Circulation of Japanese Encephalitis Virus in Pigs and Mosquito Vectors within Can Tho City, Vietnam

Circulation of Japanese Encephalitis Virus in Pigs and
Mosquito Vectors within Can Tho City, Vietnam
Johanna F. Lindahl1, Karl Sta˚hl2¤, Jan Chirico3, Sofia Boqvist2, Ho Thi Viet Thu4, Ulf Magnusson1*
1Department of Clinical Sciences, Division of Reproduction, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2Department of Biomedicine and Veterinary
Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden, 3Department of Virology, Immunology and Parasitology, National Veterinary Institute,
National Veterinary Institute, SVA, Uppsala, Sweden, 4Department of Veterinary Medicine, Can Tho University, Can Tho City, Vietnam
Abstract
Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic flavivirus causing encephalitis in humans and reproductive
disorder in pigs. JEV is present in large parts of Asia, where urbanization is high. Households within and outside Can Tho
city, South Vietnam, were selected to monitor circulation of JEV. A nested RT-PCR was established to detect the presence of
JEV in mosquitoes whereas sera from pigs belonging to households within the province were analyzed for the presence of
antibodies to JEV. A total of 7885 mosquitoes were collected and divided into 352 pools whereof seven were JEV-positive,
six of which were collected within the city. Fragments from four pools clustered with JEV genotype III and three with
genotype I. Of the 43 pigs sampled inside the city 100% had JEV antibodies. Our study demonstrates exposure to JEV in
pigs, and co-circulation of JEV genotype I and III in mosquitoes within an urban environment in South Vietnam. Thus,
although JEV has mainly been considered a rural disease, the potential for transmission in urban areas cannot be ignored.
Citation: Lindahl JF, Sta˚hl K, Chirico J, Boqvist S, Thu HTV, et al. (2013) Circulation of Japanese Encephalitis Virus in Pigs and Mosquito Vectors within Can Tho
City, Vietnam. PLoS Negl Trop Dis 7(4): e2153. doi:10.1371/journal.pntd.0002153
Editor: Michael J. Turell, United States Army Medical Research Institute of Infectious Diseases, United States of America
Received October 2, 2012; Accepted February 26, 2013; Published April 4, 2013
Copyright:  2013 Lindahl 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 Swedish International Development Cooperation Agency/Department of Research Cooperation (Sida/SAREC). 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: Ulf.Magnusson@slu.se
¤ Current address: Department of Disease Control and Epidemiology, National Veterinary Institute, SVA, Uppsala, Sweden.
Introduction
Japanese encephalitis (JE) is a zoonotic disease spread over large
parts of Asia. It is one of the most important arboviral encephalitis
in humans, with an estimated 10 million cases over the last 60
years, with 30% case fatality [1]. Pigs and wading birds are
amplifying hosts of the causative Japanese encephalitis virus (JEV),
and do not display clinical signs, except for pregnant sows that
may abort or have stillborn piglets [2,3]. Japanese encephalitis
virus is a mosquito-borne flavivirus which is divided into five
genotypes [4], and the virus has been isolated from more than 25
mosquito species, although not all are equally important in the
epidemiology of JEV [5]. One of the most important vectors is
Culex tritaeniorhynchus, a zoophilic mosquito that commonly breeds
in irrigated rice fields, and therefore the disease is mainly
considered rural [1,3,6].
Keiser et al. [6] calculated that 1.9 billion people live in rural
areas with endemic or epidemic JE. Today, however, more than
half of the world’s population live in cities [7,8] and the
urbanization, especially in low-income countries, creates needs
and possibilities for urban animal keeping to supply city
inhabitants with food. Therefore, transmission of emerging
zoonotic diseases in urban areas is increasingly important. Since
some of the most populated cities in the world are in JE infested
countries, the number of people at risk would increase dramat-
ically if JEV is also transmitted in urban areas. It has been shown
that two prerequisites for spreading JEV, the presence of
competent vectors and the main amplifying host (the pig), are
met in urban settings [9,10]. In an urban area, the vector Cx.
tritaeniorhynchus has been shown to increase in number by the
presence of pigs, whereas the number of another vector, Culex
quinquefasciatus, increases by the presence of humans [10].
However, the presence of JEV in urban areas has not been
studied extensively previously, although previous studies in other
cities in Asia have shown seropositivity in humans [11,12].
In Vietnam, the land area used for rice production is increasing
along with pig production, two factors likely to contribute to
increased transmission of JEV [13]. Cases of encephalitis in
humans are usually reported as acute encephalitis syndrome in
Vietnam, and the incidence in south Vietnam has been 1.9 cases
annually per 100 000 inhabitants between 1998 and 2007, with a
mean case fatality of 6.4% [14]. One of the regions with the most
JE cases, the Mekong Delta region [9,15], has both extensive pig
farming and rice production and disease is present all year [16,17].
Although JEV is known to circulate in the rice-producing rural
areas here, little is known about the circulation of JEV within
urban areas.
The aim of the present study was to investigate the presence of
JEV in pigs and vectors in a city in an endemic area, in order to
contribute to the risk assessment of JE in humans ... he samplings, which may explain the different results,
and in addition, two different ELISA methods were used. The
competitive IgG ELISA used in the present study has been shown to be
cross-reactive with other flaviviruses in the JEV serological group, such
as Murray Valley encephalitis virus and Kunjin virus [31,32], but none
of these flaviviruses have been demonstrated in southern Vietnam.
Apart from JEV, dengue virus is the only vector-borne flavivirus in the
region that infects mammals, and serological cross-reactions with JEV
have been demonstrated [44]. However, cross-reactions with dengue
virus occur to a lesser extent than with viruses in the JEV serological
group [45,46] and we therefore consider it unlikely that the positive
results in our study would be due to cross reactions. Another flavivirus
which could cause cross-reactions with JEV is Tembusu virus, which is
present in Southeast Asia [47], although there are no reports of the
virus in the Mekong delta region in Vietnam. More than half of the
female pigs in the urban households were born at the farm where they
were sampled and must thus have been infected at their present
location. The negative and inconclusive results using the IgM ELISA
could indicate that the JEV infections are not recent, or that infection
with JEV occurred while the pigs were still partly protected by
maternal IgG antibodies, thus inducing less IgM production [48]. JEV
seropositivity has mainly been studied in humans in urban areas
[35,12] but since humans tend be mobile, pigs born in the city may be
better indicators for JEV transmission within an urban environment.
Notably, half of the female pigs had experienced reproductive
symptoms that could be related to JEV infection. However, there are
many other pathogens circulating in the Mekong Delta [49,50] that
can cause similar reproductive symptoms, although a previous study in
Can Tho province could find an association between seropositivity to
JEV and reduced reproductive performance in female pigs less than 1.5
years [44].
Whether humans are infected in the urban area or not, is not
known. In the entire Can Tho city province the reported incidence
of acute encephalitis has been on average 2.4 cases per 100 000
inhabitants during 2009–2012, with the majority of cases being in
children under 6 months of age [51]. As in other endemic areas
the number of clinical cases in adults is relatively low, due to the
acquired natural immunity in the adult population [52], but the
risk for clinical disease may be much higher for non-immune
visitors from non-endemic areas. Vaccination against JEV is
increasing in Vietnam although not all children are covered yet
[14]. With increasing indications of risks for urban transmission of
JEV there may be cause to revise vaccination policies.
In conclusion, the present study demonstrates the presence of
JEV within an urban area by finding both serological evidence of
widespread infections in pigs and mosquitoes PCR-positive for the
virus.
Acknowledgments
The authors want to thank Luu Huu Manh, Can Tho University, for
providing excellent working facilities in Can Tho. Virus was provided by
Sirkka Vene, Swedish Institute for Control of Communicable Diseases.
The authors want to thank Nguyen Thanh Thu, Truong Van Nho and
Huynh Ngoc Trang for their assistance with the field work in Can Tho,
translations and practical issues. The authors want to acknowledge Alyssa
Pyke, Queensland Health, Australia and Jianning Wang, Australian
Animal Health Laboratory, Australia, for helpful advice and Mikael Berg
and Giorgi Metreveli, Swedish University of Agricultural Sciences,
Department of Biomedicine and Veterinary Public Health, for their input
in the laboratory work. We would like to thank three anonymous reviewers
for constructive criticism.
Author Contributions
Conceived and designed the experiments: JFL KS JC HTVT UM.
Performed the experiments: JFL HTVT. Analyzed the data: JFL KS.
Contributed reagents/materials/analysis tools: KS. Wrote the paper: JFL
KS UM SB JC HTVT.
JEV in Pigs and Vector in Can Tho City, Vietnam
PLOS Neglected Tropical Diseases | www.plosntds.org 7 April 2013 | Volume 7 | Issue 4 | e2153
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