The Incidence of Japanese Encephalitis in Taiwan—A Population-Based Study

<div><p>Background</p><p>A mass Japanese encephalitis (JE) vaccination program targeting children was launched in Taiwan in 1968, and the number of pediatric JE cases substantially decreased thereafter. The aim of this study was to elucidate the long-term trend of JE incidence, and to investigate the age-specific seroprevalence of JE-neutralizing antibodies.</p><p>Methodology/Principal Findings</p><p>A total of 2,948 laboratory-confirmed JE cases that occurred between 1966 and 2012 were analyzed using a mandatory notification system managed by the Centers for Disease Control, Taiwan. A total of 6,594 randomly-sampled serum specimens obtained in a nationwide population-based survey in 2002 were analyzed to estimate the seroprevalence of JE-neutralizing antibodies in the general population. The average annual JE incidence rate of the group aged 30 years and older was 0.167 cases per 100,000 people between 2001 and 2012, which was higher than the 0.052 cases per 100,000 people among those aged under 30 years. These seroepidemiological findings indicate that the cohort born between 1963 and 1975, who generally received two or three doses of the vaccine and were administered the last booster dose more than 20 years ago, exhibited the lowest positive rate of JE-neutralizing antibodies (54%). The highest and second highest antibody rates were observed, respectively, in the oldest unvaccinated cohort (86%) and in the youngest cohort born between 1981 and 1986, who received four doses 10–15 years ago (74%).</p><p>Conclusion/Significance</p><p>Over the past decade, the main age group of the confirmed JE cases in Taiwan shifted from young children to adults over 30 years of age. People who were born between 1963 and 1975 exhibited the lowest seroprevalence of JE-neutralizing antibodies. Thus, the key issue for JE control in Taiwan is to reduce adult JE cases through a cost-effective analysis of various immunization strategies.</p></div

Incidence rates and onset age distributions of Japanese Encephalitis confirmed cases in Taiwan, 2002–2012.

a<p>The incidence rate is the number of JE confirmed cases per 100,000 population at risk.</p>b<p>There were two JE confirmed cases have been received 1 dose of vaccine in 2008 and one has been vaccinated with 3 doses, in 2012.</p

The cases number of Japanese Encephalitis and incidence rate in Taiwan during 1966–2012.

<p>The cases number of Japanese Encephalitis and incidence rate in Taiwan during 1966–2012.</p

Japanese Encephalitis incidence rates among different birth cohorts during 1966–2012.

<p>Japanese Encephalitis incidence rates among different birth cohorts during 1966–2012.</p

Age-specific incidence rates of Japanese Encephalitis during 1966–2012.

<p>Age-specific incidence rates of Japanese Encephalitis during 1966–2012.</p

Binding specificity and predominant epitope recognized by anti-E Abs in serum from a DENV1 case.

<p>(A) Binding specificity was examined by Western blot analysis as described in Methods. Lysates of 293T cells transfected with pCB-D1 (D1 tr) were also included. (B) Dot blot assay presented as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd-0001447-g001" target="_blank">Figure 1A and 1C to 1E</a> (except that WT dot in row 8C and 153NA dot in row 2H were omitted) was probed with the tested serum or mixed sera, which consisted of a pool of 9 sera from confirmed dengue patients <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Lai1" target="_blank">[44]</a>. The relative intensities of two-fold dilutions of WT dots in row 1 were presented as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd-0001447-g001" target="_blank">Figure 1D</a>. R.I. of each mutant was shown as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd-0001447-g001" target="_blank">Figure 1E</a>. One representative experiment of two was shown. (C) Capture ELISA using WT or mutant VLPs was presented as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd-0001447-g001" target="_blank">Figure 1F</a>. Upper graph in panel C shows comparable amounts of WT and mutant VLPs added.</p

Specificity and epitope of flavivirus GR mAb DEN2-12.

<p>(A) Layout of the dot blot assay. (B) Binding specificity was examined by Western blot analysis as described in Methods. Lysates of 293T cells transfected with pCB-D1 (D1 tr) were also included. (C) Dot blot assay using lysates from 293T cells transfected with the WT pCB-D1 or each of the 67 alanine E mutants. Each membrane was probed with mAb DEN2-12 or mixed mAbs (a pool of mAbs recognizing different epitopes). The dots containing mutations in domains I, II and III are underlined by red, yellow and blue lines, respectively; the ID of each dot is shown in panel A. Two-fold dilutions of the WT lysates were dotted on row 1 to assess the exposure of each membrane. Arrows indicate mutants of epitope residues, which showed severe reduction (R.I.≤0.3) in binding by dot blot assay. One representative experiment of two was shown. (D) The relative intensities of WT dots in row 1 showed a linear decrease from 1× to 1∶16 dilution for membranes probed with mixed mAbs (open bar) and mAb DEN2-12 (closed bar). (E) The intensities of each dot were quantified to determine the R.I. as described in Methods. Data are means and standard errors of two experiments. (F) Capture ELISA was performed by using WT or mutant VLPs <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Crill3" target="_blank">[28]</a>, which had severe reduction in binding by dot blot and Western blot analyses. Data are means and standard errors of duplicates from one representative experiment of two. Lower graph shows the amounts of mutant VLPs added, which were not less than that of WT. (G) Structure-based analysis by UCSF chimera program to determine the locations of and distance (°A) between epitope residues from the same (shaded) or adjacent monomer.</p

Summary of predominant epitopes recognized by anti-E Abs in human sera after DENV infection.

<p>*Primary or secondary infection was determined by PRNT₅₀ as described in Methods.</p><p>**DF, dengue fever according to WHO case definition <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-World1" target="_blank">[2]</a>.</p>†<p>Predominant epitopes recognized by anti-E Abs in polyclonal human sera were identified by dot blot assay and residues with severe (reduction in R.I.≥70%) or moderate (50%≤reduction in R.I.<70%, shown in parenthesis) impairment in binding were shown.</p

Summary of the specificity, binding to alanine E mutants and PRNT₅₀ of 12 mAbs.

<p>*Classes of mAbs include GR (group-reactive), CR (complex-reactive), sCR (subcomplex-reactive) and TS (type-specific). Specificity was determined by Western blot (WB) analysis using lysates derived form DENV1, 2, 3, 4 or JEV-infected C6/36 cells as described in Methods.</p><p>**Alanine E mutants with consistent reduction in binding by dot blot and WB analyses are shown. ++: severe reduction in binding (reduction in R.I.≥70%);</p><p>+: moderate reduction in binding (50%≤reduction in R.I.<70%); underlined: reduced binding tested by VLP-capture ELISA.</p>†<p>Different regions (strand, loop, etc.) of domains II and III are defined based on the X-ray structure of DENV E protein <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Modis1" target="_blank">[7]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Modis2" target="_blank">[24]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Shrestha1" target="_blank">[33]</a>.</p>‡<p>PRNT₅₀ was presented as the lowest concentration that inhibited ≥50% of plaques <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001447#pntd.0001447-Chen1" target="_blank">[38]</a>.</p

Location of epitope residues on E protein recognized by potent neutralizing mAbs.

<p>Epitope residues of mAbs against DENV1 (A) and DENV2 (B). Epitope residues are highlighted with dark blue (DENV1 residues), purple (DENV2 residues), or magenta (DENV1 and DENV2 residues at the same position). Top view of E-E dimers (upper) and side view of domain III (lower right) with ribbon presentation of ß-strands and loops (lower left) are shown by the program UCSF chimera.</p