Epidemiological concordance of Japanese encephalitis virus infection among mosquito vectors, amplifying hosts and humans in India
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Epidemiol. Infect. (2013), 141, 74–80. f Cambridge University Press 2012
doi:10.1017/S0950268812000258
Epidemiological concordance of Japanese encephalitis virus
infection among mosquito vectors, amplifying hosts and humans
in India
J. B O R A H , P. DU T T A *, S. A. K HA N AND J. M A H A N T A
Regional Medical Research Centre, ICMR, Northeast Region, Dibrugarh, Assam, India
Received 10 July 2011; Final revision 6 January 2012; Accepted 1 February 2012;
first published online 24 February 2012
SUMMARY
A temporal relationship of Japanese encephalitis virus (JEV) transmission in pigs, mosquitoes
and humans revealed that sentinel pig seroconversions were significantly associated with human
cases 4 weeks before (P=0.04) their occurrence, highly correlated during the same time and
2 weeks before case occurrence (PTemporal concordance of JEV infection 75
Assam
Lahoal
Barbaruah
Dibrugarh district
Fig. 1. Map showing the location of Assam in India and Barbaruah and Lahoal in Dibrugarh district of Assam.
In Assam, pigs are likely to be the major amplifying prevention programmes not only in the state of
host as there is a high prevalence of JEV antibody in Assam but also in other JE endemic areas of the
swine sera [9]. The high occurrence of JE cases country.
in Assam may be related to the increasing number of
pigs in recent years. Pigs are currently not vaccinated
against JEV in India, including Assam. Reliable pre- METHODS
diction of epidemiological concordance of JEV infec-
Study area
tion in swine, mosquito and human populations
during any specified period in a specific endemic The study was conducted during 2009 and 2010 in two
locality is important for practical reasons that include areas, Barbaruah and Lahoal in Dibrugarh district,
initiation of mosquito control and vaccination pro- Assam, India (Fig. 1). These two areas were selected
grammes in pigs and humans and to analyse the due to JEV endemicity, abundance of breeding places
mechanism of seasonal spread of infection from one for JE vector mosquitoes and extensive pig rearing by
host to another. So far, no systematic study of this the villagers [9]. Table 1 provides details of the in-
nature has been conducted in India, the outcome of creasing pig population in the study areas. Rice is the
which is very important in order to develop a strategic major crop cultivated between May and August and
plan for JE prevention and control and to assess the rice fields are generally located within a 2 km radius of
success of these programmes in endemic areas. human habitation.
Therefore, the present study aims at identifying the
temporal transmission pattern of JEV infection by
Sentinel pig seroconversion
tracking the sentinel pigs, mosquito vectors and inci-
dence of human JE cases in Assam. This information Twenty locally procured, post-weaned pigs (aged 3–6
will provide baseline data to design and implement JE months) of both sexes and JEV antibody-negative by
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https://doi.org/10.1017/S095026881200025876 J. Borah and others
Table 1. Increasing trend of pig population in Lahoal Each pool was triturated mechanically in a sterile
and Barbaruah primary health centre, 2006–2009 mortar and pestle in 3–5 ml of 2 % fetal calf serum in
minimum essential medium containing 50 mg genta-
Years
micin, 50 IU penicillin, 50 mg streptomycin and 50 mg
Study area 2006 2007 2008 2009 amphotericin B per ml and centrifuged at 1100 g for
15 min at 4 xC. The supernatant was filtered through
Lahoal 4936 5765 9897 13 796 a 0.2 mm filter, aliquoted and tested for the presence
Barbaruah 5674 7973 12 463 18 775
of JEV antigen using an antigen-capture ELISA
Source : Directorate of Animal Husbandry and Veterinary [19]. The supernatant was subjected to RNA
Department, Government of Assam. extraction by using a QIAmp viral RNA mini kit
(Qiagen, Germany). Purified RNA was used as
template for cDNA synthesis using First Strand
haemagglutination inhibition (HI) test were dis- cDNA Synthesis kit (Fermentas, USA). Reverse
tributed to locally identified households for rearing in transcriptase–polymerase chain reaction (RT–PCR)
each area in January 2009. A similar process was fol- was performed to amplify the 348 bp C-prM gene re-
lowed in the next year after allowing the beneficiaries gion [20] of JEV by using cDNA (2 ml) as template.
to dispose of the first set of pigs in December
2009. Blood samples were collected every 2 weeks
from the sentinel pigs. JEV antibody seroconversion Minimum infection rate (MIR)
in pigs was determined by HI test [13]. Sera were The virus infection rate in mosquitoes was expressed
serially diluted twofold from 1:10 to 1:20480. Sera as MIR/1000 mosquitoes of a particular species
with an HI titre of o1:20 were considered as tested [21].
positive [14].
no: of mosquito pools positive
MIR=1000= r1000:
total no: of mosquitoes tested
JEV antigen preparation
Formalin inactivated antigen was prepared from cell
culture propagated virus using the BHK-21 cell line Human JE cases
[15] at the Regional Medical Research Centre, Collection of samples from the study area (Barbaruah
ICMR, Dibrugarh, Assam, India. The maximum and Lahoal) was limited to those that matched the
haemagglutination titre of the prepared antigen World Health Organization clinical case definition of
usually reached 1:10240. This antigen was used to test Acute Encephalitis Syndrome [22] and which were
pig sera by HI [13]. laboratory-confirmed, typically by detecting IgM
antibodies in sera or cerebrospinal fluid (MAC
ELISA kit ; NIV, India) as well as through a fourfold
Mosquitoes
rise of antibody titre in paired sera. A second sample
Adult mosquitoes were collected bi-weekly around was collected from patients with JEV infection 14-21
sentinel pig sheds and in human habitats during dusk days after the first sample had been collected. The
hours by using suction tubes (aspirators). Species fourfold rise in neutralizing antibody titre in paired
identification was done by using the mosquito identi- sera was determined by a microseroneutralization
fication keys [16–18]. Mosquitoes were stored, in assay [23]. Written informed consent was obtained
species order, in Eppendorf vials (2 ml) in pools of up from the patient or guardian before collection of the
to 50 mosquitoes in each vial. Species-wise per man sample. Ethical clearance for the present study was
hour density (PMHD) of mosquitoes was calculated obtained from the institutional ethics committee of
according to the formula : the Regional Medical Research Centre, ICMR, NE
Region, Dibrugarh, Assam, India.
Nr60
PMHD=
TrP
where, N=number of mosquitoes collected ; T=time Data analysis
spent (in minutes) ; P=number of persons involved in JEV infection in mosquitoes, i.e. MIR/1000 mos-
collection. quitoes, was calculated for each month. Cross-
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https://doi.org/10.1017/S0950268812000258Temporal concordance of JEV infection 77
(a)
250 16
No. of pig seroconverted /
14
No. of human JE cases
200 New set of pigs
introduced 12
150 10
PMHD
8
100 6
4
50
2
0 0
J F M A M J J A S O N D J F M A M J J A S O N D
2009 2010
Month
Sentinel pig seroconversion No. of human cases Cx. triaeniorhynchus
Cx. vishnui Cx. pseudovishnui
(b)
4·5 16
4·0 14
No. of pig seroconverted /
No. of human JE cases
3·5 12
3·0 New set of pigs 10
MIR/1000
2·5 introduced
8
2·0
6
1·5
1·0 4
0·5 2
0 0
J F M A M J J A S O N D J F M A M J J A S O N D
2009 2010
Month
Fig. 2. Relationship between pig seroconversion, (a) abundance and (b) JEV infection in Cx. tritaeniorhynchus, Cx. vishnui
and Cx. pseudovishnui vector mosquitoes. PMHD, Per man hour density ; MIR, minimum infection rate.
correlation analysis was applied to detect the lag over 2 years. Nineteen of 281 Cx. tritaeniorhynchus
period between pig seroconversion and occurrence of pools, 7/198 Cx. vishnui pools and 3/107 Cx.
human JE cases. A survival analysis was performed pseudovishnui pools collected during March to
on the pig serology results stratified by the presence September were found positive by antigen-capture
or absence of JEV-positive mosquito pools and a ELISA and RT–PCR. The results of this study clearly
Kaplan–Meier survival curve was plotted. Data were indicated the seasonal variation in occurrence of JEV
electronically analysed using statistical software infection in Cx. tritaeniorhynchus, Cx. vishnui and Cx.
(SPSS version 13, USA). pseudovishnui, the principal JE vectors of India, in
both PMHD and MIR peaks (Fig. 2). The MIR of
Cx. tritaeniorhynchus was found to be highest with an
RESULTS
average of 0.9 (range 0.0–4.3) during the 24-month
JEV seroconversion was seen in all 40 sentinel pigs study period followed by Cx. vishnui (average 0.4,
during 2009 and 2010, starting from March and April range 0–2.9) and Cx. pseudovishnui (average 0.2,
of the first and second year, respectively. Most of range 0–2.1)
the sentinel pigs became positive within 3 months, The monthly number of pig seroconversions during
i.e. June–August, with a peak in July in both years 2009 and 2010 compared to the monthly abundance
(Fig. 2). of the Cx. vishnui subgroup of mosquitoes (PMHD)
A total of 28503 mosquitoes, i.e. Cx. tritae- and JEV infection (MIR/1000) is shown in Figure 2.
niorhynchus, Cx. vishnui and Cx. pseudovishnui col- In 2009, the peak in pig seroconversions coincided
lected and stored in 586 pools were screened for JEV with the increase in abundance of all three Cx. vishnui
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https://doi.org/10.1017/S095026881200025878 J. Borah and others
1·0 1·0
Survival distribution function
0·8
0·8
No postitive mosquito 0·6
Correlation coefficient
0·6 detected
0·4
0·4
0·2
0·2 Postitive mosquito 0·0
detected
–6 –4 –2 0 2 4 6
0·0 –0·2
0 100 200 300 400 –0·4
Weeks
Days in study
–0·6
Fig. 3. Kaplan–Meier analysis to pig seroconversion strati-
Fig. 4. Relationship between human case counts and lagged
fied by mosquito positivity (PTemporal concordance of JEV infection 79
better assessed by shortening the time interval of administrator for management of any future outbreak
blood sampling from 2 weeks to 1 week [30]. of JE in areas under their jurisdiction.
Our study revealed that both MHD and MIR in the
Cx. vishnui subgroup of mosquitoes, the principal JE
vector in India [31] showed seasonal variation [8, 32, ACKNOWLEDGEMENTS
33]. There were three distinct peaks of MIR in June, J. Borah acknowledges the Indian Council of Medical
July and August in both years. It is noteworthy that Research, Government of India, for providing a se-
JEV infection was detected in the same month in pigs nior research fellowship (SRF) to carry out the study.
and mosquitoes, and peaks of pig seroconversion We thank the Directorate of Animal Husbandry and
preceded by 1–2 months the peaks of JEV infection in Veterinary Department, Government of Assam, India
vectors. The present study revealed that the median for providing pig population data. Technical assist-
time to seroconversion in pigs is significantly associ- ance received from N. K. Baruah, P. K. Doloi, R.
ated with the MIR of vector mosquitoes. More par- Doloi, L. J. Borah, P. Chowdhury and R. Topno in
ticularly, the MIR of vector mosquitoes can be used laboratory and field work is gratefully acknowledged.
as a tool to predict the appearance of JEV infection in
pigs [24, 34].
Our study gives a clear understanding of the tem- D E C L A R A T I O N OF IN T E R E S T
poral transmission patterns of JE in mosquitoes, pigs
None.
and humans. This information will be extremely
helpful in developing a strategic plan for JE preven-
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