEV is
maintained in a natural cycle of transmission involving mosqui-
toes, wading birds, and pigs, with occasional infection of humans
as dead-end hosts. Domestic pigs are the most common source of
infection for mosquitoes, which then transmit JEV to humans.
Culex mosquitoes, especially Cx. tritaeniorhynchus, are the principal
vector for both zoonotic and human JEV transmission
[12,13,24,25,26]. The majority of mosquitoes trapped from JE
case household settings were Cx. Tritaeniorhynchus, and a high
prevalence of antibodies to JEV was detected in piglets. All these
data together indicate that the Wuhan area is still a natural
epidemic focus of JE.
The immunization status of JE cases indicated that 9 of 31 cases
had clear immunization history of the JE vaccine. Among these
nine patients, three received two doses of vaccine and six received
one dose of vaccine. Eleven cases were not immunized with the JE
vaccine, and the immunization history for the remaining 11 cases
was unclear. All patients who received two doses of vaccine
completely recovered, one patient who only received one dose of
vaccine developed dementia, two patients who were not vaccinat-
ed developed dementia, and one child who had not been
vaccinated died. In addition, all patients with unclear immuniza-
tion history recovered. These data indicate that JE recurrence was
associated with either failure of vaccination or missed vaccination.
In Wuhan, JE live attenuated vaccine (LAV) of the SA14-14-2
virus was used for preventing disease. Studies have shown that this
vaccine is safe, well tolerated, and highly immunogenic, and a
single dose of JE vaccine is highly efficacious [27,28]. In addition,
a test in mice for evaluating the protective efficacy of this vaccine
has shown that the protection efficacy against intraperitoneal
challenge with 16 virus strains (both genotype 1 and genotype 3)
was 80–100% [29]. There are two JEV strains circulating in
China: genotype 3 and genotype 1. Genotype 1 JEV was first
isolated from mosquitoes in Yunnan Province in 1979 [30], and it
was also detected from mosquitoes in the provinces of Liaoning,
Heilongjiang, Fujian, Hunan, and Shanxi [30,31,32]. In the
Wuhan area, a genotype 3 JEV strain was isolated from
mosquitoes in 1988 (Genbank accession number AY849939),
and no genotype 1 JEV strain has been reported. In the present
study, we identified two new JEV strains for the first time, and
both of these strains belong to genotype 1.
To understand if these two new JEV strains led to the vaccine
losing its protective efficacy and could be the cause of the 9
children who had received the JE vaccination and were
subsequently infected with JE, we compared the homology of
the JEV envelope gene between WHJX09-09, WHJX09-10, and
SA14-14-2 strains and found that there were 15 mutated amino
acids (3.10%) in either the WHJX09-09 or the WHJX09-10 strain.
Thirteen of the15 mutants were located in the E gene antigen
determinant region, which plays an important role in viral
attachment, virulence, and immune response induction. JEV
E138, which was mutated from Glu to Lys in SA14-14-2 for the
purpose of reducing vaccine toxicity, still contained a Glu residue
in both WHJX09-09 and WHJX09-10 strains, meaning that new
strains of JEV would have strong viral virulence to patients.
However, there were no mutations in the E protein domain III
residues (E307–E309, E327–E333, and E386–E390). Previous
studies have shown that these three domain III residues of E
protein could form a novel cis-proline turn structure and are
important in eliciting JEV-specific neutralizing antibodies [33].
Based on this information, it seems that the current vaccine still
possesses the ability to protect children from JEV infection, at least
in theory. The nine children who had received the JE vaccine but
still acquired infection may be associated with a change in the
immune response, since the mutation of new isolates in Wuhan
affected the antigen determinants of the JEV envelop protein. A
similar JE case was also reported from Yunnan Province, where
genotype 1 JEV was obtained from the cerebrospinal fluid sample
of a 2-year-old boy who had been immunized with one dose of JE
live attenuated vaccine [1].
Eleven of 31 JE cases had not been immunized with the JE
vaccine, and 11 cases did not have a clear immunization record,
indicating that the JE vaccination program was not widespread in
the JE infection area. Thus, we performed a retrospective study of
the JE immunization strategy in Wuhan. Before 2008, all District
CDCs implemented a booster vaccination program for susceptible
children aged younger than 7 years old in the spring, before the
mosquito population increased. Starting in 2008, the live
attenuated JE vaccine was included in the expanded immunization
program in China. As a result, local CDCs no longer carried out
the mass campaign against JE. However, due to limited knowledge
and information on the vaccine, caregivers in migrant populations,
especially in the countryside, had little or no access to JE
vaccination, leading to children losing the opportunity to be
immunized. This might be the primary reason that children living
in rural areas have a low JE vaccination coverage.
Due to lack of a specific antiviral treatment for JE, JE continues
to be an important public health problem. Vaccination is still the
most important strategy for preventing the disease. Currently in
China, the JEV vaccine is included in the routine immunization
protocol and is free for children. However, some caregivers do not
realize the serious consequences of JE infection, and this leads to
some children not being immunized, especially in rural areas. For
Table 5. Comparison of the SA14-14-2 strain with two newly isolated JEV strains, WHJX09-09 and WHJX09-10, at the amino acid
level of E protein.
Strain E56 E107 E129 E138 E176 E177 E222 E244 E264 E279 E315 E327 E366 E439 E447
SA14-14-2 Val Phe Thr Lys Val Ala Ala Gly His Met Val Ser Ala Arg Asp
WHJX09-09 Ile Leu Met Glu Ile Thr Ser Glu Gln Lys Ala Thr Ser Lys Gly
WHJX10-09 Ile Leu Met Glu Ile Thr Ser Glu Gln Lys Ala Thr Ser Lys Gly
doi:10.1371/journal.pone.0052687.t005
Japanese Encephalitis Recurrence, Wuhan, China
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improving the JE immunization coverage rate in poor and remote
areas, the local CDC still needs to organize a mass campaign
against JE, and more seminars need to be provided for caregivers
to better understand JE. In addition, new JE vaccines may need to
be developed against new strains of JEV.
Supporting Information
Table S1 The status of vaccination and the outcome of the
disease.
(DOC)
Acknowledgments
The authors would like to thank Wuhan Children’s Hospital, Dongxihu,
Jiangxia, Huangpi, Caidian, Hanyang, Xinzhou, Jiangan, Hongsan,
Hannan Centre for Disease Control and Prevention in Hubei Province,
China for sample collection. The authors thank Sandra Clark for editing
the manuscript, and specially appreciate Michael M. Engelgau for advice
on manuscript writing.
Author Contributions
Conceived and designed the experiments: QH BC. Performed the
experiments: BC ZZ JT YZ. Analyzed the data: BC X. Zhang.
Contributed reagents/materials/analysis tools: ZZ JT YZ. Wrote the
paper: QH BC X. Zheng ZZ.
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